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Expression of Cathepsin Proteinases by Mouse Trophoblast
In Vivo and In Vitro
Department of Cell Biology and Neuroscience, Nelson Labs, Busch Campus, Rutgers University, Piscataway, New Jersey
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
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.
Grant Sponsor: National Institutes of Health; Grant Number:
*Correspondence to: Bruce Babiarz, Department of Cell Biology and
Neuroscience, Nelson Labs, Busch Campus, Rutgers University, Piscataway, NJ 08855. E-mail:
Received 25 March 1999; Accepted 2 September 1999
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.
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.
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
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
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
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).
This work has shown that the cathepsin proteinases
CB, CL, and CD are specifically upregulated in second-
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
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).
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.
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
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
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.
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.
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.
The authors would like to thank Drs. S. J. Chan, D. T.
Denhardt, B. Sloane, R. Hamilton, and D. Sensibar for
the cDNA probes and antibodies used in these studies,
and Chris Tovar for his technical assistance. This work
was supported by NIH grant HD 20581 (BB).
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