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Journal of Virology, December 2001, p. 11319-11327, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11319-11327.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Expression of Endogenous Betaretroviruses in the
Ovine Uterus: Effects of Neonatal Age, Estrous Cycle, Pregnancy,
and Progesterone
Massimo
Palmarini,1
C. Allison
Gray,2
Karen
Carpenter,2
Hung
Fan,3
Fuller W.
Bazer,2 and
Thomas E.
Spencer2,*
Department of Medical Microbiology and
Parasitology, College of Veterinary Medicine, The University of
Georgia, Athens, Georgia 306021; Center
for Animal Biotechnology and Genomics and Department of Animal
Science, Texas A&M University, College Station, Texas
778432; and Cancer Research Institute
and Department of Molecular Biology and Biochemistry, University of
California, Irvine, California 926973
Received 27 July 2001/Accepted 28 August 2001
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ABSTRACT |
The ovine genome contains 15 to 20 copies of endogenous
retroviruses (enJSRVs) highly related to the oncogenic jaagsiekte sheep
retrovirus (JSRV) and enzootic nasal tumor virus. enJSRVs are highly
expressed in the endometrial lumenal epithelia (LE) and glandular
epithelia (GE) of the ovine uterus. The effects of neonatal age,
estrous cycle, pregnancy, and progesterone on expression of enJSRVs in
the ovine uterus were determined. Expression of enJSRV RNAs was absent
from the uterus of ewes at birth, but enJSRV RNAs were expressed
specifically in the LE and developing GE from postnatal day (PND) 7 to
PND 56. In adult ewes, enJSRV RNAs were detected only in the epithelia
of the uterine endometrium, as well as epithelia of the oviduct,
cervix, and vagina. In cyclic ewes, endometrial enJSRV RNA abundance
was lowest on day 1, increased 12-fold between days 1 and 13, and then
decreased to day 15. In pregnant ewes, levels of endometrial enJSRV
RNAs were high on day 11, increased to day 13, and then decreased to
day 19. In day 17 and 19 conceptuses, enJSRV RNAs were also detected in
binucleate cells of the trophectoderm. Immunoreactive JSRV capsid and
envelope proteins were detected in the endometrial LE and GE, as well
as in the binucleate cells of the conceptus. In transfection assays utilizing ovine endometrial LE cells, progesterone increased
transcriptional activity of several enJSRV long terminal repeats.
Collectively, these results indicate that transcription of enJSRVs in
the endometrial epithelia of the ovine uterus is increased by
progesterone and might support a role for enJSRVs in
conceptus-endometrium interactions during the peri-implantation period
and early placental morphogenesis.
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INTRODUCTION |
A distinctive feature of
retroviruses is their presence as inherited elements in the germ line
of most eukaryotes. These elements, known as endogenous retroviruses
(ERVs), are transmitted through the germ line as stable Mendelian
genes, yet they exhibit structural and sequence similarities to
infectious exogenous retroviruses (9). It is assumed that
ERVs were derived from integration events during the evolution of
ancient exogenous retroviruses (e.g., transmitted horizontally) into
the germ line of host animal species. In recent years, considerable
effort has been directed toward understanding the biological
significance of ERVs, particularly those present in the human germ line
(8, 30, 31). Generally, endogenous proviruses are
transcriptionally silent and are often defective, typically differing
from the exogenous counterpart by deletions or point mutations that
render them incapable of forming infectious virus (9, 11).
However, several ERVs maintain at least some intact open reading frames
with expression associated with either beneficial or detrimental
effects to the host (9). Specific expression of some ERVs
in the placenta has lead to various hypotheses that these elements play
a role in mammalian reproduction (6, 7, 23, 33, 42, 43, 54,
55).
Sheep represent an interesting model with which to study the biology of
ERVs and their interaction with host species. The ovine genome contains
15 to 20 copies of endogenous retroviruses (enJSRVs) (3, 24, 25,
36, 57) that are highly related to two oncogenic exogenous
betaretroviruses, Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal
tumor virus (ENTV) (14, 40). enJSRV RNAs are highly
expressed in the epithelium of the uterus (38, 48), while
the exogenous pathogenic viruses JSRV and ENTV appear to have a strict
tropism for secretory cells of the respiratory tract (16, 36,
37). Expression of enJSRV RNAs in the ovine uterus was initially
identified by differential display PCR and PCR-based subtraction
hybridization experiments (48). In situ hybridization
analyses discovered that enJSRV RNAs were restricted in expression
to the endometrial lumenal epithelium (LE) and glandular
epithelium (GE) (48). Indeed, the expression level of
the enJSRVs in the uterine endometrial epithelia is very high
relative to a number of other genes expressed in the same epithelia, as
well as expression of enJSRVs in other sheep tissues (35,
38). The high level and specificity of temporal and spatial
expression of enJSRVs in the endometrium of the ovine uterus might
suggest physiological functions of these elements in regulation of
conceptus-endometrium interactions, as well as placental morphogenesis,
during the peri-implantation period. On the other hand, tropism for the
genital tract of the exogenous viral ancestors of the current enJSRVs
might also explain the specific expression of the endogenous loci in
the epithelium of the uterus.
To further investigate the role of enJSRVs in sheep uterine biology,
studies were conducted to determine effects of neonatal age, day of the
estrous cycle and early pregnancy, and progesterone on expression of
enJSRVs in the ovine uterus. Expression of enJSRV RNAs was detected
only in epithelia of oviduct, uterus, cervix, and vagina. Changes in
abundance of enJSRV RNAs in the endometrium during the estrous cycle
and early pregnancy suggested that progesterone, acting via the
progesterone receptor, increases transcription of enJSRV genes.
Transient transfection assays established that progesterone increased
transcriptional activity of several enJSRV long terminal repeats
(LTRs). Immunoreactive JSRV capsid and envelope proteins were detected
in the endometrial LE and GE as well as in binucleate cells of the
conceptus trophectoderm that form syncytia with the epithelium of the uterus.
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MATERIALS AND METHODS |
Animals.
Mature ewes of primarily Rambouillet or Suffolk
breeding were observed daily for estrus by using vasectomized rams. All
ewes exhibited at least two estrous cycles of normal duration (~16 to
18 days). Experimental and surgical procedures involving animals were
approved by the Institutional Agricultural Animal Care and Use
Committee of Texas A&M University.
Study 1: expression of enJSRVs mRNAs in the uterus of neonatal
ewe lambs.
Rambouillet-Suffolk ewe lambs were assigned randomly at
birth (postnatal day [PND] 0) to be necropsied on PND 1 (n = 6), 7 (n = 5), 14 (n = 5), 21 (n = 4), 28 (n = 5), 42 (n = 5), or 56 (n = 5) as described previously (51). The
uterus was obtained and trimmed free of the broad ligament, oviduct,
and cervix. Sections (~1 cm) from the midportion of each uterine horn
were fixed in fresh 4% paraformaldehyde in phosphate-buffered saline
(PBS) (pH 7.2) and embedded in Paraplast Plus (Oxford Labware, St.
Louis, Mo.).
Study 2: expression of enJSRV RNAs in reproductive tract tissues
of adult ewes.
Adult, cyclic ewes (n = 6) were
synchronized to estrus and bred with fertile rams as described
previously (20). On day 9 postmating, ewes were
necropsied, and the entire reproductive tract was excised. Sections of
the oviduct (ampulla and isthmus), cervix, and vagina (anterior and
posterior) were fixed in fresh 4% paraformaldehyde for histology. Each
uterine horn was flushed with 10 ml of Dulbecco's modified Eagle's
medium (DMEM)-F-12 medium (Sigma Chemical Co., St. Louis, Mo.) and
examined for the presence of a hatched blastocyst to confirm pregnancy.
The uterine horns were then separated, and sections from each uterine
horn (near the utero-tubal junction, the midportion, and uterine body)
were fixed in 4% paraformaldehyde and then processed and embedded in Paraplast Plus.
Study 3: expression of enJSRV RNAs and protein in uterus of
cyclic and pregnant ewes.
At estrus, ewes were assigned randomly
to cyclic or pregnant status. Ewes assigned to pregnant status were
bred with intact rams at estrus (day 0) and then ovariohysterectomized
(n = 4 ewes/day) on day 1, 3, 5, 7, 9, 11, 13, or 15 of
the estrous cycle and day 9, 11, 13, 15, 17, or 19 of pregnancy (day
0 = estrus/mating) as described previously (45).
Pregnancy was confirmed by the presence of an apparently normal
conceptus in uterine flushings. At hysterectomy, several sections from
the middle of each uterine horn were fixed in fresh 4%
paraformaldehyde and then embedded in Paraplast Plus.
Several sections of each uterine horn were also embedded in Tissue-Tek
Optimal Cutting Temperature (OCT) compound (Miles, Inc., Oneonta,
N.Y.), frozen in liquid nitrogen vapor, and stored at 
80°C. The
remaining endometrium was physically dissected from myometrium, frozen
in liquid nitrogen, and stored at 80°C for RNA extraction. In
monovulatory pregnant ewes, uterine tissue samples were marked as
contralateral or ipsilateral to the ovary bearing the corpus luteum. No
contralateral uterine samples were used in RNA and protein analyses.
RNA isolation and slot blot analyses.
Total cellular RNA was
isolated from endometrium by using Trizol (Gibco-BRL, Bethesda, Md.).
The quantity of RNA was assessed spectrophotometrically, and the
integrity of RNA was examined by gel electrophoresis in a denaturing
1% agarose gel.
Steady-state levels of enJSRV RNAs were assessed by slot blot
hybridization as described previously (
50). Denatured
total
endometrial RNA (20 µg) from each ewe was analyzed with a
radiolabeled
antisense ovine enJSRV cRNA probe generated from a partial
ovine
endometrial enJSRV cDNA (DD54) that was cloned by differential
display (DD)-PCR as previously described (
48). To
correct for
variation in total RNA loading, a duplicate RNA slot
membrane
was hybridized with radiolabeled antisense 18S rRNA and cRNA
(pT718S;
Ambion, Austin, Tex.). Following washing, nonspecific
hybridization
was removed by RNase A digestion (
2). The
radioactivity associated
with each slot was quantified by electronic
autoradiography with
an Instant Imager (Packard Instrument Company,
Meridian, Conn.)
and expressed as total counts
(TCs).
In situ hybridization analysis.
The enJSRV RNAs were
localized in uterine tissue sections (5 µm) by in situ hybridization
analysis as described previously (48). Deparaffinized,
rehydrated, and deproteinated uterine tissue sections were hybridized
with radiolabeled antisense or sense cRNA probes generated from
linearized ovine endometrial enJSRV cDNA (DD54) (48) by in
vitro transcription with [
-35S]UTP. After
hybridization, washing, and RNase A digestion, slides were dipped in
NTB-2 liquid photographic emulsion (Kodak, Rochester, N.Y.), stored at
4°C for 1 week, and developed in Kodak D-19 developer. Slides were
then counterstained with Harris modified hematoxylin (Fisher
Scientific, Fairlawn, N.J.), dehydrated through a graded series of
alcohol to xylene, and protected with a coverslip.
Immunofluorescence analyses.
JSRV protein was localized in
frozen uterine tissue sections by immunofluorescence by methods similar
to those described previously (26). Briefly, uterine
tissues embedded in OCT compound from study 3 were sectioned with a
cryostat (8 µm) and mounted on Superfrost/Plus microscope slides
(Fisher Scientific, Pittsburgh, Pa.). Frozen sections were fixed in
20°C methanol for 10 min, permeabilized with 0.3% Tween 20 in 0.02 M PBS, and then blocked in antibody dilution buffer (2 parts 0.02 M
PBS, 1.0% bovine serum albumin, 0.3% Tween 20 [pH 8.0], 1 part
glycerol) containing 5% normal goat serum for 1 h at room
temperature. Sections were rinsed in PBS and incubated overnight at
4°C with the primary antibodies towards the JSRV major capsid protein
or the envelope protein described below. Antibodies towards the JSRV
capsid protein (no. 1520) were kindly provided by J. M. Sharp
(Moredun Research Institute, Midlothian, Scotland) and produced as
described previously (37). Antiserum towards JSRV envelope
(no. 1721 and 1723) was obtained by injecting rabbits with synthetic
peptides derived from the envelope region of JSRV (M. Palmarini and H. Fan, unpublished results). Substitution of primary antibody with normal
rabbit serum (Sigma-Aldrich, St. Louis, Mo.) at the same concentration was used as the negative control. Following three rinses in PBS for 10 min each, sections were incubated with fluorescein-conjugated goat
anti-rabbit immunoglobulin G (IgG) (Zymed, San Francisco, Calif.) for
1 h at room temperature and again washed in PBS three times for 10 min each. Coverslips were placed over a layer of Prolong antifade
mounting reagent (Molecular Probes, Eugene, Oreg.).
Transient transfection and luciferase assays.
Immortalized
ovine endometrial LE cells described previously (27) were
grown in DMEM with the addition of 10% charcoal-stripped fetal bovine
serum. Transient transfections were performed on ovine LE cells (2 × 105 to 4 × 105)
plated on six-well plates approximately 24 h prior to
transfection. Each well of subcultured ovine LE cells was cotransfected
with 300 ng of firefly luciferase (luc gene) reporter
plasmid, 1,000 ng of a human progesterone receptor (PR) mammalian
overexpression vector (53), and 50 ng of pRL-null
(Promega, Madison, Wis.), a construct that expresses the renilla
luciferase gene, by using Fugene (Roche, Basel, Switzerland).
Luciferase reporter plasmids were described previously (36,
38). Briefly, pJS21-luc was constructed by placing the
JSRV21 LTR in front of luc. For
construction of penJS56A1-luc, penJS5F16-luc, and penJS59A1-luc, the
LTRs of the endogenous proviral clones enJS56A1, enJS5F16, and enJS59A1 were placed in front of the luc gene. The ENTV-luc has the
ENTV LTR placed in front of the luc gene. The ENTV LTR was
kindly provided by Cristina Cousens (Moredun Research Institute).
Transfected cells were then treated with vehicle as a control or with
progesterone (0.02 M) for 48 h. Cells were then harvested, and
luciferase activity was determined by using the dual luciferase
reporter system (Promega) and a TD 20/20 luminometer (Turner Designs,
Sunnyvale, Calif.). All assays were conducted in triplicate with at
least two independent experiments. Firefly luciferase data were
normalized by using pRL-null luciferase values. Data are expressed as
fold increase in relative light units of progesterone-treated cells
compared to values for untreated cells.
Photomicroscopy and digital imaging.
Images of
representative fields of in situ hybridization and immunofluorescence
slides were recorded with a Zeiss Axioplan2 microscope (Carl Zeiss,
Thornwood, N.Y.) fitted with a Hamamatsu C-5810 chilled three-color
charge-coupled device (CCD) camera (Hamamatsu Corporation, Bridgewater,
N.J.). Digital images were captured and/or assembled with Adobe
Photoshop 4.0 (Adobe Systems, Seattle, Wash.) and a MacIntosh PowerMac
G3 computer (Apple Computer, Cupertino, Calif.). Black-and-white prints
were generated electronically with a Kodak DS8650 color printer.
Statistical analyses.
Data from slot blot hybridization
analyses were subjected to least-squares analysis of variance
(LS-ANOVA) by the general linear models (GLM) procedures of Statistical
Analysis System version 8.1 for Windows (SAS Institute, Cary, N.C.).
Slot blot hybridization data for enJSRV RNAs (total counts) were
corrected for differences in sample loading by using the 18S rRNA data
as a covariate in LS-ANOVA. Data from study 3 were analyzed for effects of day and pregnancy status (cyclic or pregnant), as well as their interaction. Within pregnancy status, LS regression analyses were used
to determine effects of day on endometrial mRNA levels. All tests of
significance were performed by using the appropriate error terms
according to the expectation of the mean squares for error
(49). Data are presented as LS mean TCs with standard errors (SE).
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RESULTS |
Expression of enJSRV RNAs in the uterus of neonatal ewe lambs
(study 1).
Previously, high levels of enJSRV RNAs were detected in
the endometrial epithelium of uteri from adult ewes (48).
In the present study, expression of enJSRV RNAs was studied in the
uteri of neonatal ewe lambs between PND 1 and PND 56, because this
represents a period of active uterine morphogenesis in which the
endometrial GE differentiates from LE (51, 52). As
illustrated in Fig. 1, the endometrial GE
differentiates and buds into the underlying stroma from the LE between
PNDs 1 and 7. Between PNDs 14 and 56, there is extensive coiling and
branching morphogenesis of nascent endometrial glands. By PND 56, the
uterine wall appears to be histoarchitecturally mature
(52). During this period, PR is expressed by the LE and
developing GE, but progesterone is absent or below detectable limits in
the peripheral blood (52).
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FIG. 1.
In situ hybridization analysis of enJSRV mRNA expression
in the developing neonatal ovine uterus. Cross-sections of the uterine
wall from neonatal ewes (PND 0 = birth) were hybridized with
-35S-labeled antisense or sense ovine enJSRV cRNA probes
as described in Materials and Methods. Protected transcripts were
visualized by liquid emulsion autoradiography for 1 week and imaged
under bright-field or dark-field illumination. S, stroma; M,
myometrium. Magnification, ×260.
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In situ hybridization analyses revealed that enJSRV RNAs were absent or
below detectable limits in all uterine cell types
on PND 1 (Fig.
1).
The white cells in the dark-field photomicrograph
of the PND 1 uterine
section are not positive for enJSRV RNAs,
but rather are darkly
pigmented melanocytes that appear white
in dark-field photomicrographs.
In contrast to PND 1, expression
of enJSRV RNAs was detected in the LE
and budding GE on PND 7
and was present in all epithelia to PND 56. Thus, enJSRVs are
expressed in uterine epithelia in the absence of
detectable progesterone
in peripheral blood. We cannot specify which
enJSRV loci are specifically
expressed, because the DD54 probe most
likely cross-reacts with
most enJSRVs, given the high degree of
homology of the known sequences
of these elements (
3,
4,
38).
Expression of enJSRVs in tissues of the adult ovine reproductive
tract (study 2).
In previous studies, enJSRV RNAs were detected in
several tissues, including the uterus and lung (36, 48),
but other tissues in the female reproductive tract were not studied. In
this study, expression of enJSRV RNAs was surveyed in adult
reproductive tract tissues of Müllerian duct origin from day 9 pregnant ewes (Fig. 2). As expected,
abundant expression of enJSRV RNAs was detected in the endometrial LE
and GE of all regions of the uterus. In addition, abundant expression
of enJSRV RNAs was detected in the epithelia of the ampulla and isthmus
regions of the oviduct, as well as in the cervix. Although expression
of enJSRV RNAs was detected in the posterior and anterior regions of
the vagina, expression was very low compared to that in other
reproductive tract tissues.
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FIG. 2.
In situ hybridization analysis of enJSRV mRNA expression
in different tissues of the adult ovine female reproductive tract.
Cross-sections of different regions of the female reproductive tract
from day 9 pregnant ewes were hybridized with
-35S-labeled antisense or sense ovine enJSRV cRNA probes
as described in Materials and Methods. Protected transcripts were
visualized by liquid emulsion autoradiography for 1 week and imaged
under bright-field or dark-field illumination. Magnification, ×260.
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Expression of enJSRV RNAs and proteins in the uterus of cyclic and
pregnant ewes (study 3).
In order to determine the effects of day
of the estrous cycle and early pregnancy on enJSRV expression in the
endometrium, steady-state levels of enJSRV RNAs were determined in
ovine endometrial total RNA by slot blot hybridization analysis with
the DD54 partial enJSRV env cDNA as a probe
(48). As previously reported, Northern blot analyses of
ovine endometrial total RNA with antisense DD54 cRNA detected two RNA
species 7.5 and 2.4 kb in size (48). In analogy with the
exogenous JSRV, the 7.5-kb RNA transcript would represent the
full-length enJSRV genome, whereas the 2.4-kb RNA transcript
corresponds to the correctly spliced env mRNA
(32).
As illustrated in Fig.
3, steady-state
levels of enJSRV RNAs in the endometrium was affected
(
P < 0.01) by day of the estrous
cycle or pregnancy,
but not by pregnancy status (
P > 0.10, day
x status). In cyclic ewes, endometrial enJSRV RNAs increased
12-fold
between days 1 and 13 and then decreased to day 15 (
P < 0.01,
cubic effect of day). The increase in
endometrial enJSRV expression
was highly correlated with changes in
peripheral blood progesterone
content (
46). In pregnant
ewes, endometrial enJSRV RNA expression
was high on day 11, increased
to day 13, and then decreased to
day 19 (
P < 0.01, cubic effect of day).
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FIG. 3.
Effects of day of the estrous cycle or early pregnancy
on expression of enJSRV mRNAs in the ovine endometrium. Slot blot
hybridization analysis of enJSRV mRNAs in endometrium from cyclic and
pregnant ewes was performed as described in Materials and Methods.
Steady-state levels of enJSRV mRNAs are presented as LSM TCs with SE.
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In situ hybridization analyses revealed that changes in the abundance
of enJSRV RNAs in the endometrial LE were similar to
changes in
steady-state levels of enJSRV RNAs in the endometrium
(Fig.
4). As expected, enJSRV RNAs were
expressed predominantly
in the LE and GE of the endometrium, whereas
expression was not
detected in the endometrial stroma or myometrium. In
pregnant
ewes, enJRSV RNAs were abundant in the LE on days 11 to 13, but
declined to almost undetectable levels between days 15 and 19.
Interestingly, the overall abundance of enJSRV RNAs was lower
or absent
in the LE that appeared to be in contact with the conceptus
trophectoderm during this peri-implantation period of pregnancy.
Expression of enJSRV RNAs was abundant in GE of the upper stratum
compactum stroma, whereas expression in the GE in the stratum
spongiosum near the myometrium was less abundant.
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FIG. 4.
In situ hybridization analysis of enJSRV mRNA expression
in the uteri of cyclic and pregnant ewes. Cross-sections of the uterine
wall from cyclic (C) and pregnant (P) ewes were hybridized with
[-35S]-labeled antisense or sense ovine enJSRV cRNA
probes as described in Materials and Methods. Protected transcripts
were visualized by liquid emulsion autoradiography for 1 week and
imaged under bright-field or dark-field illumination. S, stroma; M,
myometrium; TE, trophectoderm. Magnification, ×260, except for the 17P
panel at the bottom (×520).
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In addition to the endometrial epithelia, expression of enJSRV RNAs was
detected in selected, specific trophectodermal cell
types of the
developing placenta. Based on their morphology, these
cells appear to
be the binucleate cells of the conceptus trophectoderm.
During
synepitheliochorial placentation in ruminants, the binucleate
cells
fuse with the endometrial epithelium and produce placental
lactogen
(
56).
Immunofluorescence analyses of uterine sections from cyclic and
pregnant ewes for JSRV envelope and capsid protein expression
are
presented in Fig.
5. As observed for
enJSRV RNA expression,
immunoreactive JSRV envelope and capsid proteins
were detected
specifically in the apical portion of the endometrial LE
and GE,
but not in the stroma or myometrium. The binucleate cells of
the
conceptus trophectoderm were also positive. Thus, the cell types
in
the ovine uterus expressing enJSRV RNAs also produce envelope
and
capsid proteins.
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FIG. 5.
Immunoreactive JSRV capsid (A) and env protein (B)
expression in the ovine endometrium. Immunofluorescence staining of
frozen uterine sections from cyclic (C) and pregnant (P) ewes was
conducted with specific antibodies or irrelevant IgG as a control as
described in Materials and Methods. TE, trophectoderm. Magnification,
×230.
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enJSRV LTRs are activated by progesterone.
In cyclic and early
pregnant ewes, expression of enJSRV genes in the endometrial epithelia
is highly correlated with circulating levels of progesterone in
peripheral blood and the presence of PR in uterine epithelia.
Therefore, a plausible hypothesis is that one or more enJSRV LTRs, in
which the retroviral promoter and enhancers are located, are regulated
by progesterone. Although 15 to 20 copies of enJSRVs are present in the
sheep genome, the expression observed in the uterus might derive from
only one or a few loci. As illustrated in Fig.
6, transient transfection experiments were utilized to determine the responsiveness of LTRs from three endogenous loci (enJS56A1, enJS5F16, and enJS59A1) and exogenous JSRV21 (40) and ENTV
(14) to progesterone. Progesterone stimulated (P < 0.01) the expression of the enJS59A1 LTR almost
10-fold and that of the enJS56A1 LTR about 4-fold. However,
progesterone had minimal effects on the enJS5F16 LTR and on the LTRs
from the exogenous viruses JSRV and ENTV. These results support the
idea that increased expression of some enJSRV genes is mediated by
increased transactivation of the LTR by liganded PR.
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FIG. 6.
Effects of progesterone on activity of several enJSRV
LTRs in transient transfection assays utilizing ovine endometrial LE
cells. Results are expressed as fold increases relative to activity of
the reporter constructs without the addition of progesterone. All
assays were conducted in triplicate with at least two independent
experiments. Results are presented as mean fold activation with SE.
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The enJSRVs LTR used in this study are derived from proviral clones.
Future studies of LTRs derived from transcriptionally
active enJSRVs
might be more appropriate in determining the response
of these loci to
hormones.
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DISCUSSION |
As a first effort to understand the significance of enJSRVs in
sheep reproductive biology, the present studies assessed the influence
of age, stage of the estrous cycle and pregnancy, and progesterone on
enJSRV expression in the ovine uterus. Available results suggest that,
during evolution, a function for these elements has been maintained
that might be beneficial to the host. At the very least, enJSRV
expression in the ovine female reproductive tract is apparently not
detrimental to reproduction.
The overall pattern of enJSRV expression in the ovine endometrial
epithelia and results of transient transfection experiments strongly
suggest that enJSRV LTR(s) are responsive to ligand-activated PR.
Levels of expression of enJSRV RNAs were low on day 1 of the estrous
cycle and increased to maximal levels on day 13 in the endometrium of
both cyclic and pregnant ewes. In situ hybridization analyses revealed
that changes in the expression of enJSRV RNA occurred in both the LE
and GE of the endometrium. Overall, these changes in steady-state
enJSRV RNA levels closely paralleled the ontogeny of ovarian
progesterone in the peripheral circulation as well as expression of the
PR in endometrial epithelia (46). The initial increase in
enJSRV RNAs between days 1 and 13 of the estrous cycle correlates with
an increase in progesterone due to formation of the corpus luteum after
ovulation. As illustrated in Fig. 7, PR
expression is abundant in the LE and GE between days 5 and 11 of the
estrous cycle in the ovine uterus. However, continuous exposure of the
ovine uterus to progesterone for 8 to 10 days down-regulates expression
of the PR (47). Expression of the PR declines in the LE to
undetectable levels between days 11 and 13, whereas PR expression in
the GE is progressively lost between days 13 and 19 of early pregnancy
(46). Transient transfection experiments presented here
indicate that the LTRs of several enJSRVs are transactivated by the
liganded PR. However, the LTRs of the same enJSRVs do not require
progesterone for basal expression. This contention is supported by the
detection of enJSRV RNAs in endometrial epithelia of the developing
uterus of neonatal ewes. Although the LE and GE express the PR
throughout neonatal endometrial development, progesterone is below
detectable limits in the peripheral circulation (52).
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FIG. 7.
Schematic representation of progesterone levels in the
peripheral blood relative to expression of the PR protein in the LE and
GE in cyclic and early pregnant ewes. The relative amount of PR protein
in the LE and GE is noted as absent (open circles), low abundance
(partially [gradient] filled circles), and abundant (solid
circles).
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During early pregnancy, maximal expression of enJSRV genes was detected
during the preimplantation period. The progressive decline in
expression of enJSRVs in the epithelium likely results from the loss of
PR expression in the LE and GE (46). In addition, loss of
the LE itself occurs during synepitheliochorial placentation as the
trophectoderm fuses with the LE and forms a syncytium beginning on day
16 of pregnancy. Of great interest for comparative physiology is the
expression of enJSRVs in the binucleate cells of the conceptus trophectoderm. Binucleate trophectodermal cells fuse with the endometrial LE in both caruncular and intercaruncular areas to form a
syncytium (56). Only the binucleate cells display invasive properties in the placenta of ruminants as well as other species. Indeed, retroviral particles have been observed in the placenta of many
animal species, including ruminants (28). The presence of
enJSRV gene expression in the developing placenta is strikingly similar
to that observed for the human endogenous retrovirus, HERV-W
(6). HERV-W is specifically expressed in the
syncytiotrophoblast of the human placenta, which is formed by fusion of
the trophoblast with the epithelium of the maternal uterus. HERV-W
envelope protein, as for many retroviral envelope proteins, is able to
induce formation of syncytia when expressed in vitro, thereby advancing
the hypothesis that HERV-W is involved in human placental morphogenesis
(7, 33). Similarly, ERV-3, another human retrovirus
(10, 12), is also abundantly expressed in the
syncytiotrophoblast, although the presence of a stop codon before the
membrane anchor domain of the ERV-3 env gene would probably
inhibit cell fusion mediated by the envelope protein (13).
Collectively, these observations support the theory that an ancient
retroviral infection had profound consequences for mammalian evolution
(23). The presence of enJSRV RNAs as well as envelope and
capsid protein expression might suggest involvement of this
betaretrovirus in synepitheliochorial placentation in sheep.
Immunofluoresence analyses in the present study indicated that
expression of enJSRV genes in the pregnant ovine uterus was not limited
to RNA, because both JSRV envelope and capsid proteins were detected in
the endometrial epithelia and trophectodermal binucleate cells. The
expression of enJSRV genes in the newborn lamb may explain some aspects
of the pathogenesis of ovine pulmonary adenocarcinoma (OPA) and
enzootic nasal tumor (ENT) induced by the highly related exogenous
viruses JSRV and ENTV. Sheep affected by OPA or ENT do not have a
humoral immune response towards JSRV or ENTV (34, 44).
Thus, it appears that the enJSRVs elements are recognized as
self-antigens due to their basal expression, and sheep are tolerized
towards infection by the exogenous JSRV. Indeed, preliminary
experiments detected expression of enJSRV RNAs in the uterus, lungs,
and gut of fetal sheep (T. E. Spencer, P. J. Griebel, and M. Palmarini, unpublished results). How the induction of tolerance
to an exogenous retrovirus could be beneficial for its host is not
readily apparent, although this might be the price of utilizing enJSRVs
in ovine uterine function. In the present study, enJSRV expression was
detected in all reproductive tract tissues of Müllerian duct
origin. This is in accord with the hypothesis that ancestors of the
modern JSRV and ENTV did not have a tropism for the respiratory
apparatus, but rather for the genital tract, and were probably
transmitted from sheep to sheep during the process of mating
(38). Thus, these exogenous viruses might have been the
cause of immunomediated disorders so that the induction of tolerance
might have proved beneficial for sheep. These speculations would be
very difficult to address experimentally.
In addition to trophectodermal binucleate cell biology, expression of
enJSRVs in the endometrial epithelia may play another role in
placental morphogenesis. Expression of enJSRVs in uterine epithelia between days 11 and 19 of pregnancy is highly correlated with
tau interferon (IFN-
) production by mononuclear cells of the
trophectoderm (19, 22). In sheep, implantation is preceded by elongation of the conceptus from a tubular to filamentous form between days 12 and 15 (19), an event that involves
trophoblast cell rearrangement and proliferation (22).
Conceptus elongation is concomitant with production of the pregnancy
recognition hormone, IFN-, which prevents transcriptional
up-regulation of the estrogen receptor and oxytocin receptor genes in
the endometrial LE (5, 46). The expression of IFN-
genes is unusual, because they are not induced by virus (15,
29), only transcribed in the mononuclear cells of the
trophectoderm (18, 22), and are sustained over several
days (1) rather than limited to a few hours
(17). Given the proliferation of the trophectoderm during
the implantation period, the enJSRV envelope proteins might also
stimulate cell division in these cells, analogous to transforming
properties induced by the exogenous JSRV envelope (32,
41), although the enJSRV sequences known so far lack the
putative phosphatidylinositol 3-kinase (PI-3K) docking site in the
transmembrane region that has been found to be necessary for
transformation in vitro (39).
In conclusion, the results of this study support the hypothesis that
enJSRVs may play key roles in the reproductive physiology of sheep
during the peri-implantation period. Sheep can be used as a model to
understand the biological significance of endogenous retroviruses with
respect to conceptus (embryo and associated extraembryonic placental
membranes) development, implantation, and pregnancy recognition signaling.
| |
ACKNOWLEDGMENTS |
We are grateful to Mike Sharp (Moredun Research Institute,
Midlothian, Scotland) for providing the rabbit antiserum towards the
major capsid protein of JSRV, to Bert W. O'Malley and Ming-Jer Tsai
(Baylor College of Medicine, Houston, Tex.) for provision of the human
PR, and to Claudio Murgia for reporter assays.
This work was supported, in part, by NRI Competitive Grants
Program/USDA grant 98-35203-6322 to T.E.S. and NIH grant P30 ES09106.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for
Animal Biotechnology and Genomics, Department of Animal Science, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX
77843-2471. Phone: (979) 845-4896. Fax: (979) 862-2662. E-mail:
tspencer{at}ansc.tamu.edu.
| |
REFERENCES |
| 1.
|
Ashworth, C. J., and F. W. Bazer.
1989.
Changes in ovine conceptus and endometrial function following asynchronous embryo transfer or administration of progesterone.
Biol. Reprod.
40:425-433[Abstract].
|
| 2.
|
Ausbel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith, and K. Struhl (ed.).
2000.
Current protocols in molecular biology.
John Wiley & Sons, New York, N.Y.
|
| 3.
|
Bai, J.,
J. V. Bishop,
J. O. Carlson, and J. C. DeMartini.
1999.
Sequence comparison of JSRV with endogenous proviruses: envelope genotypes and a novel ORF with similarity to a G-protein-coupled receptor.
Virology
258:333-343[CrossRef][Medline].
|
| 4.
|
Bai, J.,
R. Y. Zhu,
K. Stedman,
C. Cousens,
J. Carlson,
J. M. Sharp, and J. C. DeMartini.
1996.
Unique long terminal repeat U3 sequences distinguish exogenous jaagsiekte sheep retroviruses associated with ovine pulmonary carcinoma from endogenous loci in the sheep genome.
J. Virol.
70:3159-3168[Abstract].
|
| 5.
|
Bazer, F. W.,
T. E. Spencer, and T. L. Ott.
1997.
Interferon tau: a novel pregnancy recognition signal.
Am. J. Reprod. Immunol.
37:412-420.
|
| 6.
|
Blond, J.-L.,
F. Bèseme,
L. Duret,
O. Bouton,
F. Bedin,
H. Perron,
B. Mandrand, and F. Mallet.
1999.
Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family.
J. Virol.
73:1175-1185[Abstract/Free Full Text].
|
| 7.
|
Blond, J. L.,
D. Lavillette,
V. Cheynet,
O. Bouton,
G. Oriol,
S. Chapel-Fernandes,
B. Mandrand,
F. Mallet, and F. L. Cosset.
2000.
An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor.
J. Virol.
74:3321-3329[Abstract/Free Full Text].
|
| 8.
|
Bock, M., and J. P. Stoye.
2000.
Endogenous retroviruses and the human germline.
Curr. Opin. Genet. Dev.
10:651-655[CrossRef][Medline].
|
| 9.
|
Boeke, J. D., and J. P. Stoye.
1997.
Retrotransposons, endogenous retroviruses and the evolution of retroelements, p. 343-436.
In
J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
|
| 10.
|
Boyd, M. T.,
C. M. Bax,
B. E. Bax,
D. L. Bloxam, and R. A. Weiss.
1993.
The human endogenous retrovirus ERV-3 is upregulated in differentiating placental trophoblast cells.
Virology
196:905-909[CrossRef][Medline].
|
| 11.
|
Coffin, J. M.
1996.
Retroviridae: the viruses and their replication, p. 763-843.
In
B.N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed. Lippincott-Raven Publishers, New York, N.Y.
|
| 12.
|
Cohen, M.,
N. Kato, and E. Larsson.
1988.
ERV3 human endogenous provirus mRNAs are expressed in normal and malignant tissues and cells, but not in choriocarcinoma tumor cells.
J. Cell Biochem.
36:121-128[CrossRef][Medline].
|
| 13.
|
Cohen, M.,
M. Powers,
C. O'Connell, and N. Kato.
1985.
The nucleotide sequence of the env gene from the human provirus ERV3 and isolation and characterization of an ERV3-specific cDNA.
Virology
147:449-458[CrossRef][Medline].
|
| 14.
|
Cousens, C.,
E. Minguijon,
R. G. Dalziel,
A. Ortin,
M. Garcia,
J. Park,
L. Gonzalez,
J. M. Sharp, and M. de las Heras.
1999.
Complete sequence of enzootic nasal tumor virus, a retrovirus associated with transmissible intranasal tumors of sheep.
J. Virol
73:3986-3993[Abstract/Free Full Text].
|
| 15.
|
Cross, J. C., and R. M. Roberts.
1991.
Constitutive and trophoblast-specific expression of a class of bovine interferon genes.
Proc. Natl. Acad. Sci. USA
88:3817-3821[Abstract/Free Full Text].
|
| 16.
|
De las Heras, M.,
J. M. Sharp,
J. A. Garcia de Jalon, and P. Dewar.
1991.
Enzootic nasal tumour of goats: demonstration of a type D-related retrovirus in nasal fluids and tumours.
J. Gen. Virol.
72:2533-2535[Abstract/Free Full Text].
|
| 17.
|
De Maeyer, E., and J. De Maeyer-Guignard.
1987.
You cannot get away from interferon.
J. Interferon Res.
7:467-470[Medline].
|
| 18.
|
Farin, C. E.,
K. Imakawa,
T. R. Hansen,
J. J. McDonnell,
C. N. Murphy,
P. W. Farin, and R. M. Roberts.
1990.
Expression of trophoblastic interferon genes in sheep and cattle.
Biol. Reprod.
43:210-218[Abstract].
|
| 19.
|
Godkin, J. D.,
F. W. Bazer,
J. Moffatt,
F. Sessions, and R. M. Roberts.
1982.
Purification and properties of a major, low molecular weight protein released by the trophoblast of sheep blastocysts at day 13-21.
J. Reprod. Fertil.
65:141-150[Abstract/Free Full Text].
|
| 20.
|
Gray, C. A.,
F. W. Bazer, and T. E. Spencer.
2001.
Effects of neonatal progestin exposure on female reproductive tract structure and function in the adult ewe.
Biol. Reprod.
64:797-804[Abstract/Free Full Text].
|
| 21.
|
Guillomot, M.
1995.
Cellular interactions during implantation in domestic ruminants.
J. Reprod. Fertil. Suppl.
49:39-51[Medline].
|
| 22.
|
Guillomot, M.,
C. Michel,
P. Gaye,
N. Charlier,
J. Trojan, and J. Martal.
1990.
Cellular localization of an embryonic interferon, ovine trophoblastin and its mRNA in sheep embryos during early pregnancy.
Biol. Cell
68:205-211[CrossRef][Medline].
|
| 23.
|
Harris, J. R.
1991.
The evolution of placental mammals.
FEBS Lett.
295:3-4[CrossRef][Medline].
|
| 24.
|
Hecht, S. J.,
J. O. Carlson, and J. C. DeMartini.
1994.
Analysis of a type D retroviral capsid gene expressed in ovine pulmonary carcinoma and present in both affected and unaffected sheep genomes.
Virology
202:480-484[CrossRef][Medline].
|
| 25.
|
Hecht, S. J.,
K. E. Stedman,
J. O. Carlson, and J. C. DeMartini.
1996.
Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals.
Proc. Natl. Acad. Sci. USA
93:3297-3302[Abstract/Free Full Text].
|
| 26.
|
Johnson, G. A.,
M. D. Stewart,
C. A. Gray,
Y. Choi,
R. C. Burghardt,
L. Y. Yu-Lee,
F. W. Bazer, and T. E. Spencer.
2001.
Effects of the estrous cycle, pregnancy, and interferon tau on 2',5'-oligoadenylate synthetase expression in the ovine uterus.
Biol. Reprod.
64:1392-1399[Abstract/Free Full Text].
|
| 27.
|
Johnson, G. A.,
R. C. Burghardt,
K. M. Taylor,
J. G. W. Fleming,
F. W. Bazer, and T. E. Spencer.
1999.
Development and characterization of immortalized ovine endometrial cell lines.
Biol. Reprod.
61:1324-1330[Abstract/Free Full Text].
|
| 28.
|
Kalter, S. S.,
R. L. Heberling,
R. J. Helmke,
M. Panigel,
G. C. Smith,
D. C. Kraemer,
A. Hellman,
A. K. Fowler, and J. E. Strickland.
1975.
A comparative study on the presence of C-type viral particles in placentas from primates and other animals.
Bibl. Haematol.
40:391-401.
|
| 29.
|
Leaman, D. W.,
J. C. Cross, and R. M. Roberts.
1994.
Multiple regulatory elements are required to direct trophoblast interferon gene expression in choriocarcinoma cells and trophectoderm.
Mol. Endocrinol.
8:456-468[Abstract/Free Full Text].
|
| 30.
|
Lower, R.
1999.
The pathogenic potential of endogenous retroviruses: facts and fantasies.
Trends Microbiol.
7:350-356[CrossRef][Medline].
|
| 31.
|
Lower, R.,
J. Lower, and R. Kurth.
1996.
The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences.
Proc. Natl. Acad. Sci. USA
93:5177-5184[Abstract/Free Full Text].
|
| 32.
|
Maeda, N.,
M. Palmarini,
C. Murgia, and H. Fan.
2001.
Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA.
Proc. Natl. Acad. Sci. USA
98:4449-4454[Abstract/Free Full Text].
|
| 33.
|
Mi, S.,
X. Lee,
X. Li,
G. M. Veldman,
H. Finnerty,
L. Racie,
E. LaVallie,
X. Y. Tang,
P. Edouard,
S. Howes,
J. C. Keith, Jr., and J. M. McCoy.
2000.
Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis.
Nature
403:785-789[CrossRef][Medline].
|
| 34.
|
Ortin, A.,
E. Minguijon,
P. Dewar,
M. Garcia,
L. M. Ferrer,
M. Palmarini,
L. Gonzalez,
J. M. Sharp, and M. De las Heras.
1998.
Lack of a specific immune response against a recombinant capsid protein of jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis.
Vet. Immunol. Immunopathol.
61:229-237[CrossRef][Medline].
|
| 35.
|
Palmarini, M.,
C. Cousens,
R. G. Dalziel,
J. Bai,
K. Stedman,
J. C. DeMartini, and J. M. Sharp.
1996.
The exogenous form of jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep.
J. Virol
70:1618-1623[Abstract].
|
| 36.
|
Palmarini, M.,
S. Datta,
R. Omid,
C. Murgia, and H. Fan.
2000.
The long terminal repeat of jaagsiekte sheep retrovirus is preferentially active in differential epithelial cells of the lungs.
J. Virol.
74:5776-5787[Abstract/Free Full Text].
|
| 37.
|
Palmarini, M.,
P. Dewar,
M. De las Heras,
N. F. Inglis,
R. G. Dalziel, and J. M. Sharp.
1995.
Epithelial tumour cells in the lungs of sheep with pulmonary adenomatosis are major sites of replication for jaagsiekte retrovirus.
J. Gen. Virol.
76:2731-2737[Abstract/Free Full Text].
|
| 38.
|
Palmarini, M.,
C. Hallwirth,
D. York,
C. Murgia,
T. de Oliveira,
T. Spencer, and H. Fan.
2000.
Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus.
J. Virol.
74:8065-8076[Abstract/Free Full Text].
|
| 39.
|
Palmarini, M.,
N. Maeda,
C. Murgia,
C. De-Fraja,
A. Hofacre, and H. Fan.
2001.
A phosphatidylinositol 3-kinase docking site in the cytoplasmic tail of the jaagsiekte sheep retrovirus transmembrane protein is essential for envelope-induced transformation of NIH 3T3 cells.
J. Virol.
75:11002-11009[Abstract/Free Full Text].
|
| 40.
|
Palmarini, M.,
J. M. Sharp,
M. De las Heras, and H. Fan.
1999.
Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer in sheep.
J. Virol.
73:6964-6972[Abstract/Free Full Text].
|
| 41.
|
Rai, S. K.,
F. M. Duh,
V. Vigdorovich,
A. Danilkovitch-Miagkova,
M. I. Lerman, and A. D. Miller.
2001.
Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation.
Proc. Natl. Acad. Sci. USA
98:4443-4448[Abstract/Free Full Text].
|
| 42.
|
Schulte, A. M.,
S. Lai,
A. Kurtz,
F. Czubayko,
A. T. Riegel, and A. Wellstein.
1996.
Human trophoblast and choriocarcinoma expression of the growth factor pleiotrophin attributable to germ-line insertion of an endogenous retrovirus.
Proc. Natl. Acad. Sci. USA
93:14759-14764[Abstract/Free Full Text].
|
| 43.
|
Schulte, A. M.,
C. Malerczyk,
R. Cabal-Manzano,
J. J. Gajarsa,
H. J. List,
A. T. Riegel, and A. Wellstein.
2000.
Influence of the human endogenous retrovirus-like element HERV-E.PTN on the expression of growth factor pleiotrophin: a critical role of a retroviral Sp1-binding site.
Oncogene
19:3988-3998[CrossRef][Medline].
|
| 44.
|
Sharp, J. M., and A. J. Herring.
1983.
Sheep pulmonary adenomatosis: demonstration of a protein which cross-reacts with the major core proteins of Mason-Pfizer monkey virus and mouse mammary tumour virus.
J. Gen. Virol.
64:2323-2327[Abstract/Free Full Text].
|
| 45.
|
Spencer, T. E.,
F. F. Bartol,
F. W. Bazer,
G. A. Johnson, and M. M. Joyce.
1999.
Identification and characterization of glycosylation-dependent cell adhesion molecule 1-like protein expression in the ovine uterus.
Biol. Reprod.
60:241-250[Abstract/Free Full Text].
|
| 46.
|
Spencer, T. E., and F. W. Bazer.
1995.
Temporal and spatial alterations in uterine estrogen receptor and progesterone receptor gene expression during the estrous cycle and early pregnancy in the ewe.
Biol. Reprod.
53:1527-1543[Abstract].
|
| 47.
|
Spencer, T. E.,
M. A. Mirando,
J. S. Mayes,
G. H. Watson,
T. L. Ott, and F. W. Bazer.
1996.
Effects of interferon-tau and progesterone on oestrogen-stimulated expression of receptors for oestrogen, progesterone and oxytocin in the endometrium of ovariectomized ewes.
Reprod. Fertil. Dev.
8:843-853[CrossRef][Medline].
|
| 48.
|
Spencer, T. E.,
A. G. Stagg,
M. M. Joyce,
G. Jenster,
C. G. Wood,
F. W. Bazer,
A. A. Wiley, and F. F. Bartol.
1999.
Discovery and characterization of endometrial epithelial messenger ribonucleic acids using the ovine uterine gland knockout model.
Endocrinology
140:4070-4080[Abstract/Free Full Text].
|
| 49.
|
Steele, R. G. D., and J. H. Torrie.
1980.
Principles and procedures of statistics.
McGraw-Hill, New York, N.Y.
|
| 50.
|
Stewart, M. D.,
G. A. Johnson,
C. A. Gray,
R. C. Burghardt,
L. A. Schuler,
M. M. Joyce,
F. W. Bazer, and T. E. Spencer.
2000.
Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy.
Biol. Reprod.
62:1779-1789[Abstract/Free Full Text].
|
| 51.
|
Taylor, K. M.,
C. Chen,
C. A. Gray,
F. W. Bazer, and T. E. Spencer.
2001.
Expression of messenger ribonucleic acids for fibroblast growth factors 7 and 10, hepatocyte growth factor, and insulin-like growth factors and their receptors in the neonatal ovine uterus.
Biol. Reprod.
64:1236-1246[Abstract/Free Full Text].
|
| 52.
|
Taylor, K. M.,
C. A. Gray,
M. M. Joyce,
M. D. Stewart,
F. W. Bazer, and T. E. Spencer.
2000.
Neonatal ovine uterine development involves alterations in expression of receptors for estrogen, progesterone, and prolactin.
Biol. Reprod.
63:1192-1204[Abstract/Free Full Text].
|
| 53.
|
Vegeto, E.,
G. F. Allan,
W. T. Schrader,
M. J. Tsai,
D. P. McDonnell, and B. W. O'Malley.
1992.
The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor.
Cell
69:703-713[CrossRef][Medline].
|
| 54.
|
Venables, P. J.,
S. M. Brookes,
D. Griffiths,
R. A. Weiss, and M. T. Boyd.
1995.
Abundance of an endogenous retroviral envelope protein in placental trophoblasts suggests a biological function.
Virology
211:589-592[CrossRef][Medline].
|
| 55.
|
Villarreal, L. P.
1997.
On viruses, sex, and motherhood.
J. Virol.
71:859-865[Medline].
|
| 56.
|
Wooding, F. B.
1982.
The role of the binucleate cell in ruminant placental structure.
J. Reprod. Fertil. Suppl.
31:31-39[Medline].
|
| 57.
|
York, D. F.,
R. Vigne,
D. W. Verwoerd, and G. Querat.
1992.
Nucleotide sequence of the jaagsiekte retrovirus, an exogenous and endogenous type D and B retrovirus of sheep and goats.
J. Virol.
66:4930-4939[Abstract/Free Full Text].
|
Journal of Virology, December 2001, p. 11319-11327, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11319-11327.2001
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-
Carlson, J., Lyon, M., Bishop, J., Vaiman, A., Cribiu, E., Mornex, J.-F., Brown, S., Knudson, D., DeMartini, J., Leroux, C.
(2003). Chromosomal Distribution of Endogenous Jaagsiekte Sheep Retrovirus Proviral Sequences in the Sheep Genome. J. Virol.
77: 9662-9668
[Abstract]
[Full Text]
-
Spencer, T. E., Mura, M., Gray, C. A., Griebel, P. J., Palmarini, M.
(2002). Receptor Usage and Fetal Expression of Ovine Endogenous Betaretroviruses: Implications for Coevolution of Endogenous and Exogenous Retroviruses. J. Virol.
77: 749-753
[Abstract]
[Full Text]
-
Alberti, A., Murgia, C., Liu, S.-L., Mura, M., Cousens, C., Sharp, M., Miller, A. D., Palmarini, M.
(2002). Envelope-Induced Cell Transformation by Ovine Betaretroviruses. J. Virol.
76: 5387-5394
[Abstract]
[Full Text]
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Abstract
Introduction
