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INTRODUCTION |
Scrapie in sheep and goats is a naturally occurring fatal
neurodegenerative disorder characterized by accumulation of the abnormally folded,
PK1-resistant prion protein
(PrP-Sc) (1, 2). In contrast to the ubiquitous distribution of the
cellular, PK-sensitive prion protein (PrP-C) (3, 4), PrP-Sc
accumulation in sheep is limited to the central and peripheral nervous
system (5, 6), the lymphoreticular system (6), and placenta (7-9).
PrP-C is synthesized primarily as a
glycosylphosphatidylinositol-anchored glycoprotein found in the
cell membrane of a variety of cell types (10). A secretory form of
PrP-C lacking the glycosylphosphatidylinositol anchor is present in
cell-free translation systems, cell cultures, human cerebrospinal
fluid, and platelets (11-15). Previous studies demonstrated that PrP-C
is the substrate for conversion to PrP-Sc and is required for scrapie
infection (16-19).
Scrapie can be transmitted experimentally through multiple routes (20).
However, natural scrapie transmission may be associated primarily with
exposure to the infectious agent in the placenta. Pattison et
al. (7, 8) reported that oral inoculation with placental membranes
derived from scrapie-infected ewes caused scrapie in both sheep and
goats. Race et al. (9) recently showed that PrP-Sc was
detected by Western blotting in the placenta from scrapie-infected ewes
and scrapie infectivity in tissue homogenates of placental membranes.
Under natural conditions, fetal fluids and placental tissues are
expelled during or shortly after parturition and contaminate the body
surface of the dam and the area where lambing takes place. Lambs may be
exposed to scrapie by ingesting the transmissible agent during contact
with contaminated wool and mammary gland of the dam. Adult sheep in the
same flock may also be exposed orally through contaminated feed sources.
In the present study, we investigated the distribution of PrP-C
and biochemical properties of PrP-C and distribution of PrP-Sc in the
ovine female reproductive, placental, and selected fetal tissues and
fetal fluids of infected and uninfected ewes. In contrast to the
ubiquitous distribution of PrP-C in the female reproductive tract and
conceptuses, PrP-Sc was present only in the uterine caruncular
endometrium and placental cotyledonary chorioallantois of pregnant
infected ewes. N-terminally truncated, glycosylated, PK-sensitive PrP-C
with apparent molecular masses of 23-37 kDa was present in
cotyledonary chorioallantois, allantoic fluid, fetal bladder, and fetal
kidney as well as endometrium, myometrium, oviduct, and ovary of
pregnant and nonpregnant uninfected ewes. PrP-C levels were low or
undetectable in intercotyledonary chorioallantois, amnion, urachus,
amniotic fluid, and fetal urine. In pregnant ewes, cotyledonary
chorioallantois, allantoic fluid, or caruncular endometrium contained
higher (p < 0.05) levels of PrP-C than did intercaruncular endometrium, myometrium, oviduct, ovary, fetal bladder,
or fetal kidney. PrP-C expression in caruncular endometrium was
up-regulated (p < 0.05) during pregnancy when compared
with caruncular endometrial PrP-C of nonpregnant ewes. Pregnancy status had no effect on PrP-C expression in the brain, myometrium, oviduct, and ovary. PrP-C detected in uninfected ewes had a protein core of
~18-22 kDa in tissues and ~16.5 kDa in allantoic fluid.
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EXPERIMENTAL PROCEDURES |
Animals and Tissue Collection--
Uninfected (n = 10) and scrapie-infected (n = 4) Suffolk sheep were
used. All uninfected ewes and rams were from flocks with no reported
history of scrapie and negative for PrP-Sc by live animal testing
(immunohistochemistry of third eyelid lymphoid tissue) prior to mating,
and no PrP-Sc was detected in brain or lymphoid tissues by
immunohistochemistry and Western blotting following necropsy (21, 22).
Scrapie-infected ewes were identified by positive third eyelid testing,
and scrapie was confirmed by immunohistochemistry and Western blotting
of brain and lymphoid tissue following necropsy. All sheep were
homozygous for glutamine at codon 171 of the PrP gene (23). Pregnant
scrapie-infected ewes (n = 4) were mated to rams at the
United States Department of Agriculture research support facility in
Pullman and estimated to be near term pregnancy at euthanasia. The
fetuses of these infected ewes were also homozygous for glutamine at
codon 171 of the PrP gene. Uninfected ewes (n = 6) were
checked daily for estrus in the presence of vasectomized rams and the
first day of exhibiting estrous behavior is referred to as Day 0 of the estrous cycle. All ewes were mated to intact rams two to three times at
12-h intervals starting on Day 0 of the second estrous cycle. All
pregnant uninfected ewes used in this study were 135-140 days pregnant
(full term pregnancy for sheep is 144-150 days) at euthanasia.
Reproductive tissues from nonpregnant uninfected ewes
(n = 4) were also examined. Sheep were fed with hay and
alfalfa and had free access to water. All sheep were euthanized
immediately prior to necropsy. Maternal tissues (brain, uterine
caruncular endometrium, and intercaruncular endometrium, uterine
myometrium, oviduct, and ovary), fetal tissues (brain, kidney, and
bladder), placenta and fetal membranes (cotyledonary and
intercotyledonary chorioallantois, amnion, and urachus), and fetal
fluids (allantoic fluid, amniotic fluid, and fetal urine) were
collected, frozen, and stored at
80 °C until use (Fig.
1). Animal care, handling, and use in
this study were approved by the Institutional Animal Care and Use
Committee of Washington State University.

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Fig. 1.
A schematic representation of the ovine
pregnant uterus with a fetus and associated membranes and the
placentome. A, pregnant uterus with fetus and
fetal-associated membranes; B, an ovine placentome. Sheep
and goats possess a synepitheliochorial placenta that is formed by
interdigitation of villi of the uterine caruncular endometrium and
placental cotyledonary chorioallantois. The intercotyledonary
portion of chorioallantois forms only diffuse attachment with the
apposing uterine intercaruncular endometrial epithelium.
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Antibodies--
mAb 97/99 (recognizing an epitope between amino
acid residues 217 and 231 near the C terminus of the ovine PrP) and mAb
8H4 (recognizing epitopes between residues 146 and 200) were used to
identify PrP in ovine reproductive, fetal, and placental tissues and
fetal fluids (22, 24, 25). mAbs 5B2 and 8B4 (recognizing epitopes
located at amino acid residues 34-52 and 36-43) were used to identify
the presence of the N terminus of PrP-C (24, 25).
Tissue Homogenate Preparation--
Tissue homogenates were
prepared with (for PrP-Sc detection) or without (for PrP-C detection)
the use of 10% Sarkosyl and ultracentrifugation. Tissue homogenates
obtained in the absence of Sarkosyl (direct tissue homogenates) were
prepared from either fresh or frozen tissues. Briefly, tissues (100 mg)
were minced with scissors in microcentrifuge tubes or
homogenized with disposable tissue grinders. Homogenates were incubated
at room temperature for 1 h in 900 µl of lysis buffer containing
10 mM Tris (pH 7.5), 0.5% Nonidet P-40, and 0.5% sodium
deoxycholate. Tissue homogenates or fetal fluids were digested with or
without PK (Roche Diagnostic Inc., Indianapolis, IN) at 20 µg/ml for
30 min at 37 °C. PK digestion was stopped by the addition of a
protease inhibitor, Pefabloc (Roche Diagnostic Inc., Indianapolis, IN),
to a final concentration of 4 mM, and the reaction mixtures
were stored at
20 °C until analyzed by electrophoresis and Western
blotting. Tissue homogenates without PK digestion were prepared in the
presence of protease inhibitor mixture following the manufacturer's
instructions (c
mplete, Roche Diagnostic Inc., Indianapolis, IN).
Tissue homogenates enriched for PrP-Sc (PrP-Sc-enriched tissue
homogenates) by differential solubilization and ultracentrifugation obtained in the presence of 10% Sarkosyl were prepared as described by
Race et al. (9) with minor modifications. Briefly, fresh or
frozen tissue (up to 1 g) was minced, homogenized, and incubated at room temperature for 1 h in 10 mM Tris-HCl buffer
(pH 7.5) containing 5 mM MgCl2. An equal volume
of Sarkosyl solution (20% Sarkosyl in 10 mM Tris-HCl
buffer (pH 7.5)) was added to tissue homogenates, which were
centrifuged at 6,000 × g for 10 min; the resultant
supernatants were collected and centrifuged at 348,000 × g for 50 min at room temperature. The pellets were dissolved in 10 mM Tris-HCl (pH 7.5) and treated with or without PK
(Roche Diagnostics Inc., Indianapolis, IN) as described above. The
preparations were centrifuged at 279,000 × g for 30 min, and the pellets were resuspended in 10 mM Tris-HCl (pH
7.5) and boiled for 5 min in sample loading buffer prior to analysis by
Western blotting.
Western Blot Analysis--
Tissue homogenates and fetal fluids
were analyzed by 14% SDS-polyacrylamide gel electrophoresis minigels
(Invitrogen) followed by transfer using a semidry transblotter to
polyvinylidene difluoride membranes (Millipore) prior to detection by
mAbs 97/99, 8H4, 8B4, or 5B2. The membranes were incubated with goat
anti-mouse IgG-horseradish peroxidase (Southern Biotechnology
Associates, Inc., Birmington, AL), developed with a chemiluminescence
substrate (Amersham Pharmacia Biotech), and exposed to x-ray film
(Eastman Kodak Co.) for 1-10 min.
The relative levels of PrP-C in tissue homogenates and fetal fluids
were determined by densitometric analysis of films of Western blotting
using mAb 99/97. Briefly, fixed amounts of tissue homogenates (1.5 mg/lane of wet tissue equivalent) and fetal fluids (7.5 µl/lane of
allantoic, amniotic, or fetal urine) were analyzed by Western blotting
as described above. Brain tissue homogenate was analyzed at 0.15 mg/lane, due to high levels of PrP-C present in the brain. All films
were exposed to Western blots for 1 min, and densitometry was performed
using a digital imaging system (ChemiImager 4000, Alpha Innotech Corp.,
San Leandro, CA). The sensitivity of the assay was determined by
detecting serial dilutions of roPrP (21) (ranging from 1 to 600 ng/lane) suspended in tissue homogenates. Twenty-five nanograms of
roPrP were loaded on one lane of each gel to normalize the relative
levels of PrP-C in tissues and fluids. Results are expressed as
relative units, representing the ratio between densitometric values of
sample PrP-C and roPrP (25 ng).
Deglycosylation--
Deglycosylation was conducted using a
commercial kit following the manufacturer's instructions (Prozyme,
Inc., San Leandro, CA). Briefly, 2 µl of 5× PNGase F buffer and 2 µl of denaturation solution were added to 1 µl of brain homogenate
plus 5 µl of water or 6 µl of extraneural tissue homogenate as
prepared above (200 mg wet tissue in 1 ml of lysis buffer), followed by
incubation at 100 °C for 5 min. After the reaction was cooled to
room temperature, 1 µl of Triton X-100 and 1 µl of PNGase F were
added and incubated at 37 °C overnight. The reaction mixture was
boiled for 5 min in sample loading buffer prior to analyses by
electrophoresis and Western blotting.
Statistics--
Data were analyzed by one-way analysis of
variance with Student-Newman-Keuls multiple comparisons test.
Probability values less than 0.05 were considered statistical significant.
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RESULTS |
PrP-C Is Detected in the Uterine Endometrium, Myometrium, Oviduct,
and Ovary of Pregnant and Nonpregnant Normal Ewes and Cotyledonary
Chorioallantois, Fetal Bladder, and Allantoic Fluid of Pregnant Normal
Ewes--
Representative Western blot results of PrP-C in brain,
reproductive, placental, and selected fetal tissues and fetal fluids of
six pregnant uninfected and four nonpregnant uninfected ewes are
shown in Fig. 2, A-C. PrP-C
was not detectable when the primary mAbs were replaced by isotype
control mAbs, nor when mAb 99/97 was preabsorbed with the immunogen
peptide to which mAb 99/97 was generated (data not shown).

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Fig. 2.
Representative Western blot analysis of PrP-C
in maternal brain and reproductive tissues of pregnant (n = 6) and nonpregnant (n = 4) ewes and in fetal
and placental tissues and fetal fluids (six fetal units were
analyzed). A, tissues and fetal fluids from a pregnant
uninfected ewe analyzed with mAb 99/97; B, tissues and fetal
fluids from a pregnant uninfected ewe analyzed with mAb 8H4;
C, tissues from a nonpregnant uninfected ewe analyzed with
mAb 99/97. m-bra, maternal brain; ca-end,
caruncular endometrium; co-cho, cotyledonary chorioallantois;
ico-cho, intercotyledonary chorioallantois;
all-fl, allantoic fluid; amn-fl, amniotic fluid;
ica-end, intercaruncular endometrium; myo,
myometrium; ovi, oviduct; ova, ovary;
amn, amnion; ura, urachus; f-uri,
fetal urine; f-bla, fetal bladder; f-kid, fetal
kidney; and f-bra, fetal brain. One to one and a half mg of
tissue homogenates (wet tissue equivalent) or 7-9 µl of fetal fluids
per lane were used.
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Fetal brain (Fig. 2, A and B, lane
f-bra) contained PrP-C at an apparent molecular mass of ~25-38
kDa, similar to that in maternal brain (Fig. 2, A
C,
lane m-bra) when probed with mAb 99/97 or 8H4. The molecular
masses of these immunoreactive bands are consistent with the predicted
molecular masses of PrP-C in sheep (4).
PrP-C isoforms with apparent molecular masses of ~23-37 kDa
were also detected by both mAbs 97/99 and 8H4 in tissue homogenates of
maternal reproductive tissues of both pregnant and nonpregnant ewes
(uterine intercaruncular and caruncular endometria, myometrium, oviduct, and ovary; Fig. 2, A
C, lanes ica-end,
ca-end, myo, ovi, and ova),
fetal placenta (cotyledonary chorioallantois; Fig. 2, A and
B, lane co-cho), fetal fluid (allantoic fluid;
Fig. 2, A and B, lane all-fl), and
selected fetal tissues (fetal bladder; Fig. 2, A and
B, lane f-bla). These PrP-C isoforms were either very low or undetectable in the intercotyledonary chorioallantois, urachus, amnion, amniotic fluid, fetal kidney, and fetal urine (Fig. 2,
A and B, lanes ico-cho,
ura, amn, amn-fl, f-kid,
and f-uri). One predominant species of PrP-C was detected in
cotyledonary chorioallantois (~26 kDa), allantoic fluid (27 kDa), and
fetal bladder (27 kDa) (Fig. 2, A and B,
lanes co-cho, all-fl, and f-bla).
Levels of PrP-C in Reproductive, Placental, and Fetal Tissues and
Fetal Fluids of Pregnant and Nonpregnant Uninfected Ewes--
Relative
levels of PrP-C in fixed amounts of tissue homogenates of maternal
brain, caruncular endometrium, cotyledonary chorioallantois, intercotyledonary chorioallantois, allantoic fluid, amniotic fluid, intercaruncular endometrium, myometrium, oviduct, ovary, amnion, urachus, fetal urine, fetal bladder, fetal kidney, and fetal brain of
pregnant ewes and brain, caruncular endometrium, myometrium, oviduct,
and ovary of nonpregnant ewes were determined by Western blotting and
densitometry (Fig. 3). The Western blot
assay was linear over a range of 3-200 ng of roPrP per lane in the
presence or absence of 10% tissue homogenates (data not shown).

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Fig. 3.
Levels of PrP-C in reproductive, placental,
fetal tissues, and fetal fluids were determined in a fixed amount of
tissue homogenates or fetal fluids of pregnant and nonpregnant
uninfected ewes. Affinity-purified roPrP (25 ng/lane), tissue
homogenates prepared from brain (0.15 mg/lane wet tissue equivalent),
all other tissues (1.5 mg/lane wet tissue equivalent), and fetal fluids
(7-9 µl/lane) were analyzed by Western blotting using mAb 99/97.
Films exposed to Western blots for 1 min were analyzed by a digital
imaging system. The results are expressed as relative units,
representing the ratio between densitometric values of sample PrP-C and
roPrP (25 ng). Data were analyzed by one-way analysis of variance with
Student-Newman-Keuls multiple comparisons test. A, levels of
PrP-C in tissues/fluids of pregnant uninfected ewes (tissues and fluids
from six ewes and fetal units were analyzed). B, levels of
PrP-C in tissues of nonpregnant ewes (tissues from four ewes were
analyzed). C, comparison of PrP-C levels in reproductive tissues of
pregnant (n = 6) and nonpregnant (n = 4) ewes. See Fig. 2 legend for explanation of abbreviations.
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Levels of PrP-C in brains of pregnant, nonpregnant, and fetal sheep
were similar (p > 0.05), but were higher
(p < 0.001) than those of PrP-C in extraneural tissues
and fetal fluids (Fig. 3, A-C). In pregnant ewes, similar
(p > 0.05) levels of PrP-C were detected in caruncular
endometrium, cotyledonary chorioallantois, and allantoic fluid (Fig.
3A). Likewise, intercaruncular endometrium, myometrium,
oviduct, ovary, fetal bladder, and fetal kidney also had similar
(p > 0.05) levels of PrP-C (Fig. 3A).
However, PrP-C levels in caruncular endometrium, cotyledonary
chorioallantois, and allantoic fluid were higher (p < 0.05) than those in intercaruncular endometrium, myometrium, oviduct,
ovary, fetal bladder, and fetal kidney. PrP-C in intercotyledonary
chorioallantois, amniotic fluid, amnion, urachus, and fetal urine was
either very low or undetectable (Fig. 3A). In nonpregnant
ewes, levels of PrP-C in endometrium, myometrium, oviduct, and ovary
were similar (p > 0.05) (Fig. 3B). PrP-C in
pregnant caruncular endometrium was higher (p < 0.05) by almost 4-fold than PrP-C in nonpregnant caruncular endometrium (Fig.
3C). Pregnancy had no effect on PrP-C levels in myometrium, oviduct, and ovary (p > 0.05) (Fig. 3C).
PrP-C of the Uterus, Oviduct, Ovary, Cotyledonary Chorioallantois,
Fetal Kidney, Fetal Bladder, and Allantoic Fluid Is Differentially
Glycosylated and N-terminal Truncated--
PrP-C in the ovine brain
was consistently detected as multiple bands with apparent molecular
masses between 25 and 38 kDa (Fig. 2). However, the apparent molecular
masses of PrP-C in reproductive, fetal, and placental tissues and fetal
fluid were variable between 23 and 37 kDa (Fig. 2). To determine
whether apparent variation of molecular masses of PrP-C isoforms in
brain, uterus, oviduct, ovary, cotyledonary chorioallantois, fetal
kidney, fetal bladder, and allantoic fluid was a result of differential
glycosylation, tissue homogenates and allantoic fluid were treated with
endoglycosidase (PNGase F) to remove the carbohydrate structures to
assess the molecular mass of PrP-C protein core (Fig.
4).

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Fig. 4.
Representative results of deglycosylation of
PrP-C in brain, reproductive, fetal, and placental tissues and
allantoic fluid of pregnant uninfected ewes (n = 6). Tissue homogenates (1-1.5 mg/lane wet tissue equivalent) or
allantoic fluid (7-9 µl/lane) was treated with or without PNGase F
at 37 °C overnight followed by Western blot analysis using mAb
99/97. Similar results were also shown for PrP-C in tissues from
nonpregnant ewes (data not shown). See Fig. 2 legend for explanation of
abbreviations.
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Treatment with endoglycosidase reduced the apparent molecular
mass of fetal and maternal brain PrP-C to ~18-22 and ~29 kDa (Fig.
4, lanes m-bra and f-bra). Endoglycosidase
treatment of tissue homogenates of the uterine caruncular endometrium,
cotyledonary chorioallantois, intercaruncular endometrium, myometrium,
oviduct, ovary, and fetal bladder reduced the apparent molecular
mass of PrP-C in these tissues to ~18-22 kDa with a minor
species of 29 kDa in some tissues (Fig. 4, lanes ca-end,
co-cho, ica-end, myo, ovi,
ova, and f-bla). Allantoic fluid PrP-C was
reduced by endoglycosidase treatment to ~16.5 kDa (Fig. 4, lane
all-fl). Similar results were also shown for PrP-C in tissues
collected from nonpregnant ewes (data not shown).
N-terminal truncation is common for PrP-C (26, 27) and, therefore, was
also investigated in this study. mAb 5B2 or 8B4, which recognizes the N
terminus of PrP, reacted to the full-length PrP-C in maternal or fetal
brain, but lacked reactivity to isoforms of PrP-C present in uterine
caruncular endometrium, cotyledonary chorioallantois, allantoic fluid,
uterine intercaruncular endometrium, myometrium, oviduct, ovary, fetal
bladder, and fetal kidney (data not shown). Neither mAb 99/97, 5B2, nor
8B4 detected PrP-C in intercotyledonary chorioallantois, amniotic
fluid, amnion, urachus, or fetal urine (data not shown), further
confirming the absence of PrP-C in these tissues/fluids.
PK Susceptibility of PrP-C and PrP-Sc in the Reproductive, Fetal,
and Placental Tissues and Fetal Fluids of Normal and Scrapie-infected
Ewes--
To determine whether the PrP detected in these tissues was
susceptible to PK digestion, all direct tissue homogenates,
PrP-Sc-enriched tissue homogenates, and fetal fluids of uninfected and
scrapie-infected ewes were digested with PK and analyzed by Western
blotting (Fig. 5). Results showed that
PrP-C from fetal fluid and direct tissue homogenates of pregnant
uninfected ewes was susceptible to PK digestion (Fig. 5A).
Similar results were also shown for PrP-C in tissues collected from
nonpregnant ewes (data not shown). No PrP-Sc was detected when tissues
and fluids of pregnant uninfected ewes were extracted with the
procedure used to prepare PrP-Sc-enriched tissue homogenates (data not
shown).

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Fig. 5.
Representative results of PK susceptibility
of PrP in brain, reproductive, fetal, and placental tissues and fetal
fluids of uninfected (n = 6) and scrapie-infected
(n = 4) ewes. Direct tissue homogenates (1-1.5
mg/lane wet tissue equivalent), PrP-Sc-enriched tissue homogenates
(15-30 mg/lane wet tissue equivalent), or allantoic fluid (7-9
µl/lane) were incubated with or without PK (20 µg/ml) at 37 °C
for 30 min, followed by Western blot analysis using mAb 99/97.
A, PK treatment and Western blot analysis of direct tissue
homogenates of reproductive, fetal, and placental tissues and allantoic
fluid from a pregnant uninfected ewe; similar results were also shown
for PrP-C in tissues from nonpregnant uninfected ewes (data not shown).
B, PK treatment and Western blot analysis of PrP-Sc-enriched
tissue homogenates of caruncular endometrium and cotyledonary
chorioallantois from a pregnant scrapie-infected ewe; PrP-Sc in other
tissue homogenates was not detected (data not shown). See Fig. 2 legend
for explanation of abbreviations.
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PK-resistant PrP-Sc was detected in enriched tissue homogenates of
maternal brain, uterine caruncular endometrium, and placental cotyledonary chorioallantois (Fig. 5B, lanes
m-bra, ca-end, and co-cho), but not in
intercaruncular endometrium, myometrium, oviduct, ovary, fetal spleen,
fetal bladder, fetal kidney, and fetal brain (data not shown) from
pregnant scrapie-infected ewes and their conceptuses. The detection of
allantoic PrP-Sc was inconsistent due to the presence of endogenous
protease inhibitors in allantoic fluid (data not shown) (28, 29).
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DISCUSSION |
The present study demonstrated that PrP-C was differentially
expressed by tissues and fluids of the female reproductive tract and
conceptuses. Despite the wide distribution of PrP-C in the reproductive, placental, and fetal tissues and fetal fluids, PrP-Sc accumulation was only detected in the caruncular endometrium and cotyledonary chorioallantois of the placentome from pregnant infected ewes. These results may implicate a role of PrP-C in reproductive and
fetal physiology and facilitate a further understanding of prenatal,
perinatal, and postnatal transmission of scrapie under field conditions.
The biochemical properties of PrP-C in ovine reproductive, fetal, and
placental tissues and fetal fluids have not been reported. This study
used mAbs recognizing nonoverlapping epitopes of PrP to characterize
PrP-C in these tissues and fluids. mAb binding showed that PrP-C in
these tissues and fluids had an intact C terminus; however, the N
terminus of PrP-C in reproductive, placental, and selected fetal
tissues and allantoic fluid was differentially truncated. After the
N-linked carbohydrate structures of PrP-C were removed, the
protein core of ~29 kDa may represent the full-length PrP-C and that
of ~18-22 kDa may represent the N-terminally truncated, potentially
PK-resistant, C-terminal fragment of PrP-C (24, 26). These results
suggest that PrP-C in these tissues and allantoic fluid is
differentially glycosylated and truncated, giving rise to various
apparent molecular masses. The results are consistent with the
intrinsic partial protease resistance of the C-terminal portion of
PrP-C in several species (27), indicating that an intermediate
C-terminal fragment is present in many of the peripheral tissues as a
result of limited protease digestion of PrP-C prior to a complete
proteolysis. The additional truncation of PrP-C in allantoic fluid may
indicate a further processing by proteases in the allantoic fluid,
which is suggested by the synthesis and secretion of a number of
proteases by the ovine placenta during pregnancy (30, 31).
The present study investigated the distribution of both PrP-C and
PrP-Sc in the ovine reproductive tract, placental and selected fetal
tissues, and fetal fluids. The findings suggest potential sites of
maternal transmission of scrapie. In sheep, the ovum is released from
the ovary at estrus, and it takes 3-4 days for the ovum to reach the
uterus through the oviduct, where the ovum is fertilized to form an
embryo. The embryo will not be hatched from the zona pellucida, a
noncellular mucoprotein structure immediately surrounding the embryo,
until it is in the uterus. The present study showed that PrP-C, but not
PrP-Sc, was present in the ovine ovary and oviduct. Our findings with
four pregnant infected ewes are consistent with the report that scrapie
infectivity was absent from ovaries of seven infected ewes (32).
However, Hourrigan et al. (33) reported that scrapie
infectivity was detected in ovaries of 4 of 14 ewes with unknown
reproductive status. The presence of PrP-C or PrP-Sc in the ova,
preimplantation embryos, and the zona pellucida with attached granulosa
cells has not been reported. Although studies using embryo transfer to
determine scrapie (34-36) or bovine spongiform encephalopathy (37)
transmission by preimplantation embryos were conducted by several
laboratories, the presence of PrP-Sc in embryos and disease
transmission through embryos remain inconclusive (20, 38, 39). More
studies with larger sample size are needed to confirm ovarian PrP-Sc
using both bioassay and Western blotting.
PrP-C was abundantly present in the ovine uterus, which confirmed
the previous finding (4); our study extended that observation by
demonstrating that PrP-C was detectable in direct tissue homogenates of
separated caruncular and intercaruncular endometria and myometrium. In
addition, higher levels of PrP-C were detected in pregnant caruncular
endometrium than in nonpregnant caruncular endometrium, suggesting that
PrP-C is up-regulated during pregnancy. Since PrP-C up-regulation was
limited to the caruncular region (caruncles) of the endometrium of
pregnant ewes, it suggests that the intimate cellular contact or
factor(s) synthesized and released by the apposing cotyledonary
chorioallantois (cotyledon) as well as pregnancy-associated hormones
and cytokines at the interface play a role in regulating PrP-C
synthesis by the maternal endometrium. Since the caruncular endometrium is glandless and PrP-C was low in intercaruncular endometrium (rich in glands), PrP-C in caruncular endometrium may be
primarily contributed by the surface endometrial epithelium. The
present study for the first time demonstrated that PrP-Sc was present
in the caruncular endometrium, but not in intercaruncular endometrium
of the uterus of pregnant scrapie-infected ewes. These findings are
consistent with the observation that low levels of scrapie infectivity
were present in the ovine uterus, although the report provided no
details on reproductive status of the animals and specific parts of the
ovine uterus tested (33).
As the allantois grows during early gestation, it fuses with the
chorion to form chorioallantois, which establishes extensive contact
with the uterine endometrium at both caruncular and intercaruncular regions. The most intimate contact between the uterus and placenta is
within the placentome (Fig. 1). In contrast to the placentation of
primates and rodents, the conceptuses (fetus and associated membranes) of ruminants establish interactions with the uterus through
noninvasive placentation within the uterine lumen. The cellular
interactions and molecular exchange are active at the fetal-maternal
interface of placentomes where leakage of maternal blood by endometrial
villi and phagocytosis of maternal blood and degenerating endometrial
cells by fetal trophoblast cells take place (40-42). The placental
trophoblast cells may acquire nonspecifically pathogens at the
interface during the physiological process of uptake nutrients during
gestation. The present study and studies by others showed that scrapie
infectivity (7-9, 43), PrP-Sc (9), and PrP-C (44) are present in the
ovine uterus and placenta; therefore, PrP-Sc in the cotyledonary
chorioallantois may be derived from direct phagocytosis of or
conversion by the maternal PrP-Sc in the caruncular endometrium of
infected ewes.
As the embryo develops, the amnion is formed and filled with amniotic
fluid in which the embryo is suspended. The fetal urine and lung liquid
secretion are the primary sources for amniotic fluid. The allantois is
formed as a result of outgrowth of the hindgut and filled with
allantoic fluid surrounding the amnion (Fig. 1). The allantoic fluid is
primarily derived from fetal urine drained from the bladder through the
urachus. Allantoic fluid may also be derived from chorioallantois
secretion (45). The present study showed that high levels of soluble
PrP-C were present in allantoic fluid, but not in other fetal fluids
such as amniotic fluid and fetal urine. The source for allantoic fluid PrP-C was not directly determined. The absence of detectable PrP-C in
fetal urine and amnion fluid shown in this study may indicate that
fetal kidney, bladder, and urachus do not secret detectable PrP-C into
the urine, although PrP-C is synthesized by the kidney and bladder.
Thus, we conclude that cotyledonary chorioallantois may be the primary
source for allantoic PrP-C. The presence of PK-resistant allantoic PrP
could not be demonstrated in the present study due to the presence of
protease inhibitors in allantoic fluid.
Taken together, it appears that PrP-Sc is only present in tissues (such
as caruncular endometrium and cotyledonary chorioallantois) expressing
high levels of PrP-C, and the embryo/fetus is physically separated from
the PrP-rich allantoic fluid and chorioallantois by the amnion, which
has no detectable PrP-C. Based on the results of this study and
previous reports, we propose that the ovine embryo/fetus may not be
exposed to scrapie prior to parturition. The oocyte or early conceptus
may not be exposed to PrP-Sc prior to hatching in the uterus, because
the presence of an intact zona pellucida at this stage may protect the
embryos from infection, as suggested by studies of a number of other
pathogens (46). The hatched, preimplantation embryo may not be exposed
to PrP-Sc due to the formation of extraembryonic membranes. After
placentation, the fetus may not be exposed to PrP-Sc due to the
presence of a PrP-negative zone, the amnion, around the developing
fetus during the gestational period. A neonate is most likely to be
exposed to PrP-Sc during or after parturition when fetal membranes are broken and chorioallantoic tissues and allantoic fluid contaminate the
birth canal, body surface, and wool of both the mother and neonate,
mammary glands, and the lambing area. PrP-Sc from both fetal membranes
and fluids may serve as a source for horizontal transmission of scrapie
under natural conditions (7). Overall, we propose that 1) the
embryos/fetuses are less likely to be exposed to scrapie in
utero, 2) lambs are likely to be exposed to scrapie at birth and
during nursing, and 3) wasted placenta and possibly allantoic fluid of
infected ewes may serve as a primary source for horizontal transmission.
The results of this study provide useful information for a
better understanding of maternal transmission of scrapie during the
prenatal, perinatal, and postnatal periods. Despite some differences in
placentation among different species, ovine scrapie represents a
valuable model for investigating PrP-Sc replication at the
fetal-maternal interface in prion diseases such as chronic wasting
disease and variant Creutzfeldt-Jakob disease, in which PrP-Sc is
distributed outside of the nervous system (47, 48). These results also contribute to our understanding of the regulation of PrP-C at different stages of pregnancy and fetal development and the role of
PrP-C in fetal-maternal interactions at the interface.