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Departments of Biochemistry (C.S.H., R.M.R), Animal Sciences (N.M., R.M.R), and Veterinary Pathobiology (R.M.R), University of Missouri, Columbia, Missouri 65211; and Department of Biology (S.W.K.), Rollins College, Winter Park, Florida 32789
Address all correspondence and requests for reprints to: R. Michael Roberts, 158 Animal Sciences Reseach Center, University of Missouri, Columbia, Missouri 65211. E-mail: michael_roberts{at}muccmail.missouri edu.
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Abstract |
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(IFN-), a type I IFN structurally related to IFN-
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is regarded as the major antiluteolytic factor secreted by the
conceptus of ruminant ungulate species before definitive trophoblast
attachment and implantation. It mediates its effects by acting on the
uterine endometrium, where it blunts the normal pulsatile production of
PGF2, presumably as a result of its binding to type I
IFN receptors. In this study, we describe the complementary DNAs for
the two known subunits, IFNAR1 and IFNAR2, of this receptor isolated
from bovine and ovine endometrial complementary DNA libraries by
homology cloning. Although there is extensive inferred amino acid
sequence similarity between bovine and ovine IFNAR1 (92% identity) and
between bovine and ovine IFNAR2 (88% identity), they have diverged
extensively from the human receptor subunits (
67% and 58%
identity, respectively). Despite these differences in primary
structure, the respective subunits from all three species are organized
similarly in their extracellular and cytoplasmic regions, and the
bovine and ovine subunits have each retained a number of polypeptide
motifs implicated in signal transduction. These uterine receptors also
appear not to be splice variants. The cloned ovine IFNAR1 subunit, for
example, possesses the expected four extracellular SD100 domains of
full-length bovine and huIFNAR1, and only the homologs of the so-called
long form (huIFNAR2c) of human IFNAR2 have so far been identified.
RT-PCR procedures indicate that the messenger RNA for both subunits are
found, not only in endometrium, but in all other tissues examined
except those of preimplantation conceptuses, which presumably cannot
respond to the IFN- they produce. Quantitative RNase protection
assays of ovine endometrial RNA show that the expression of neither
subunit changes greatly during the estrous cycle or pregnancy. These
data suggest that the type I IFN receptor, which is expressed by the
endometrium and binds IFN-, is probably not a structurally unusual
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Introduction |
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, -ß,
-
, and - (1, 2). The first three of these are virally inducible,
although they demonstrate cell type specificity and may also be
expressed in response to stimuli not originating from viral infection
(1, 3). All four subtypes share many properties in common. For example,
they have antiviral, antiproliferative, and immunomodulatory properties
(1, 3) and compete with one another for binding to the same type I IFN
receptor (4, 5). Although in the human there are only single functional
IFN-ß and - genes, there are at least 21 IFN- genes, of which
13 are believed to be active (6). In cattle, the situation is even more
complex, as each of the subtypes is represented by multiple genes (2, 7, 8, 9). It remains unexplained why so many type I genes have arisen,
although they differ in their inducibility and hence may provide a
flexible response to infection by different pathogens (10). It is also
becoming clear that the properties of individual type I IFN, although
generally similar, are not identical. Comparable antiviral potency, for
example, does not necessarily predict equivalent antiproliferative
activities (11, 12, 13). Different human IFN- can also differ by as much
as three orders of magnitude in their antiviral potency (14). Moreover
cells that are responsive to one IFN, may respond poorly to another,
even of the same subtype (1, 15, 16). Together, such data suggest that
there may be multiple type I receptor complexes linked to different
signal transducing components. The recent observation that receptors
containing splice variants of one of the subunits (IFNAR1) of the human
type I receptor are differentially responsive to different IFN subtypes
(17) is consistent with such a hypothesis. Alternatively, similar
receptor complexes may be capable of initiating alternative signal
transducing pathways, depending on which IFN binds.
About 10 yr ago, a fourth type I IFN subtype, the IFN-, was
described (18, 19). Unlike the other three type I IFNs, IFN- is not
virally inducible (20, 21); nor is it believed to be important as a
response to infection (19). Instead, it is expressed in what may be a
constitutive manner during normal preimplantation development of
embryos of ruminant ungulate species, where it plays a pivotal role in
maternal recognition of pregnancy (19, 22). Specifically, IFN- acts
on the uterine endometrium and blocks the normal pulsatile output of
the uterine luteolysin, PGF2. When released in such a
pulsatile manner toward the end of the estrous cycle,
PGF2 causes the demise of the corpus luteum (23). The
mechanism of IFN- action is incompletely understood but may involve
a down-regulation of the estrogen receptor and a subsequent failure of
the uterine epithelial cells to express the oxytocin receptor (24, 25, 26).
Oxytocin, originating from either the pituitary or the corpus luteum,
regulates PGF2 release (23). As a result, during
pregnancy, the lifespan of the corpus luteum is extended and
progesterone production maintained.
Although IFN- has comparable antiviral activity to boIFN-1 on
bovine (Mabin Darby bovine kidney) cells, and the two have almost
identical affinities for the endometrial type I IFN receptor (27),
IFN- is considerably more potent as an antiluteolysin, again raising
questions about the nature of the type I receptor expressed by the
uterus.
Molecular cloning and expression studies have so far indicated that a
functional type I receptor consists of a minimum of two subunits, both
of which are in the so-called class II cytokine receptor family (28, 5). The first to be identified, IFNAR1, has been cloned as its
complementary DNA (cDNA) from human (29), mouse (30), and bovine (31, 32) cells. Its extracellular part consists of two distinct
approximately 200 amino acid domains (D200), each of which is divided
further into two shorter subdomains (SD100) with structural similarity
to the constant region of immunoglobulins (28). Its cytoplasmic domain
contains binding sites for the tyrosine kinase, Tyk2, and the
transcription factor signal-transducer and activator of transcription
(STAT) 2 (33, 34, 35) and 3 (36). Three splice variants have been
described. One of these lacks the second SD100 domain (17), whereas the
other two are soluble forms without the single transmembrane domain
(37). IFNAR1, alone, generally appears not to bind type I IFN
efficiently (29), and a second subunit (IFNAR2) is necessary to allow
IFN to bind with high affinity to the receptor (30, 38). Three variant
forms of IFNAR2 have been recognized. One is soluble (IFNAR2a) and was
first purified from human urine (38). The two membrane-associated forms
from human tissues (IFNAR2b and IFNAR2c) have identical extracellular
regions, but, as the result of alternative splicing have different
length cytoplasmic domains (38, 39). IFNAR2b, the so-called short form,
lacks the signal-transducing capacity of the long form IFNAR2c, whose
cytoplasmic domain may provide docking sites for JAK1 and STAT2 (39). A
functional type I receptor, capable of binding IFN- and -ß and
participating in signal transduction, seems only to be reconstituted by
coexpressing human IFNAR1 (huIFNAR1) and IFNAR2c (39).
In this study we set out to determine whether the full-length forms of
IFNAR1 and IFNAR2 were expressed together in the endometrial tissues of
sheep and cattle, and whether they differed significantly from their
human counterparts. In addition, we wished to determine whether the
expression of these two receptor subunits changed either as the estrous
cycle progressed or as pregnancy was established, and whether the
conceptus, as the source of the IFN-, possessed type I IFN
receptors. Preliminary reports of these findings were published in an
abstract form (40).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(two 5-mg
injections given 3 h apart). Ewes were either then bred to intact
rams or allowed to progress to selected days of their estrous cycle.
Recovery of embryos from pregnant ewes and the procedure for
hysterectomy have been described previously (41). All animals were
maintained with the highest standards of humane animal care. Study
protocols were approved by the University of Missouri Animal Care and
Use Committee. Total RNA and poly(A)+ messenger RNA (mRNA) extraction from sheep tissues followed standard procedures (42). Total RNA and mRNA isolated from pooled tissues of five to six animals were used in RT-PCR, as well as for preparation of the cDNA libraries, whereas total RNA from single ewes was used in RNase protection assays.
Oligonucleotides, enzymes, and other reagents
The oligonucleotides synthesized for use in RT-PCR, rapid
amplification of cDNA ends (RACE), subcloning, and labeling of cDNA
probes are shown in Table 1
. Products
produced by these PCR procedures were sequenced to confirm identity and
hence specificity. All restriction enzymes, T4 DNA ligase and avian
myeloblastosis virus reverse transcriptase were purchased from Promega
(Madison, WI); Taq DNA polymerase and terminal
deoxynucleotide transferase were obtained from GIBCO BRL (Gaithersburg,
MD); Sequenase and [-35S]deoxy-ATP (dATP) were from
Amersham (Arlington Heights, IL); [-32P]dATP was from
DuPont NEN (Boston, MA); other chemical reagents were mainly purchased
from Fisher Scientific Company (Pittsburgh, PA). Bovine IFNAR1
(boIFNAR1) cDNA plasmid (pMD56) was kindly provided by Dr. G. Uzé
from Institute de Génétique Moléculaire, CNRS,
Montpellier, France.
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ZAP II vector (Stratagene, La Jolla,
CA), respectively. The libraries were amplified in XL-1 blue cells and
used for screening by standard procedures (43). For cloning ovine
IFNAR1 (ovIFNAR1), about 3.2 x105 plaque-forming units of
the d12 sheep endometrial cDNA library was screened with a 1.2 kb
HincII cut fragment of boIFNAR1 as probe. This probe spans
1208 bp from 357–1565 of the IFNAR1 cDNA and represents most of the
coding region (31). Six positive clones were plaque purified. The
plasmids were excised from the phage by in vivo excision and
sequenced in both directions. The insert sizes of the six clones were
in the range of 0.65–2.2 kb. The merged sequences of these clones gave
the full-length sequence of ovIFNAR1 (2914 bp), though none of them
contained the entire coding region, which was subsequently cloned from
the same source by RT-PCR.
For cloning ovIFNAR2, the same cDNA library was screened with a
huIFNAR2 cDNA probe. This huIFNAR2 cDNA probe, representing part of the
extracellular domain (amino acids 74–204) of the protein, was
amplified by RT-PCR from Daudi cells (38). Only a single positive clone
(length 1890 bp) was isolated out of 7.5 x 105
plaques screened. This clone lacked the entire 5' untranslated region
(UTR) as well as the 5' end of the open reading frame (ORF) of the
cDNA. A RACE procedure was therefore employed (Fig. 1) to clone the 5' end of the transcript.
The RACE procedure involved RT coupled with two rounds of PCR. The RT
step converted the mRNA to a single-strand cDNA. Terminal
deoxynucleotide transferase was then used to add a poly(A)+
tail to the 3' end (42). In the first round of PCR, ovIFNAR2-specific
antisense primer 2A and primer T17 (Table 1
) were used. The PCR
products obtained appeared as a smear in an ethidium bromide-stained
agarose gel. In the second round of amplification, ovIFNAR2-specific
antisense primer 7A (Fig. 1 and Table 1) was used in combination with
T17. First-round PCR product (1 µl) was used as template. This time,
a single PCR product band was seen in the agarose gel and was
subsequently cloned and sequenced.
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The cDNA probes used to screen the libraries were labeled by PCR in the
presence of [-32P]dATP (for PCR conditions, see
below). As a result, high specific activity probes (sp. act.
1.3 x 109 cpm/µg) were produced. The
hybridization solution contained either 30 or 50% formamide, 0.5 mg/ml
herring sperm DNA, 5 x SSC, 5 x Denhardt’s solution, 50
mM sodium phosphate, and 0.1% SDS. Because formamide (30%
when using the human cDNA probe to screen ovine library; 50% when
using bovine/ovine probes to screen ovine/bovine libraries) was
included, the hybridization temperature was always kept at 42 C (42).
After positive clones were identified by the first round of screening,
they were further purified by another two rounds of screening. They
were then subcloned by in vivo excision from the
phagmid.
RT-PCR and Southern blot analysis of PCR products
Total RNA extracted from various tissues of ewes and from Daudi
cells were used for RT-PCR. All RT reactions were performed as follows.
Approximately 2 µg total RNA and 1 µg oligo(dT) primer, T17, in 20
µl sterile water was incubated at 70 C for 3 min and cooled to room
temperature. Premixed reaction mixture (30 µl), containing 10 µl
5 x RT buffer, 5 µl deoxynucleotide triphosphate mixture (10
mM each in the mixture), 40 U recombinant RNasin, 2 µl
100 mM sodium pyrophosphate, and 30 U AMV reverse
transcriptase, was added to the RNA-oligo(dT) mixture and incubated at
42 C for 1 h to synthesize the first strand cDNA. The reactions
were terminated by heating at 70 C for 3 min. For subsequent PCR, 1
µl of the above RT product was used as template in a 20-µl reaction
containing 60 ng sense and antisense primers, 20 µM of
each deoxynucleotide triphosphate, 50 mM KCl, 2.5
mM MgCl, 10 mM Tris-HCl, pH 8.3, and 0.5 U
Taq DNA polymerase. PCR was carried out under the following
conditions: 94 C for 1 min, 55 C for 1 min, and 72 C for 1 min for 30
cycles in a Perkin-Elmer thermocycler (Perkin-Elmer Corp., Norwalk,
CT).
The PCR products were resolved by electrophoresis in 1% (wt/vol)
agarose gels in the presence of ethidium bromide (1 µg/ml) (43).
After electrophoresis, agarose gels were photographed under UV
illumination, and the PCR products blotted onto nitrocellulose filters
that were hybridized with 32P-labeled probes in 50%
formamide, 0.5 mg/ml herring sperm DNA, 5 x SSC, 5 x
Denhardt’s solution, 50 mM sodium phosphate, and 0.1% SDS
at 42 C. The filters were washed with 5 x SSC and 0.1% SDS at 42
C for 15 min and subjected to autoradiography. The probes used for
confirming the identities of PCR procedures always nested inside the
expected PCR fragments so that they would not hybridize to any
nonspecific PCR products that contained the 5' and 3' primer sequences
(Fig. 4A).
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RNase protection assay and densitometry
Rase protection assays were carried out by
using a High-Speed Hybridization Ribonuclease Protection Assay Kit
(Ambion, Austin, TX) according to the manufacturer’s protocol. Animal
treatment and RNA isolation were as described earlier. For each time
point, one, two, or three animals were used, depending on availability.
Each analysis was repeated at least twice. Briefly, 20 µg total RNA
and 105 cpm cRNA probe (generated by in vitro
transcription with [-32P]CTP) were coprecipitated by
ethanol, resuspended in 10 µl hybridization buffer, and denatured at
95 C for 5 min. Hybridization was carried out by incubating at 68 C for
10 min. An RNase A/T1 mixture was then added and incubated for 30 min
at 37 C to digest the unannealed RNA. The RNases were inactivated, and
the probe-RNA duplex ethanol precipitated. The precipitate was
resuspended in RNA loading buffer and analyzed by electrophoresis on a
5% polyacrylamide gel containing urea (43). The gel was dried at 80 C,
and the protected bands were detected by autoradiography. The
autoradiographs were scanned, and the densities of the bands were
measured by using the software GPTOOLS (Biophotonics, Ann Arbor, MI).
The statistical analysis of data and plot were done by using the
Cricket Graph software (Cricket Software, Malvern, PA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A cDNA for ovIFNAR2 was also obtained from the day 12 ovine endometrial
cDNA library by homology screening using a cDNA probe representing part
of the extracellular domain (amino acids 74–204) of huIFNAR2 and
obtained from human Daudi cells by RT-PCR. A single ovine clone (H1,
Fig. 1) of 1890 bp was isolated out of 7.5 x 105
plaques screened. This cDNA, when compared with the huIFNAR2, lacked
the entire 5' UTR as well as the 5' end of the ORF. This missing
sequence was rescued by using the RACE procedure. The complete ovIFNAR2
cDNA (GenBank Accession number U65979) was 2309 bp in length and
contained a 1560-bp ORF, a 129-bp 5' UTR, and a 620-bp 3' UTR.
A boIFNAR2 cDNA was also cloned from a bovine endometrial cDNA library (48) by screening with the 32P-labeled ovIFNAR2 cDNA described above. Eight positive clones were purified out of 4.2 x 105 plaques screened, and three of them were sequenced. The longest clone was 2714 bp in length and contained an entire 1593-bp ORF, 266 bp 5' UTR, and 855 bp 3' UTR (GenBank Accession number U75304).
General comparison of primary structures
The inferred amino acid sequences of the IFNAR1 and IFNAR2
polypeptides from human, cow, and sheep are shown in Figs. 2 and 3. As expected, there is extensive
sequence similarity between ov- and boIFNAR1 (92% identity) reflecting
the close evolutionary relationship of these two species. Differences
between ovIFNAR1 and its human (29) and murine (30) counterparts were
extensive (67% and 47% amino and sequence identities, respectively)
(Table 2). The placement of the eight
extracellular cysteine residues is identical in ov-, bo-, and huIFNAR1,
however, and consistent with cytokine receptor family membership, as is
the single 23 amino acid transmembrane sequence and a proline-rich
Box-1-like motif in the cytoplasmic region adjacent to the membrane
(Figs. 2 and 3).
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) that are considered necessary for the cytoplasmic
segment of the receptor to associate with the tyrosine kinase Tyk2
(34). The tyrosine residue, which on huIFNAR1 becomes phosphorylated by
Tyk2 following ligand binding (35) and which is placed between the
Box-1-like motif and the transmembrane domain, is also conserved in the
bovine and ovine receptors. This residue, once it is phosphorylated,
becomes the docking site for STAT2 (35). The two tyrosine residues
Tyr327 and Tyr538 of huIFNAR1, which are required for recruiting STAT3
to the receptor upon ligand-dependent phosphorylation (36), are also
conserved across the three species (Fig. 2).
The ov- and boIFNAR2 subunits exhibit 88% overall amino and
sequence identity with each other, but less than 60% identity with
their human homolog. The placement of the four cysteines in the
extracellular domain is strictly conserved. The extracellular domain of
the hu-, bo-, and ovIFNAR2 proteins have three common, conserved
potential sites for N-glycosylation. The proteins in sheep and cattle
share two additional such sites (Fig. 3).
Between the transmembrane domain and the Box-1-like motif, there is
also a conserved proline (marked # in Fig. 3), which has been
interpreted to be important in binding of the JAK1 kinase to the
interferon-
receptor subunit 1 (IFNGR1) (49). Finally, there are six
relatively conserved acidic domains (marked A1 through A6 in Fig. 3)
whose functions are unknown.
In addition to the above features shared between the huIFNAR2c and its
bovine and ovine homologs, there are some clear differences. For
example, bo- and ovIFNAR2 are slightly longer, having an eight amino
acid extension at their carboxyl ends. Both possess a five amino acid
SLEDC (ser-leu-asp-glu-cys) motif, repeated three times in ovIFNAR2 and
twice in boIFNAR2, which is absent in the human protein (shaded
boxes in Fig. 3). The significance of this sequence is
unknown.
Finally, the inferred lengths of the signal sequences as determined by the method of von Heijne (50) differ between the human and bovine/ovine polypeptides. Compared with their human homolog, the bo- and ovIFNAR1 would appear to have a shorter signal peptide due to a favorable cleavage site following Gly 17. In the case of IFNAR2, the most optimal position for signal cleavage is found further downstream than in the bovine and ovine sequences. In the absence of amino terminal sequence data from the purified subunits, these inferences must, of course, remain tentative.
Expression of ovIFNAR1 and ovIFNAR2 in the ewe
RNA from a day 12 endometrium had been chosen as a starting point
for cloning the cDNA for the two subunits of the ovine type I IFN
receptors because high-affinity receptors for IFN- had clearly been
demonstrated at this stage (45, 51). RT-PCR was employed to determine
whether the same mRNA was expressed at other stages of development, as
well as in other tissues of ewes. To ensure that the amplified DNA
represented the IFNAR1 and IFNAR2 transcripts, Southern blotting was
performed with probes nested within the anticipated products. As a
positive control, primers designed to amplify cDNA for the housekeeping
gene glyceraldehyde 3-phosphate dehydrogenase (G3PD) were included in a
separate PCR reaction. The data confirmed that mRNA for both subunits
was present in day 12 uterine endometrium but was absent in day 15
conceptuses (Fig. 4). These observations
have been confirmed in three separate experiments. Whereas the
concentrations of IFNAR1 mRNA seemed comparable in endometrium, kidney,
liver, and pituitary, IFNAR2 mRNA seemed to be more highly represented
in the latter three tissues than in endometrium.
The mRNA for both subunits could be detected in endometrium at days 3, 7, 10, and 16 of the estrous cycle. The significance of the low amount of amplified IFNAR1 transcript at day 16 of pregnancy in this experiment is unclear in the light of the RNA protection assays described next.
Quantitative RNase protection assays of IFNAR1 and IFNAR2 mRNA in
endometrium
To determine the relative changes in expression pattern of IFNAR
mRNA during the estrous cycle and early pregnancy, quantitative RNase
protection assays were performed. In this case, the cRNA probe used for
ovIFNAR1 corresponded to the first 480 nt of the ovIFNAR1 cDNA and
therefore largely represented 5' UTR. The probe for IFNAR2 mRNA was
defined by primers 8S and 3A (Fig. 4A) and represented part (258 nt) of
the cytoplasmic domain. Because the protected fragments of ovIFNAR2
(258 nt) and that of the internal control G3PD (280 nt) have similar
lengths and are not easily separable by electrophoresis, the expression
of ovIFNAR2 was normalized relative to ovIFNAR1 mRNA (Fig. 5B), and the latter was measured relative
to the content of G3PD mRNA (Fig. 5A), which is assumed to remain
relatively constant through the cycle and during pregnancy. The RNase
protection assays that used these probes gave well-defined protected
bands (Fig. 5), but additional smaller fragments were invariably
present. Although the origin of these fragments is not clear, they
may have resulted from mRNA species that were partially, but not fully
protected by the probes.
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, the quantities of neither subunit mRNA changed significantly
relative to the G3PD control between days 3–16 of the estrous cycle
(Fig. 6). If it is assumed that the
specific activities per unit length of each probe were identical, the
protected fragment for IFNAR1 would be expected to contain
approximately 1.9x (480 nt/258 nt) as much 32P as that for
IFNAR2. On this basis it would appear that the relative concentrations
of the two mRNA were quite similar on each day of the cycle that was
examined.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, two groups have described splice variants of the huIFNAR1
(17, 37). One of these splice variants, a form lacking the second SD100
domain, was responsive to certain type I IFN but not to all (17). This
discovery raised the possibility that the endometrium might express a
form of the receptor uniquely adapted to IFN- signaling. Although no
systematic effort has so far been made to define splice variants of
IFNAR1 in ovine or bovine endometrium, the amplification of reverse
transcribed mRNA with primers selected from within the 3' and 5' UTR,
for example, has revealed no evidence for short forms similar to those
described by Cook et al. (17). Consequently, it seems likely
that the major form of the type I IFN receptor in the endometrium, and
hence used by IFN-, has the full-length IFNAR1 as one of its
subunits. Kaluz et al. (47), using Northern blotting,
reported one major (6.5 kb) and two minor bands (4.3 and 3.7 kb). It is
unclear which of these bands corresponds to the IFNAR1 subunit cloned
in our study and whether any represent splice variant forms. Smaller
splice variants might be overlooked by PCR amplification techniques if
the full-length form is present in much greater abundance.
In the human, IFNAR1 alone has very low affinity for type I IFN, such
as IFN-, and requires the presence of the second subunit IFNAR2c to
bind IFN and to participate in IFN signaling (39). huIFNAR2, on the
other hand, can bind both IFN- and -ß with high affinity, but
needs IFNAR1 as a signaling partner. This study provides the first
report of the structures of IFNAR2 from cattle and sheep. Only the
human sequence has previously been published (39). The boIFNAR2 was
fairly well conserved (88% amino acid identity) compared with its
ovine homolog but had diverged extensively from huIFNAR2c (62% and
56% identity in the extracellular and intracellular regions,
respectively). This result was somewhat surprising, because most
huIFN- (3, 7, 10) are at least as active on bovine cells as
boIFN- and presumably bind the bovine receptor with high affinity. A
comparison of the sequences of the extracellular region reveals that
the most distal segment of the first SD100 domain on the three IFNAR2
proteins is well conserved. This portion of the molecule, plus a few
other regions of conserved sequence in the second SD100 domain, may
form the primary contacts with ligand. The intracellular regions of bo-
and ovIFNAR2 also retain most of the general features of the human
protein, including the six acidic domains and a proline-rich Box-1-like
motif.
As reviewed in the Introduction, at least three forms of
IFNAR2 have now been recognized, a soluble form representing only the
extracellular portion of the molecule, a membrane-bound form with a
truncated cytoplasmic domain, and the full-length receptor. Screening
of both ovine and bovine cDNA libraries has so far only revealed the
presence of the long form (comparable to huIFNAR2c), but again no
systematic study has yet been conducted to detect transcripts for
shorter splice variants. For example, the oligonucleotides used to
amplify IFNAR2-related transcripts (Fig. 4A) would not have detected
either of the two shorter forms discussed above. They would also have
failed to detect mRNA encoding polypeptides that had alterations in
their extracellular regions. Therefore, it is not yet possible to
decide whether some of the endometrial receptor complexes contain
unusual splice variant forms of IFNAR2.
The experiments have corroborated results from binding studies (51)
that there is not great variability in the expression of the type I
receptor during the estrous cycle. The concentrations of both mRNA were
comparable and remained relatively constant on the 5 days of the cycle
examined. Their expression would appear not, therefore, to be strongly
influenced by sex steroid hormones. A more complete analysis will
require in situ hybridization to distinguish receptor mRNA
present in the uterine epithelium from that in stroma, blood vessels,
and other cell types. Rather than decreasing during pregnancy, when
IFN- concentrations in the uterine lumen are high, there appeared to
be an up-regulation of receptor mRNA concentrations, although this
result needs to be confirmed by using more animals.
The one tissue examined that was completely devoid of receptors was the
day 15 conceptus, which is comprised largely of trophoblast and which
in vivo is producing very large quantities of IFN- at
that stage of pregnancy. Perhaps this absence of receptor expression is
necessary to avoid cytotoxic or other side effects on the embryo during
the period in which IFN- is being produced. It will be of interest
to determine precisely when during pregnancy the trophoblast becomes
responsive to type I IFN and potentially capable of resisting viral
infection. During the time of IFN- production, the uterine
epithelium is likely to act as an effective barrier to virus as well as
the site at which IFN- effects are transduced to prevent luteolysis
(19).
In conclusion, these experiments provided the primary structures of
IFNAR1 and IFNAR2 in sheep and IFNAR2 in cattle. They indicate that the
full-length forms of the receptor subunits are expressed in the
endometrium but not in the conceptus during the time of IFN-
production by embryonic trophectoderm. There was no indication that the
endometrial forms of the receptor complex contained splice variants of
either subunit, but the presence of such forms has not been ruled
out.
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Acknowledgments |
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Footnotes |
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Received May 27, 1997.
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  J. F. Oliveira, L. E. Henkes, R. L. Ashley, S. H. Purcell, N. P. Smirnova, D. N. R. Veeramachaneni, R. V. Anthony, and T. R. Hansen Expression of Interferon (IFN)-Stimulated Genes in Extrauterine Tissues during Early Pregnancy in Sheep Is the Consequence of Endocrine IFN-{tau} Release from the Uterine Vein Endocrinology, March 1, 2008; 149(3): 1252 - 1259. [Abstract] [Full Text] [PDF]
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Y. Chen, J. A. Green, E. Antoniou, A. D. Ealy, N. Mathialagan, A. M. Walker, M. P. Avalle, C. S. Rosenfeld, L. B. Hearne, and R. M. Roberts Effect of Interferon-{tau} Administration on Endometrium of Nonpregnant Ewes: A Comparison with Pregnant Ewes Endocrinology, May 1, 2006; 147(5): 2127 - 2137. [Abstract] [Full Text] [PDF]
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 C. Klein, S. Bauersachs, S. E. Ulbrich, R. Einspanier, H. H.D. Meyer, S. E.M. Schmidt, H.-D. Reichenbach, M. Vermehren, F. Sinowatz, H. Blum, et al. Monozygotic Twin Model Reveals Novel Embryo-Induced Transcriptome Changes of Bovine Endometrium in the Preattachment Period Biol Reprod, February 1, 2006; 74(2): 253 - 264. [Abstract] [Full Text] [PDF]
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M. M. Joyce, F. J. White, R. C. Burghardt, J. J. Muniz, T. E. Spencer, F. W. Bazer, and G. A. Johnson Interferon Stimulated Gene 15 Conjugates to Endometrial Cytosolic Proteins and Is Expressed at the Uterine-Placental Interface throughout Pregnancy in Sheep Endocrinology, February 1, 2005; 146(2): 675 - 684. [Abstract] [Full Text] [PDF]
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S.-Z. Wang and R. M. Roberts Interaction of Stress-Activated Protein Kinase-Interacting Protein-1 with the Interferon Receptor Subunit IFNAR2 in Uterine Endometrium Endocrinology, December 1, 2004; 145(12): 5820 - 5831. [Abstract] [Full Text] [PDF]
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C. S. Rosenfeld, C.-S. Han, A. P. Alexenko, T. E. Spencer, and R. M. Roberts Expression of Interferon Receptor Subunits, IFNAR1 and IFNAR2, in the Ovine Uterus Biol Reprod, September 1, 2002; 67(3): 847 - 853. [Abstract] [Full Text] [PDF]
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 X. Wu, A. Blanck, G. Norstedt, L. Sahlin, and A. Flores-Morales Identification of genes with higher expression in human uterine leiomyomas than in the corresponding myometrium Mol. Hum. Reprod., March 1, 2002; 8(3): 246 - 254. [Abstract] [Full Text] [PDF]
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M. D. Stewart, Y. Choi, G. A. Johnson, L.-y. Yu-Lee, F. W. Bazer, and T. E. Spencer Roles of Stat1, Stat2, and Interferon Regulatory Factor-9 (IRF-9) in Interferon Tau Regulation of IRF-1 Biol Reprod, February 1, 2002; 66(2): 393 - 400. [Abstract] [Full Text] [PDF]
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 C.-S. Han, Y. Chen, T. Ezashi, and R. M. Roberts Antiviral activities of the soluble extracellular domains of type I interferon receptors PNAS, May 3, 2001; (2001) 111139598. [Abstract] [Full Text]
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M. D. Stewart, G. A. Johnson, F. W. Bazer, and T. E. Spencer Interferon-{{tau}} (IFN{{tau}}) Regulation of IFN-Stimulated Gene Expression in Cell Lines Lacking Specific IFN-Signaling Components Endocrinology, May 1, 2001; 142(5): 1786 - 1794. [Abstract] [Full Text]
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 M. A. Larson and H. M. Kubisch The effects of group size on development and interferon-{tau} secretion by in-vitro fertilized and cultured bovine blastocysts Hum. Reprod., August 1, 1999; 14(8): 2075 - 2079. [Abstract] [Full Text] [PDF]
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C.-S. Han, Y. Chen, T. Ezashi, and R. M. Roberts Antiviral activities of the soluble extracellular domains of type I interferon receptors PNAS, May 22, 2001; 98(11): 6138 - 6143. [Abstract] [Full Text] [PDF]
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(only conserved within bovine
and ovine species). Sequences resembling a Pro-rich Box-1-motif and a
strictly conserved Tyr residue (*), which, when phosphorylated, is the
docking site for STAT2 (35), are marked. Boxes labeled
$ are three conserved clusters of amino acids which in
huIFNAR1 are required for binding of Tyk2 (34).
