The Expression and Function of Cystatin C and Cathepsin B and Cathepsin L During Mouse Embryo Implantation and Placentation

Development 124, 3415-3425 (1997) 3415 Printed in Great Britain © The Company of Biologists Limited 1997 DEV4896

The expression and function of cystatin C and cathepsin B and cathepsin L during mouse embryo implantation and placentation

Suzanne Afonso*, Linda Romagnano and Bruce Babiarz Department of Cell, Developmental and Neurobiology, Nelson Labs, Busch Campus, Rutgers University, Piscataway, NJ 08855, USA *Author for correspondence (e-mail: [email protected])

SUMMARY

The implantation of the mouse embryo requires the con- product of the decidualizing stroma. Message levels first trolled invasion of the uterine stroma by the embryonic tro- increased in peripheral decidualizing cells, with the protein phoblast. This event is dependent, in part, on the secretion localizing close to the embryo during implantation (days of matrix metalloproteinases and serine proteinases for the 5.5-8.5). With the regression of the decidua beginning on extracellular degradation of the uterine matrix. Proteinase day 9.5, a coordinated upregulation of both cathepsin B activity is controlled by stromal decidualization and and cystatin C was observed implying a role for controlled specific proteinase inhibitors. This work adds to our under- cathepsin expression during apoptosis. E-64, a synthetic standing of implantation and placentation by reporting the inhibitor of cathepsins B and L, was injected into pregnant expression and function of another class of females at the stage of blastocyst attachment (days 4.5-5.5). proteinases/inhibitors closely related to invasive cell High doses resulted in the complete failure of implantation behavior. We focused on the cysteine proteinases, cathep- while lower doses resulted in stunted embryos and a sins B and L, and their inhibitor cystatin C. Northern blots reduced decidual reaction. These results suggested that showed that trophoblast expressed cathepsin B throughout cathepsins B and L are necessary for normal embryo devel- the invasive period (days 5.5-10.5). Both cathepsin B opment and uterine decidualization, and that decidua con- message and cathepsin L protein were localized to the tributes to their control by a coordinated expression of mature, invasive trophoblast giant cells. Substrate gel elec- cystatin C within the implantation site. trophoresis showed an increase in giant cell cathepsin activity with enzyme profiles changing at the end of the invasive period. Northern and western blotting showed that Key words: mouse, implantation, placentation, trophoblast, decidua, cystatin C, the main inhibitor of cathepsins, was a major cathepsin B, cathepsin L, cystatin C.

INTRODUCTION invasion is due, in part, to the decidualizing stroma. Decidua is transient tissue, which ﬁrst develops antimesometrially in the Mouse embryo implantation is a highly controlled event uterine chamber. The antimesometrial zone (AMZ) begins requiring invasion of the uterine stroma by the extraembryonic differentiation at the time of blastocyst attachment on day 4.5. trophoblast (for review see Cross et al., 1994). On day 4.5 of Decidualization spreads to the mesometrial zone (MZ), which gestation, the blastocyst attaches to the uterine epithelium and begins its differentiation 2 days later at the time of secondary initiates implantation. The outer cells of the blastocyst, the trophoblast giant cell invasion. Decidualizing cells increase in mural trophectoderm, begin differentiation into invasive size and form extensive junctional complexes (O’Shea et al., primary trophoblast giant cells. These cells breach the uterine 1983; Ono et al., 1989). They remodel the matrix by phago- epithelium and degrade the underlying extracellular matrix to cytosis of collagen type I and produce basement membrane connect with the maternal blood supply. By day 7.5, the components including laminin and collagen type IV (Zorn et implantation site is established. Concurrently, the cells al., 1986; Wewer et al., 1986). These changes allow decidua to adjacent to the inner cell mass, the polar trophectoderm, serve as a thick tissue barrier to uncontrolled trophoblast continue to proliferate and form the ectoplacental cone (EPC). invasion. However, beginning on day 6.5, the decidua cells in On day 6.5, the outer cells of the EPC begin differentiation into close proximity to the invasive trophoblast undergo apoptosis invasive secondary trophoblast giant cells. Placentation is and are removed by trophoblast cell phagocytosis, thus dependent upon the invasion of the uterine stroma by these providing room for the growing embryo (Bevilacqua and Abra- cells from days 7.5-10.5. hamsohn, 1989; Alexander et al., 1996). By the end of the In ectopic sites, trophoblast invasion is uncontrolled and invasive period (day 10.5), the remaining AMZ decidua and destructive but is strictly limited within the microenvironment portions of the MZ decidua totally regress, leaving the mature of the uterus (Kirby, 1965). Uterine control of trophoblast placenta and decidua basalis. 3416 S. Afonso, L. Romagnano and B. Babiarz

Trophoblast invasion is dependent on proteinases for the CA). Restriction enzymes were purchased from New England Biolabs extracellular degradation and phagocytosis of maternal cells (Beverly, MA). The digoxigenin labeling mix and immunodetection and matrix (Bevilacqua and Abrahamsohn, 1989). Mouse tro- kit were from Boehringer Mannheim (Indianapolis, IN). The DAB (3- phoblast has been shown to synthesize and secrete the metal- 3′ diaminobenzidine) substrate kit was from Vector (Burlingame, loproteinases, gelatinase and stromelysin (Harvey et al., 1995; CA). The cystatin C cDNA probe from American Type Culture Col- Lefebvre et al., 1995; Alexander et al., 1996) the serine pro- lection (Rockville, MD) was subcloned into Bluescript II KS+ (Strat- agene, La Jolla, CA). Mouse cathepsin L probe was purchased from teinase, plasminogen activator (Teesalu et al., 1996; Zhang et University Technologies International, Inc. (Calgary, Alberta, al., 1996) and the cysteine proteinase, cathepsin L (Hamilton Canada). Preprocathepsin B cDNA probe was the generous gift of S. et al., 1990, 1991). The decidua participates in the control of J. Chan, University of Chicago, IL. The IgG fraction of normal rabbit these proteinases by secreting plasminogen activator inhibitor serum and the 18s rRNA probe were kindly provided by D. T. and tissue inhibitors of metalloproteinases (TIMP1, TIMP2 Denhardt, Rutgers University, NJ. Cystatin C antiserum was and TIMP3) in specific temporal and spatial patterns (Water- purchased from DAKO (Carpinteria, CA) and cathepsin L antiserum house et al., 1993; Apte et al., 1994; Harvey et al., 1995; was the generous gift of R. Hamilton, Iowa State University, IA. Alexander et al., 1996). Inhibitors of cysteine proteinases have Mice not been well characterized in mouse decidua. However, in vitro, cysteine proteinase inhibitors were shown to prevent Naturally mated mice (CF1) were killed on days 4.5-13.5 of development (day 0 = morning of vaginal plug). Stroma was enzymatically EPC trophoblast invasion (Babiarz et al., 1992). This suggests isolated from virgin female endometrium and females on day 4.5 of that the control of cysteine proteinases are important during pregnancy as described (Romagnano et al., 1996). Decidual capsules mouse implantation. (days 5.5-13.5) were split open and the embryo and trophoblast teased Cysteine proteinases are controlled by members of the free. Decidual tissue was collected from whole decidual capsules or cystatin superfamily, which include both intracellular (cystatin separated into antimesometrial and mesometrial regions by dissection A and cystatin B) and secreted (cystatin C) forms. Cystatin C at the level of the ectoplacental cone (Babiarz et al., 1996). Tro- is a low molecular mass protein (14 kDa) that is expressed in phoblast tissue was collected by microdissection of the EPC from day several tissues and is present in high concentrations in a 7.5 embryos (Romagnano and Babiarz, 1990) and, using fine forceps, number of biological fluids (Abrahamson et al., 1986; Tavera the embryo was withdrawn from the surrounding sheath of primary et al., 1990; Huh et al., 1995). It is known to be an inhibitor of trophoblast. Sheets of primary trophoblast were peeled from days 9.5 and 11.5 implantation sites with fine needles. Whole placentae were the cysteine proteinases cathepsin B and cathepsin L (Barrett collected on days 9.5-13.5. et al., 1984; Abrahamson et al., 1986), which are present in the lysosomal apparatus of phagocytic cells (Everts et al., 1996). Northern analysis The invasive phenotype of many metastatic cell types is asso- Tissues were collected as described above and total RNA was isolated ciated with the increased production and secretion of cysteine using Tri-Reagent. Samples (5 µg) were denatured with glyoxal and proteinases and aberrant regulation of cystatin C (Lah et al., dimethylsulfoxide, resolved on a 1.2% agarose gel and transferred to 1989; Gottesman, 1990; Sloane et al., 1990; Chambers and GeneScreen Plus membrane. Membranes were hybridized with 32P- Tuck, 1993). Cystatin C is also the principle cathepsin inhibitor labeled cystatin C or preprocathepsin B (CB) cDNA probes according regulated in chronic inflammatory diseases (Lenarcic et al., to standard protocol and exposed to X-ray film for 24-48 hours. Sub- 1988; Billing et al., 1992; Lah et al., 1993; Henskens et al., sequently, membranes were stripped and rehybridized with the 18s rRNA loading control. Scanning densitometry was used to quantify 1994) and tissue remodeling events (Lerner and Grubb, 1992). relative mRNA levels by normalizing band intensity to the 18s rRNA. In this paper, we analyze the expression and function of All experiments were repeated in triplicate and analyzed using Image- cystatin C and cathepsins B and L during mouse implantation Quant software (Molecular Dynamics, Sunnyvale, CA). and placentation in vivo. The results show that cystatin C message and protein levels are regulated in a specific spatial Western analysis and temporal pattern that correlates with cathepsin B and L Tissue samples were collected as described above and incubated in production. We also show that perturbation of cysteine pro- 0.02% NP-40 with 0.01% phenylmethyl sulfonyl fluoride (PMSF), 10 teinase activity during early implantation causes abnormal µg/ml aprotinin and 10 µg/ml soybean trypsin inhibitor at 37¡C for embryo development and uterine decidualization. 30 minutes, sonicated on ice and spun at high speed to remove unsol- ubilized material. Total protein from each sample (100 µg) was analyzed by SDS-PAGE on 5-15% gradient gels under reducing con- ditions and transferred to Immobilon-P membranes. Membranes were MATERIALS AND METHODS blocked with 5% Carnation milk in phosphate-buffered saline (PBS), then incubated with rabbit anti-human cystatin C diluted 1:2,000 in Materials 1% Carnation milk in PBS. After washing, the membrane was Tissue dissociation enzymes, proteinase inhibitors, SDS-PAGE incubated with goat anti-rabbit IgG-peroxidase conjugate (1:2,000). supplies, laminin antiserum, secondary antibody conjugates and in All incubations were performed for 1 hour at room temperature. situ hybridization reagents were from Sigma (St Louis, MO). All Bands were visualized by chemiluminescence. Experiments were culture media, serum and supplements were from Gibco Laboratories repeated three times and relative intensities of the bands were quan- (Grand Island, NY). Tri-Reagent was from Molecular Research tified by scanning densitometry as above. For cathepsin L (CL) Center Inc. (Cincinnati, OH) and the Megaprime random priming kit detection in EPC-conditioned media (CM), 12-14 EPCs from day 7.5 was from Amersham (Arlington Heights, IL). GeneScreen Plus nylon embryos were cultured in Dulbecco’s Modified Eagle Medium membrane, Renaissance chemiluminescence kit and [α-32P]ATP were (DMEM) supplemented with 10% bovine calf serum in a single well from DuPont NEN (Boston, MA). The Lowry-based DC Protein of a 96-well plate. After 48 hours, cultures were rinsed in DMEM to Assay kit was from BioRad (Hercules, CA) and the western blotting remove serum proteins and incubated a further 8 hours in 40 µl of medium was Immobilon-P from Millipore (Bedford, MA). Non- DMEM supplemented with 0.2% lactalbumin hydrolysate. The reduced protein standards (Mark 12) were from Novex (San Diego, medium was then collected and added to 10 µl of 4× nonreducing Cystatin and cathepsins in mouse development 3417 treatment buffer. Aliquots of 20 µl were analyzed on 10% SDS-PAGE sodium acetate at pH 5.2 to reactivate enzymes by removal of SDS. minigels. The separated proteins were transferred to Immobilon-P Gels were then incubated overnight in 0.1 M sodium acetate membrane, blocked and incubated overnight with rabbit anti-mouse substrate buffer at pH 5.2 containing 2 mM dithiothreitol, a cysteine cathepsin L diluted 1:200. After washing, the membrane was proteinase-specific enhancer (Dowd et al., 1994). To demonstrate incubated for 2 hours with goat anti-rabbit IgG peroxidase conjugate specific proteinase activities, gels were incubated in substrate buffer (1:200). Bands were visualized by staining with 4-chloro-napthol. with class-specific enzyme inhibitors. These included 1 mM PMSF, 5 mM disodium ethylenediamine tetraacetic acid (EDTA), 0.5 mM In situ hybridization trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane (E-64), 20 The CB cDNA plasmid was linearized with NheI or SalI and the CL µg/ml leupeptin and 100 µM pepstatin A. To detect areas of protease cDNA was linearized with BamHI or HindIII to provide template for digestion (indicated by clear bands against a dark background), gels antisense or sense riboprobes, respectively. The cystatin C cDNA was were stained in Coomassie Blue for 45 minutes, destained in 50% subcloned into Bluescript II KS +, purified and linearized with either methanol/10% acetic acid for 5-10 minutes, followed by rehydration SpeI or SalI to generate antisense or sense cRNA probes, respectively. in 5% methanol/7% acetic acid. Zymograms were replicated a Linearized CB, CL or cystatin C template (1 µg) was used to synthe- minimum of three times to ensure reproducibility of results. size T7 or T3 polymerase-directed digoxigenin-labeled riboprobes with DIG RNA labeling mix. The in situ procedure followed was In vivo cathepsin perturbations described in Schaeren-Wiemers and Gerfin-Moser (1993). Briefly, E-64 was diluted in PBS and injected intraperitoneally at concentra- samples were frozen in liquid nitrogen. Cryosections (15 µm) were tions of either 10, 30 or 50 mg/kg. Pregnant mice were injected once cut and mounted, fixed with cold phosphate-buffered 4% on days 4.5 and 5.5, then killed on day 7.5. Control mice were injected paraformaldehyde, then acetylated with 0.1 M triethanolamine, pH 8, with PBS. Uterine capsules were separated, fixed in Bouin’s and 0.25% acetic anhydride. The slides were prehybridized at room tem- embedded in paraffin by standard protocol. Serial 7 µm sections were perature for 4-6 hours in hybridization buffer (50% formamide, 5× stained with haematoxylin and eosin. Measurements of capsule length SSC, 2% blocking solution provided by Boehringer Mannheim, 0.1% and width were taken from longitudinal serial sections through the N-laurylsarcosine, 0.02% SDS, 250 µg/ml tRNA and 500 µg/ml center of implantation sites. Embryo and EPC lengths were calculated sheared salmon sperm DNA). Hybridization mixture was prepared by from serial cross sections from 6 capsules/treatment. Statistical sig- adding 200 ng of DIG riboprobe per ml of hybridization solution, nificance was analyzed using the Student’s t-test with P<0.01. heating to 85¡C, then chilling on ice. Sections were hybridized overnight at 72¡C, washed with 2× SSC buffer and incubated with anti-digoxigenin antibody. Labeled probe was detected using a col- RESULTS orimetric immunodetection kit containing NBT (4-nitroblue tetra- zolium chloride) and X-phosphate (BCIP, 5-bromo-4-chloro-3- indolyl-phosphate). For negative controls, sections were incubated Cystatin C expression in decidualizing tissue with sense strand cRNA or without probe. Northern analysis showed that cystatin C was expressed and upregulated during stromal decidualization (Fig. 1A). A 0.7 kb Immunohistochemistry transcript was observed in total RNA samples isolated from To localize cystatin C, 7 µm cryosections of uterine tissue from virgin non-pregnant and pregnant uteri on days 5.5-10.5 of gestation. or pregnant mice were fixed in cold acetone for 10 minutes and rehy- The relative levels of cystatin C mRNA in whole decidual drated with PBS. Sections were blocked in 5% Carnation milk in PBS, capsules increased by a factor of 5 from days 5.5-10.5 (Fig. incubated in anti-human cystatin C antiserum diluted 1:50 in 1% 1A). Message levels accumulated slowly during days 5.5-7.5, Carnation milk in PBS. After washing, slides were incubated in goat anti-rabbit IgG-FITC conjugate (1:100). All incubations were then increased sharply at day 8.5 (Fig. 1A). Western analysis performed for 1 hour at room temperature and signal was visualized also identified an increase of cystatin C protein (Fig. 1B). In by epifluorescence using a Nikon Diaphot 300 inverted microscope. non-pregnant uteri, cystatin C was only detected in overex- To localize extracellular laminin, 7 µm cryosections of normal and E- posed films (data not shown). By the time of blastocyst attach- 64-treated day 7.5 capsules were fixed in methanol for 10 minutes, ment on day 4.5, a band of 14 kDa was observed. From days rehydrated in PBS, and digested for 10 minutes in 0.5% Triton X-100 4.5-10.5, cystatin C levels showed a 20-fold increase with the (Glasser et al., 1987). Sections were blocked as above, then incubated largest change in protein accumulation occurring at day 7.5. in laminin antiserum (1:100) followed by goat anti-rabbit IgG-FITC Regional differences in cystatin C protein expression were conjugate (1:100). Antibody incubations were performed at 37¡C for analyzed in decidual capsules dissected into the two zones of 1 hour and signal was visualized by epifluorescence as above. To differentiation. The relative levels of cystatin C in the AMZ localize CL, decidual capsules were fixed with Bouin’s, embedded in paraffin and sectioned at 7 µm. Tissue sections were incubated with decidua increased in a linear fashion from days 6.5-10.5 (Fig. CL antibody (1:600). The signal was visualized with peroxidase-con- 1C). MZ decidua levels remained fairly consistent until late in jugated goat anti-rabbit IgG (1:200) using the DAB Substrate kit for development, with a dramatic increase at day 10.5 (Fig. 1C). peroxidase (Hamilton et al., 1991). Negative control experiments Control western blots using only secondary antibody showed included incubation with the IgG fraction of normal rabbit serum or no labeling (data not shown). secondary antibody alone. Cystatin C and cathepsin B expression in Zymography trophoblast Acidic proteinase activity was detected in CM (20 µl) or tissue µ To assess whether invasion was associated with the production lysates (25 g total protein) after separation on 10% SDS-poly- of trophoblast-derived cystatin C, we examined its expression acrylamide minigels that were copolymerized with 1 mg/ml gelatin. CM was collected from day 7.5 EPCs cultured for 48 hours (see in day 7.5 EPC lysates and placentae from days 9.5, 10.5 and above). Tissue samples including day 7.5 EPCs, primary trophoblast 13.5. Trophoblast-derived cystatin C mRNA was barely from days 7.5, 9.5 and 11.5, and 48-hour day 7.5 EPC outgrowths detectable on days 7.5-10.5 but, on day 13.5, the noninvasive were lysed in 2× non-reducing treatment buffer. After electrophore- placental trophoblast showed increased cystatin C expression sis, gels were washed 2× 15 minutes in 2.5% Triton X-100 in 0.1 M (Fig. 2A). During this time, the invasive cells were found to 3418 S. Afonso, L. Romagnano and B. Babiarz

Fig. 2. Northern analysis of cystatin C or cathepsin B expression in developing placentae. Total RNA was analyzed from days 7.5-8.5 ectoplacental cones and placentae from days 9.5-11.5 and day 13.5. (A) A single 0.7 kb cystatin C band was barely detectable on Fig. 1. Cystatin C expression in pregnant and non-pregnant uteri. days7.5-10.5. High levels were observed in the postimplantation (A) Northern blot of total RNA isolated from stroma of non-pregnant placenta on day 13.5. (B) Hybridization with CB probe detected a uteri and whole decidual capsules on days 5.5-10.5. A 0.7 kb cystatin single transcript at 2.2 kb showing that levels were maintained from C transcript (arrow) was identified. The same blot was stripped and days 7.5-11.5. Both blots were stripped and reprobed with the 18s reprobed with the 18s rRNA control probe (lower panel). Relative rRNA control probe (lower panels). levels of cystatin C expression were measured by scanning densitometry and normalized to the control probe, and showed a 5- fold increase in cystatin C from days 5.5-10.5. (B) Western blot of the implantation site on day 5.5 showed that CB expression cystatin C protein from lysates of non-pregnant uteri and whole was restricted to the uterine epithelium and glands, and tro- decidual capsules on days 4.5-10.5. Cystatin C antibody detected a phoblast giant cells (Fig. 3E). Control sections hybridized with predominant band at 14 kDa. Relative protein levels were quantified using scanning densitometry and an increase of 20-fold in cystatin C sense strand riboprobes of either cystatin C or CB showed no was observed by day 10.5. (C) Western blot of cystatin C protein labeling (Fig. 3F,G, respectively). from lysates of AMZ and MZ samples. In the AMZ, cystatin C levels In situ localization of cystatin C message in day 7.5 decidual steadily increased from days 6.5-10.5. In the MZ, protein levels capsules showed higher levels in the AMZ cells relative to the remained fairly constant except for a large increase on day 10.5 of MZ cells (Fig. 4A). Lower levels of expression were seen in development. st, non-pregnant uteri; AMZ, antimesometrial zone; the visceral endoderm (Fig. 4A). The invasive trophoblast giant MZ, mesometrial zone. cells remained negative while the adjacent decidua showed the highest cystatin C expression (Fig. 4B). Concomitantly, CB message was localized to the differentiated trophoblast giant express mRNA for CB. A 2.2 kb transcript was expressed in cells and not to the immature cells of the developing EPC (Fig. EPCs from days 7.5 and 8.5 and placentae from days 9.5-11.5 4C,D). High levels of CB were also expressed in the visceral (Fig. 2B). endoderm and uterine glands but not in the decidua (Fig. 4C,D). Localization of cystatin C and cathepsin B As development continued, decidual cell regression along In situ hybridization was used to elucidate the uterine cell-type- with extracellular matrix degradation occurred in order to make specific expression patterns of cystatin C and its target enzyme, room for the growing conceptus. From days 9.5-11.5, high CB. In non-pregnant uteri, cystatin C message was restricted levels of cystatin C mRNA were observed throughout the to the uterine glands and epithelium (Fig. 3A). On day 4.5, decidual capsule with increased expression in the apoptotic cystatin C was present in small “islands” of expression that decidua adjacent to the invading trophoblast (Fig. 5A). As the were randomly distributed in the surrounding stroma (Fig. 3B). trophoblast reached the end of the invasive period, giant cells No localized increase in synthesis was invoked at the site of began upregulating the synthesis of cystatin C message (Fig. blastocyst attachment. Sections through the center of the day 5B). These cells continued to express high levels of CB (Fig. 5.5 implantation site showed cystatin C localized to the periph- 5C,D). In addition, high levels of CB were also seen in the eral decidualizing cells of the AMZ and MZ (Fig. 3C). The visceral yolk sac and were observed for the first time in the primary decidual zone (PDZ), which is the avascular region regressing AMZ decidua (Fig. 5C,D). adjacent to the implanting embryo and consists of the most dif- Immunofluorescence using cystatin C antiserum revealed ferentiated stromal cells, showed less intense labeling at this that, on day 4.5, cystatin C protein was restricted to uterine stage (Fig. 3C). The undifferentiated basal stem cell layer, epithelium and glands and scattered cells within the uterine adjacent to the inner myometrium, did not express cystatin C stroma (Fig. 6A). This was similar to the distribution of (Fig. 3C). CB was expressed in the uterine glands and epi- cystatin C mRNA (Fig. 3B). Blastocyst attachment did not thelium in the non-pregnant uterus (Fig. 3D). This was similar appear to induce an increase in cystatin C around the attach- to the pattern seen for cystatin C (Fig. 3A). Sections through ment site. However, on day 5.5, higher concentrations of Cystatin and cathepsins in mouse development 3419

Fig. 4. In situ hybridization of cystatin C (A,B) and CB (C,D) on day 7.5. (A) High levels of cystatin C message Fig. 3. In situ hybridization of cystatin C (A-C) and CB (D,E) during early were detected in the antimesometrial zone relative to the implantation. (A) In non-pregnant uteri, cystatin C mRNA was localized to the mesometrial zone and low levels were seen in the uterine epithelium and glands (arrowheads). (B) On day 4.5 at the site of blastocyst visceral endoderm (arrowhead). Elevated levels were attachment, low levels of cystatin C were observed randomly distributed in the observed in the decidua cells adjacent to the embryo surrounding stroma cells (arrows). (C) Within the center of the day 5.5 implantation (arrow) while the ectoplacental cone remained devoid of site, cystatin C was localized in the peripheral decidualizing stroma while the cystatin C signal. (B) Higher magnification of the primary decidual zone and basal stem cell layer (arrowheads) remained negative. decidual/trophoblast interface showed that the invasive (D) CB expression in the non-pregnant uterus showed colocalized expression with giant cells did not express cystatin C. (C) CB expression cystatin C in the uterine epithelium and glands (arrowheads). (E) By day 5.5, CB was detected in the uterine glands (arrowheads), visceral expression was confined to the primary trophoblast giant cells, the uterine endoderm and differentiated trophoblast giant cells epithelium and glands (arrowheads). Inset: high magnification of embryo, (arrows), but was lacking in the antimesometrial and surrounding giant cells and uterine epithelium. (F) Negative control sections probed mesometrial decidua. (D) Higher magnification showed with sense strand RNA of cystatin C or (G) CB. Bars indicate 100 µm in A,C,D,G, the details of visceral endoderm and trophoblast giant 20 µm in B,E inset, F and 50 µm in E. ul, uterine lumen; b, blastocyst; pdz, primary cell expression of CB. Bars indicate 100 µm in A and C decidual zone; e, embryo; amz, antimesometrial zone; mz, mesometrial zone; gc, and 10 µm in B and D. amz, antimesometrial zone; mz, giant cells. mesometrial zone; epc, ectoplacental cone; gc, giant cells; ve, visceral endoderm; dec, decidua. cystatin C protein accumulated in the PDZ (Fig. 6B), whereas increased throughout the AMZ, with slight expression mRNA was localized to the peripheral decidualizing cells of detectable in the visceral yolk sac (Fig. 6F). At day 13.5, the the implantation chamber (Fig. 3C). Sections incubated with visceral yolk sac (Fig. 6G) and the trophoblast giant cells (Fig. the IgG fraction of normal rabbit serum served as the negative 6H) labeled intensely for cystatin C. control and showed minimal background labeling (Fig. 6C). By day 7.5, cystatin C protein immunolocalization in the PDZ Detection of cathepsin activity showed a higher concentration versus the outer decidua (Fig. To determine if trophoblast cells synthesized or secreted acidic 6D). In the MZ, highest protein levels were seen in the decidua cysteine proteinases, substrate gel electrophoresis was cells adjacent to the developing EPC and in endothelial cells performed. Lysates of day 7.5 EPCs and primary trophoblast lining the decidual sinuses (Fig. 6E). By day 9.5, cystatin C from days 7.5, 9.5 and 11.5 all possessed bands of gelatinase 3420 S. Afonso, L. Romagnano and B. Babiarz

effect on activity was observed with an aspartic proteinase (pepstatin A), serine proteinase (PMSF) or metalloproteinase (EDTA) inhibitor. E-64, a specific inhibitor of CB and CL (Barrett et al., 1982), completely inactivated proteinase activity as did the cysteine proteinase inhibitor, leupeptin (Fig. 7B). This indicated that the observed zymogens were members of the cysteine proteinase family. In vitro differentiating EPCs continued to synthesize the 34 and 37/38 kDa species (Fig. 7C). In EPC-conditioned media, secreted forms of proteinases with molecular masses 31 and 32 kDa were observed (Fig. 7C) suggesting that further processing occurred. These enzymes were also inhibited by E-64 and leupeptin but not by the other class-specific inhibitors (data not shown). Localization and detection of cathepsin L In situ analysis localized CL to the trophoblast giant cells and visceral and parietal endoderm on day 7.5 of development (Fig. 7D). CL message was also observed within the decidualizing stroma with the most intense expression in the lateral sinuses of the mesometrial decidua (Fig. 7D). Since a suitable antibody to CB was not available, we analyzed CL protein expression. CL protein was immunolocalized to trophoblast giant cells and visceral and parietal endoderm in the day 7.5 implantation site (Fig. 7E). Within the decidua, CL expression was highest in the lateral decidualizing zone (Fig. 7E). Higher magnification showed intense staining in small cytoplasmic vesicles of invasive trophoblast giant cells (Fig. 7F). This suggested that increased lysosomal activity occurred in the most differentiated cells compared to the immature cells of the EPC core. Further confirming the secretion of trophoblast cathepsins, western blotting detected CL bands at 35 and 36 kDa in EPC- conditioned media (Fig. 7C). Fig. 5. In situ localization of cystatin C (A,B) and CB (C,D) transcripts on days 9.5 and 11.5. (A) On day 9.5, cystatin C In vivo cathepsin perturbations expression was high throughout the decidual capsule and trophoblast The E-64 inhibitor of cysteine proteinases has been used to giant cell-derived cystatin C was first observed. (B) By day 11.5, study the role(s) of cathepsin enzymes in development. Studies cystatin C mRNA was significantly upregulated in the decidua as in rat have shown that injections of E-64 during organogene- well as the giant cells, and lower expression was detected in the sis produced defects in neural development and embryonic parietal yolk sac and visceral yolk sac. (C) High expression levels of growth (Tachikura, 1990). We used E-64 to determine the CB were detected in the antimesometrial zone, uterine glands (arrowhead), trophoblast giant cells and visceral yolk sac. (D) CB effects of cysteine proteinase inhibition on implantation. Since levels remained high in giant cells and visceral yolk sac on day 11.5. cysteine proteinase inhibitors have been shown to inhibit blas- However, the parietal yolk sac remained negative. Bars indicate 100 tocyst hatching (Ichikawa et al. 1985), intraperitoneal injec- µm in A,C and 10 µm in B,D. amz, antimesometrial zone; mz, tions were made after blastocyst attachment (days 4.5 and 5.5). mesometrial zone; gc, giant cells; pys, parietal yolk sac; vys, visceral Females killed on day 7.5 showed a dose-dependent effect on yolk sac; e, embryo. development. Decidual capsules of control mice and those treated with 10 mg/kg of E-64 were indistinguishable. Normal day 7.5 capsules were egg-shaped and the embryo had activity at pH 5.2 (Fig. 7A). The major enzyme for EPCs, and undergone gastrulation, forming the primitive streak and primary trophoblast from days 7.5 and 9.5 was observed at 34 mesoderm (Fig. 8A). When animals were treated with 30 kDa. Increased levels of the 34 kDa zymogen were apparent in mg/kg of E-64, significant differences were observed when primary trophoblast tissue from days 7.5 and 9.5 compared to compared to the controls (Table 1). Over one-third of the the undifferentiated day 7.5 EPC core. Days 7.5 and 9.5 also capsules contained resorbed embryos and 53% were stunted at expressed minor bands of activity seen as a doublet at 37/38 the egg cylinder stage showing no mesoderm formation (Fig. kDa and a single band at 62 kDa (Fig. 7A). Primary trophoblast 8B). Serial cross-sectional analysis showed that 30 mg/kg of from day 11.5 showed a change in zymogen activity. Most E-64 decreased embryo and EPC length by 40% and 54%, activity was associated with the 62 kDa zymogen with a respectively. Effects were also observed on stromal decidual- decrease in activity of the bands at 34 and 37/38 kDa (Fig. 7A). ization, with abnormalities in the size and shape of the decidual This suggested that, at this stage of development, acidic pro- capsule (Fig. 8B). When measured through the mid-plane, the teinases were in an unprocessed (precursor) or aggregate form. treated decidual capsules were significantly shorter in length Class-specific proteinase inhibitors were used to identify the (77% of the control) and the capsule area was decreased to 69% gelatinases showing activity at pH 5.2 (Fig. 7B). No inhibitory of the controls. Contributing to the observed size and shape dif- Cystatin and cathepsins in mouse development 3421

Fig. 6. Immunofluorescent localization of cystatin C during development. (A) On day 4.5, cystatin C protein was limited to the uterine epithelium (arrow) with no apparent upregulation of the protein at the site of blastocyst attachment. (B) By day 5.5, cystatin C protein appeared in the primary decidual zone surrounding the embryo. (C) Negative control of day 5.5 implantation site labeled with the IgG fraction of normal rabbit serum. (D) On day 7.5, uterine glands were positive for cystatin C (arrowhead) and pericellular accumulation of the protein in the primary decidual zone was observed. (E) In the MZ on day 7.5, the ectoplacental cone did not stain for cystatin C. High levels were observed in the decidua cells adjacent to the ectoplacental cone as well as in the uterine sinuses (arrows). (F) By day 9.5, cystatin C was present throughout the decidual capsule and low levels of label in the visceral yolk sac first appeared. (G) Cystatin C became a predominant product of the visceral yolk sac, but not of the parietal yolk sac by day 13.5. (H) Non-invasive giant cells from day 13.5 stained positive for cystatin C. Bars indicate 15 µm in A, 30 µm in B,C, 24 µm in D-F and 10 µm in G,H. b, blastocyst; pdz, primary decidual zone; e, embryo; epc, ectoplacental cone; vys, visceral yolk sac; pys, parietal yolk sac; gc, giant cell; dec, decidua. ferences was the lack of lateral sinus development in the MZ observed in laminin deposition between control and treated day region of E-64-treated capsules (Fig. 8A,B). Light microscopic 7.5 capsules (Fig. 9). However, laminin organization within the analysis of treated capsules showed that all expected layers MZ clearly delineated the elongated sinuses in normal capsules were present, but reduced in size when compared to controls (Fig. 9A). In treated capsules, the lack of these structures was (Fig. 8C,D). No evidence of abnormal cell death or invasion of reflected by the presence of only small round sinuses (Fig. 9B). lymphatic cells was observed in non-resorbing capsules. When animals were treated with 50 mg/kg of E-64, total resorption of all implantation sites was observed (Table 1). DISCUSSION To further characterize the E-64-induced decidual defect, we used immunofluorescence to examine the pattern of extracel- We have shown that the cathepsin proteinases B and L are lular laminin deposition. No qualitative differences were important regulators of normal development in the mouse and

Table 1. Analysis of day 7.5 decidual capsules from mice treated with the cysteine proteinase inhibitor, E-64 Embryonic stage Decidua Treatment No. of Prim. Stunted Width Length‡ Area Shape¤ (No. of mice) capsules streak* EC† Resorbed (mm) (mm) (mm2) (w/l) Control 45 45 0 0 2.48±0.11 3.32±0.02 6.46±0.92 0.74±0.01 (4) E-64 (10 mg/kg) 11 11 0 0 2.46±0.005 3.13±0.01 6.07±0.14 0.78±0.01 (2) (P>0.9)¦ (P<0.02)¦ E-64 (30 mg/kg) 38 5 20 13 2.18±0.06 2.57±0.11 4.46±0.98 0.85±0.01 (4) (P>0.015)¦ (PӶ0.001)¦ (PӶ0.001)¦ (P<0.006)¦ E-64 (50 mg/kg) 34 − − 34 (4)

*Primitive streak staged embryos with mesoderm formation. †Stunted egg cylinder staged embryos lacking mesoderm. ‡Mean length in millimeters (mm) ± the standard deviation calculated from serial longitudinal sections measured through the center of 6-9 capsules/treatment. ¤Shape determined by the ratio of capsule width to length (w/l) measured from longitudinal sections through the center of 6-9 capsules/treatment. ¶Signiﬁcance was determined using the Student’s t-test where P<0.01. 3422 S. Afonso, L. Romagnano and B. Babiarz

Fig. 7. Detection of cysteine proteinase activity. (A) Gelatin zymogram of acidic proteinase activity from day 7.5 EPCs (lane 1) and primary trophoblast from days 7.5, 9.5 and 11.5 (lanes 2, 3 and 4, respectively). A major 34 kDa band was observed in EPCs, which was upregulated in primary trophoblast giant cells. Minor bands at 37/38 kDa and 62 kDa were also observed in giant cell samples. By day 11.5, high zymogen activity was detected at 62 kDa with decreased activity of the 34 kDa and 37/38 kDa species. (B) Class-specific inhibitors pepstatin A (lane 1), PMSF (lane 2) and EDTA (lane 3) did not inhibit proteinase activity of day 9.5 primary trophoblast. Complete inactivation of proteinase activity was seen in the presence of cathepsin inhibitors E-64 and leupeptin (lanes 4 and 5, respectively). The same results were obtained with day 7.5 EPCs and days 7.5 and 11.5 primary trophoblast (not shown). (C) Zymography of lysates of in vitro differentiating EPCs showed the synthesis of the 34 kDa and 37/38 kDa forms of cysteine proteinases (lane 1). Faint bands at 31 and 32 kDa were detected from EPC- conditioned media (lane 2). CL antiserum detected bands at 35 and 36 kDa in a western blot of EPC-conditioned media (lane 3). (D) In situ localization of CL in the day 7.5 implantation site detected high message levels in the mature trophoblast cells of the ectoplacental cone (arrow), parietal (arrowheads) and visceral endoderm, and decidual cells surrounding the lateral sinuses. (E) Similarly, immunolocalization in the day 7.5 implantation site detected CL in the outer trophoblast cells of the ectoplacental cone, parietal and visceral endoderm, and the lateral decidual region. (F) Higher magnification showed intense labeling of small cytoplasmic vesicles (arrowheads) in the trophoblast giant cells. Bars indicate 40 µm in D, 30 µm in E, and 50 µm in F. mz, mesometrial zone; s, lateral sinus; e, embryo; ld, lateral decidual region; epc, ectoplacental cone; ve, visceral endoderm; pe, parietal endoderm; pdz, primary decidual zone. that their expression is coordinated spatially and temporally found to express CB. A single 2.2 kb CB transcript was detected with their inhibitor, cystatin C, during implantation and pla- which is typical of non-malignant cellular phenotypes. Aggres- centation. Cystatin C message and protein increased continu- sive malignant cell types express additional transcripts of 4-5 kb ally in decidualizing stroma. The increase of protein on day 7.5 (Qian et al., 1989). Like other proteinases such as gelatinase B preceded the increase in message at day 8.5 suggesting post- (Alexander et al., 1996), we have shown that CB and CL are transcriptional regulation of cystatin C. On day 5.5, high levels detected in the outer, more mature trophoblast cells. Both CB of cystatin C message were localized to the peripheral decidua message and CL message and protein were localized to these cells within the implantation chamber and CB message was cells, with CL protein accumulating in small cytoplasmic first detectable in trophoblast giant cells. No increases in vesicles suggesting increased lysosomal activity. Zymography cystatin C or cathepsin B were observed in the basal fibroblast identified cathepsin activity at 34 and 37/38 kDa. Others have layer. Although cystatin C message was highest in the periph- reported active or mature CB and CL species in the same eral decidua cells, the protein appeared localized in the PDZ. molecular mass range (Hamilton et al., 1991; Gong et al., 1993; This localization suggested a role for the PDZ in regulating tro- Mach et al., 1993; Ryan et al., 1995). Cathepsin activity was phoblast cathepsins. From days 7.5 to 9.5, when both AMZ and greater in day 9.5 giant cells versus the undifferentiated day 7.5 MZ regions are actively invaded by giant cells, cystatin C EPCs. The increased production of cathepsins coincided with the message levels increased dramatically and protein continued to period of trophoblast phagocytosis of epithelial cells, maternal be localized close to the invading cells. After the completion blood cells and decidual cells (Bevilacqua and Abrahamsohn, of placental trophoblast invasion (day 11.5), the trophoblast 1989). Non-invasive giant cells showed activity primarily asso- cells expressed high levels of cystatin C. This suggested that ciated with high molecular mass zymogens, most likely repre- they are programmed to regulate their invasive capacity as they senting unprocessed or aggregate forms (Chan et al., 1986). This switch from an invasive cell type to one that provides nour- suggested that the more aggressive trophoblast giant cells ishment and protection to the growing embryo. process cathepsin precursors into the active or mature forms of Throughout the invasive period, trophoblast giant cells were enzymes to facilitate invasion. Cystatin and cathepsins in mouse development 3423

Fig. 9. Immunoﬂuorescent localization of extracellular laminin on day 7.5 of development in normal and E-64-treated capsules. (A) In the normal day 7.5 implantation site, elongated sinuses develop in the lateral region of the MZ surrounded by a well-organized laminin basement membrane (arrows). (B) Day 7.5 capsules treated with E- 64 lacked the elongated lateral sinuses and contained only small round sinuses in the MZ region (arrows). Bars indicate 130 µm.

that soluble cysteine proteinase digests liberated three proteolyt- ically active fibronectin fragments, suggesting that these enzymes can activate latent proteinase activity from the basement membrane and thus initiate a novel proteolytic cascade. In summary, cysteine proteinases may contribute to invasion by the Fig. 8. Comparison of control and E-64-treated day 7.5 implantation digestion of matrix molecules, the extracellular activation of other sites. (A) The control capsule was egg-shaped and the embryo had pro-enzymes and the intracellular breakdown of molecules gastrulated forming mesoderm. (B) Treatment with 30 mg/kg of E-64 caused a decrease in capsule size and a stunting of the embryo phagocytosed by cells. suggesting that CB and CL activity were necessary for In addition to its role in invasion, cysteine proteinases may decidualization, embryo growth and differentiation. Higher contribute to the apoptosis that is necessary for normal devel- magnification of day 7.5 AMZ of normal (C) and E-64-treated (D) opment. At attachment and early implantation, the trophoblast capsules showed three distinct regions: the primary decidual zone, and uterine epithelium are in direct contact until the epithelial the lateral decidual region and the basal stem cell layer. Treated cells undergo apoptosis (Parr et al., 1987; Abrahamsohn and capsules (D) showed a marked decrease in size of all three layers. Zorn, 1993). We have shown that high levels of CB were Length of arrows indicates size of individual layers. Bars indicate expressed in the uterine epithelium on day 5.5 suggesting that µ µ 300 m in A,B and 50 m in C,D. mz, mesometrial zone; pdz, it was one of the genes upregulated during uterine epithelial primary decidual zone; ld, lateral decidual region; bs, basal stem cell cell death. Increased production of CB has been associated layer. with secretory epithelial cell death during mammary gland involution (Tenniswood et al., 1994; Guenette et al., 1994). Decidual cell regression also occurs by apoptosis. This pro- Both CB and CL are capable of digesting matrix molecules grammed cell death begins on day 6 in the PDZ with apoptotic (Burleigh et al., 1974; Mason et al., 1986), including laminin (Lah decidual cells cleared by giant cell phagocytosis to allow for et al., 1989), collagen IV and fibronectin (Guinec et al., 1993). expansion of the implantation site through day 8.5 (Alexander Increases in cathepsin synthesis and secretion are associated with et al., 1996). Shortly after, AMZ decidua begins a program of numerous invasive carcinomas and ras-transformed fibroblasts total regression on day 9.5 spreading in a wave-like fashion to (Sloane et al., 1990; Chambers et al., 1992). Lower molecular the MZ on day 10.5 (Akcali et al., 1996; Gu et al., 1994). mass forms of cysteine proteinase zymogen activity were Electron microscopic analysis of regressing decidua showed an observed in EPC-conditioned media compared to EPC lysates accumulation of autophagosomes and lysosomes (Abraham- suggesting that further processing occurs when these enzymes are sohn, 1983). Our data showed that CB expression significantly secreted. Detection of CL in trophoblast-conditioned media increased in the AMZ and MZ decidua after day 9.5 of confirmed the secretion of this enzyme by mouse trophoblast gestation, suggesting that this enzyme may play a role in their (Hamilton et al., 1991) and supported a possible role in extra- apoptotic program. Cystatin C levels also increased in these cellular matrix degradation. CB and CL also activate other pro- regions and cystatin C expression was co-localized with CB teinases involved in matrix degradation. 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