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Regular Article |
a Departments of Animal Sciences,
b Veterinary Biomedical Sciences, and
c Biochemistry, University of Missouri, Columbia, Missouri 65211
d Center for Animal Biotechnology and Genomics, Department of Animal Science, Texas A & M University, College Station, Texas 77843
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) is the antiluteolytic factor released by concepti of ruminant ungulate species prior to implantation. All type I interferons, including IFN-, exert their action through a common receptor, which consists of two subunits, IFNAR1 and IFNAR2c, but the distribution of the two polypeptides in uterine endometrium has not been examined. In situ hybridization and immunohistochemistry on sections from pregnant and nonpregnant ovine uteri at Days 14 and 15 after estrus and mating showed that both IFNAR1 and IFNAR2 mRNA and protein were strongly expressed in endometrial luminal epithelium (LE), superficial glandular epithelium (GE), and stromal cells, within but not outside caruncles. Similar staining patterns were noted in pregnant and nonpregnant uteri for both subunits. Western blot analysis of membrane fractions from cell lines derived from endometrial LE, GE, and stromal cells, and affinity cross-linking experiments with radioactively labeled IFN- performed on crude endometrial membranes indicated the presence of both high (
110 kDa) and low (75–80 kDa) molecular mass forms of the two receptor subunits. To localize where IFN- binds when it is introduced into the uterine lumen, immunohistochemistry with an antiserum against IFN- was performed on sections of uteri from Day 14 nonpregnant ewes whose uteri had previously been infused with IFN-. Staining was concentrated on the LE and superficial GE cells, and was absent from the deeper regions of the glands and from the stromal tissues. These studies demonstrate the heavy concentration of IFNAR1 and IFNAR2 in cells of the LE and superficial GE, which appear to be the main targets for IFN-.
cytokines, female reproductive tract, implantation, polypeptide receptors, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) [1]. In nonpregnant animals, the uterus releases the luteolysin, PGF2
, in a series of pulses, as a response to oxytocin from the ovary and pituitary gland. IFN- is believed to prevent the release of luteolytic PGF2 pulses from the endometrium by inhibiting increases in endometrial estrogen receptor- expression, which in turn prevents the required increase expression of uterine oxytocin receptor [2, 3]. Although evidence exists for this model, the precise actions of IFN- on the uterus have yet to be characterized.
IFN- is a member of the type I family of interferons, whose other members include IFN-, IFN-ß, IFN-
, IFN-
, IFN-
, and limitin [4–6]. With the possible exception of IFN-, these other interferons are virally induced, and thus are probably involved in different aspects of pathological responses to viral infection. Although IFN- is expressed by porcine conceptuses, it does not appear to have antiluteolytic effects [7, 8]. Although IFN- is structurally similar to other type I interferons, particularly IFN- and IFN-, it is unique to ruminant ungulate species and is induced weakly, if at all, by virus [9]. Available studies indicate that IFN- is expressed only by the mononuclear cells of the trophectoderm, and then for only a short period corresponding to the critical time when the corpus luteum must be maintained, if the mother is to remain pregnant. The only maternal tissue immediately exposed to IFN- is the epithelium that lines the endometrium.
All type I interferons, including IFN-, act through a common receptor, (IFNAR), which consists of two polypeptide subunits, IFNAR1 and IFNAR2c. Both subunits are associated with the class II cytokine receptor superfamily [10, 11]. Alternatively spliced forms of both exist, but they are either soluble extracellular proteins or relatively minor membrane anchored variants whose role in signal transduction remains unclear [12–14]. Ovine IFNAR1 consists of four extracellular, structurally related SD100 domains [11], a transmembrane domain, and an intracellular domain of 97 amino acids, which constitutively binds the kinase Tyk2 and a variety of other molecules involved in signal transduction [15, 16]. Ovine IFNAR2c has an extracellular region of only two SD100 domains but is considered to be the subunit that contributes most to interferon binding [17–19]. Its long intracellular region of 268 amino acids [20] binds Jak1 and Stat2 [21–25] and possibly other signal transduction components [26]. Even though crystallographic data are still lacking, much is known about the structures of the receptors through modeling and other types of inference [27–30]. However, scant data exist on their expression patterns within various organs, possibly because it has been assumed that the receptors, much like the major histocompatibility complex, are ubiquitously expressed.
Because IFN- does not become detectable in the systemic blood circulation of early pregnant ewes, it was presumed that the uterus, and specifically the uterine endometrium, is its main target [31]. Consistent with this hypothesis, a high concentration of binding sites were noted in ovine and bovine endometrial membranes [32–34]. However, no studies have been performed to localize where the IFNAR subunits are expressed within the endometrium.
The objectives of these experiments were 1) to demonstrate where within the uterus the mRNA and protein for IFNAR1 and IFNAR2 were concentrated and whether their expression was influenced by the presence of a conceptus; 2) to determine whether cell lines derived from the ovine uterine endometrium exhibited a similar distribution of receptors as the intact tissue; and 3) to determine the localization of IFN- after it had been introduced into the uterine lumen, with the hypothesis that it would be localized to the luminal surface of the uterine epithelium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The University of Missouri Animal Care and Use Committee approved all animal procedures for these experiments. Estrus was induced in ewes by injecting 15 mg of PGF2 [35]. Some ewes were bred to intact rams; others were not and formed the nonpregnant group. Six pregnant (Days 14 through 15) and six nonpregnant ewes (also Days 14 through 15) were used for localization experiments. Ewes were killed with pentobarbital, and reproductive organs were collected and either fixed in Bouin solution (Sigma, St. Louis, MO) or frozen in liquid nitrogen.
Infusion of Recombinant IFN- into Uterine Lumen
This procedure was performed on nonbred ewes as described previously [35]. Briefly, on Day 5 postestrus, uterine catheters were surgically placed into the uterine horn ipsilateral to the functional corpus luteum. Either ovine IFN-4 (n = 4) (100 µg; specific antiviral activity of 3 x 108 IU/mg) or 1x PBS solution (n = 4) was introduced twice daily from Day 11 to Day 15 into the uteri through the implanted cannulae. On Days 14 and 15, ewes were killed, and uterine tissues were fixed in Bouin solution and embedded in paraffin. Immunohistochemistry was performed with a rabbit antiserum raised against IFN-4.
Generation of Recombinant IFNAR1 and IFNAR2 Proteins
The coding region for the entire cytoplasmic domains of ovine IFNAR1 and IFNAR2 [20] were amplified by polymerase chain reaction and inserted into PGEX-2T vector (Pharmacia Biotech, Piscataway, NJ). The primer pairs used for ovine IFNAR1 were 5'-GATCGGATCCTCTACACAGTTTATACACAA-3' and 5'-GATCGAATTCTCACACACTGTCCTGCTGCA-3', and those employed for ovine IFNAR2 were 5'-ATTCGAGGATCCAAACGGATTGGTTATATATG-3' and 5'-GTCTCAGAATTCTTAATTAAAATTTTTCAGAT-3'. Glutathione S-transferase (GST) fusion proteins were produced in soluble form in Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI). The soluble products were affinity purified on GST-Sepharose, and assessed for purity by SDS-PAGE.
Immunization of Rabbits Against IFNAR1 and IFNAR2 Recombinant Proteins
Rabbits were immunized as described previously [36] with recombinant proteins (400 µg) mixed with Freund complete adjuvant (Sigma). Subsequent boosts with 200 µg of recombinant protein with Freund incomplete adjuvant were performed every 4–6 wk. At the time of each boost, blood (25 ml) was collected from the marginal ear vein. After clotting, samples were centrifuged at 3000 x g, and the resulting serum was collected. Preimmune serum was collected before the first immunization.
In Situ Hybridization
This procedure was performed as described previously [36]. Briefly, cDNA fragments spanning the cytoplasmic domain of IFNAR1 and IFNAR2 were cloned into the PGEM-T Easy vector (Promega, Madison, WI). Antisense probes for each subunit were generated by restriction digest with SalI and in vitro transcription with SP6 polymerase (Promega). The sense probes for each subunit were generated by restriction digestion with SacII and in vitro transcription with T7 polymerase (Promega). The probes were biotinylated with 2 µl of 10x 16-UTP (Roche Molecular Biomedicals, Indianapolis, IN) Tissue sections were incubated overnight at 55°C with 200 µl of hybridization buffer containing 100 ng of probe. The next day, the sections were washed at 37°C in the buffer from the DAKO (Carpinteria, CA) In Situ Hybridization Detection System. Regions of uterine sections that hybridized to the biotinylated probes were detected by streptavidin alkaline phosphatase and by using the chromogens 5-bromo-4-chloro-3-inoyl phosphate (5-BCIP) and nitroblue tetrazolium (NBT) (DAKO). The sections were counterstained with nuclear fast red. In situ hybridization was repeated three times for each section of uterine tissues.
Preparation of Ovine Uterine Cell Lysates
Ovine uterine cells, LE, GE, and S cells (which are primarily from the intercaruncular region) were cultured in Dulbecco modified Eagle medium/Ham F12 and 10% fetal bovine serum [37] in either T75 or T150 flasks until the cells had undergone 30–40 passages. Cells were divided (1:5) every 2 days to maintain the cultures below 75% confluency. Cell membranes were isolated as described previously [38]. Cells from the three lines (eight T-75 flasks per line) were resuspended in lysis buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl2, and 0.1 mM PMSF), and passed 30 times through an 18.5-gauge needle. The larger cellular fractions were removed by low-speed centrifugation (2000 x g). The resulting supernatant was diluted with lysis buffer to a final volume of 30 ml and was then centrifuged in a Beckman L8-55 ultra centrifuge (Beckman Instruments, Inc., Palo Alto, CA) for 2 h at 100 000 x g.
Western Blot Analysis
Ovine uterine membrane proteins (30 µg) were separated by electrophoresis through 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. For the Western blots, the membranes were blocked in 2% bovine serum albumin; 1% nonfat dry milk in 0.1 M NaHCO3 pH 9.0; reacted with either IFNAR1 antiserum (1:1000), IFNAR2 antiserum (1:1000), or preimmune serum (1:1000); washed with TBS-Tween-20 (0.05%); and reacted with alkaline-phosphatase-conjugated anti-rabbit immunoglobulin G (IgG; 1:5000; Promega). The blots were washed and stained in a mixture of NBT and 5-BCIP (Promega).
Cross-Linking Studies with 32P-Labeled IFN-
A form of IFN-4 (IFN-4-P) with a peptide tag at its amino terminus that can be phosphorylated (RRASV) was expressed as a soluble GST fusion protein in the pGEX-2T-K vector (Pharmacia Biotech). After purification on GST-Sepharose [35], a phosphate group was introduced by using cAMP-dependent protein kinase (Sigma) as described previously [39] with some modifications [40].
Briefly, 250 µCi of [
-32P]ATP, 6000 Ci/mmol (NEN, Boston, MA) and 30 units of the catalytic subunit of cAMP-dependent protein kinase were used to label 1 µg of IFN- preparation. The unincorporated ATP was removed by centrifugation at 3000 x g in a Centricon-10 (Amicon Inc., Beverly, MA) with three changes of 10 mM sodium pyrophosphate buffer pH 6.7 (1 ml each) at 4°C for 4–5 h. Incorporation of 32P was measured as counts of precipitated trichloroacetic acid.
Ovine endometrial membranes were obtained from a nonpregnant ewe (Day 11 of the estrous cycle) as described previously [41]. Protein concentration in the membrane preparation was adjusted to 4 mg/ml. Binding and cross-linking of sheep endometrial membranes with [32P]ovIFN-4-P was performed as described previously [40]. A 20-ng aliquot (0.315 µCi) of [32P]IFN-4-P was incubated in the presence or absence of 500 molar excess (10 µg) of unlabeled ovIFN-4, 250 µl (1 mg protein) of endometrial membrane suspension, and 50 µl of 10x buffer (250 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM CaCl2, and 1% BSA), on ice for 1.5 h with occasional mixing. Membrane-bound interferon was separated by centrifugation (15 000 x g for 5 min). After three washes with 0.5 ml of PBS pH 7.5, the pellet was resuspended in 0.5 ml of PBS pH 7.5 containing 5 mM MgCl2. For cross-linking, disuccinimidyl suberate was added to final concentration of 5 mM. After 30 min of incubation on ice, the reaction was stopped by adding 30 µl of 1 M Tris-HCl pH 7.5. Reaction mixtures were centrifuged at 15 000 x g for 10 min, resuspended in 30 µl of extracting buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 100 mM NaCl, 0.1 mM PMSF, and 1% NP-40) and then incubated on ice for 30 min. Insoluble fractions were pelleted by centrifugation at 15 000 x g for 10 min, and the supernatants were loaded on 7.5% SDS-polyacrylamide gels. Bands corresponding to interferon/receptor cross-linked complexes were visualized by autoradiography.
Immunohistochemistry
Immunohistochemistry was performed as described previously [42]. The sections were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide:methanol to quench endogenous peroxidase activity. Normal goat serum was used to reduce nonspecific binding. Antisera to the intracellular domains of IFNAR1 and IFNAR2 were diluted to 1:250, and incubated overnight on the sections at 4°C. Negative controls included serial sections incubated with either preimmunized rabbit serum or antiserum that had been previously incubated with its corresponding recombinant protein of the cytoplasmic domain of each subunit. Immunohistochemistry for IFN-4 in ovine uterine sections was performed as described above.
The next day, slides were washed three times for 5 min each with Tris-buffered saline. The secondary anti-rabbit IgG antibody, followed by avidin and biotin were incubated on the tissues for 30 min each. The chromagen 3,3'-diaminobenzidine (DAKO) was used to detect binding. The slides were counterstained with Harris hematoxylin for 30 sec. Immunohistochemistry was repeated three times for each section.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovine IFNAR1 and IFNAR2 mRNA were present in most uterine cell types, but were most concentrated in LE and GE, caruncular stromal connective tissue (Fig. 1, A and C), the tunica media of blood vessels, and the myometrium (data not shown). Similar staining was evident in uterine sections from pregnant sheep that were incubated with antisense probes against either IFNAR1 or IFNAR2 (data not shown). No signal was detected when uterine sections from pregnant and nonpregnant sheep were hybridized with sense probes against IFNAR1 (Fig. 1B) or IFNAR2 (Fig. 1D).
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Immunohistochemistry
Both IFNAR1 and IFNAR2 polypeptide subunits were predominantly expressed in the LE (Fig. 2, A, C, E, and F) and superficial GE (Fig. 2, A, E, and F). Glandular epithelial cells near the myometrium were negative for both subunits (data not shown). The caruncular stromal cells also expressed both IFNAR1 and IFNAR2 polypeptides (Fig. 2, A and C), although expression in the remaining stroma was low. Smooth muscle fibers of the myometrium and tunica media of arterioles were positive for both subunits (data not shown).
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Western Blot Analysis
When the IFNAR1 antiserum was employed in conjunction with Western blotting, the anticipated [43, 44] but weakly staining band of 110 kDa (arrowhead) was observed in LE and GE cells, but was barely evident in stromal cells (Fig. 3A). The IFNAR1 antiserum unexpectedly recognized an intensely staining, lower molecular mass (80 kDa) protein in the LE cells and a slightly smaller component in GE cells. IFNAR2 antiserum revealed a weak band of 110 kDa (arrowhead) in each cell type (Fig. 3B). Again, however, a lower molecular weight component was present in the LE and GE lanes. In LE cells, a band of 80 kDa with strong staining was evident, whereas GE cells appeared to express a 75-kDa band (Fig. 3B). No bands were detected when the preimmune serum was used (data not shown). The broad, faster migrating band noted in lane 6 (stromal cells) was inconsistent and is probably an artifact.
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Cross-Linking Studies with 32P-Labeled IFN-
To determine whether the lower molecular weight (75- to 80-kDa forms) of IFNAR1 and IFNAR2 recognized by the antisera on Western blots were artifacts, we performed affinity cross-linking experiments with 32P-labeled IFN- on membrane fractions isolated from Day 11, nonpregnant ovine endometrium. The experiment revealed three major radioactive bands, a lower one with a molecular mass of 32 kDa, and two slower migrating components of 120 kDa and 80 kDa (Fig. 4). The two upper bands were broad, suggesting that they might contain a mixture of components. Although the cross-linking to IFN- appeared not to increase the apparent molecular mass of the receptor polypeptides, such an effect has been noted previously [14]. A likely explanation is that the presence of cross-links prevents SDS and 2-mercaptoethanol from completely unfolding the complex. The two upper bands did not become evident when an excess of unlabeled IFN-4 was added to the reaction mixture in which cross-linking was performed (Fig. 4). They are likely therefore to be specific IFN- binding components. The intense lower band of 32 kDa, on the other hand, was present under both reaction conditions and was judged to be nonspecific. It may represent a cross-linked complex of IFN-, most probably a dimer. This experiment indicated that the bands revealed by Western blotting in Figure 3 were probably not artifacts, but were each forms of IFNAR1 and IFNAR2.
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IFN Infusion and Binding Within the Uterus
To localize where in the uterus IFN- binds when it is introduced into the uterine lumen, we performed immunohistochemistry on sections of uteri from Day 14 nonpregnant ewes whose uteri had been infused with interferon. Staining was concentrated on the LE and superficial GE cells, and was absent from the deeper regions of the glands and from the stromal tissues (Fig. 5A) This pattern of interferon binding in the uterus mirrors that for the distribution of the interferon receptor subunits, except that blood vessels, caruncular stromal cells, and the myometrium were not stained with the antiserum to IFN-. Tissue sections from ewes that had been infused with PBS rather than IFN- exhibited no labeling (Fig. 5B). Nor was any staining present when nonimmunized rabbit serum was incubated with uterine sections from IFN- (Fig. 5C) or PBS-infused sheep (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[45], and are thus considered to be the prime targets for the paracrine actions of IFN-. This conclusion is strengthened by the results of the IFN- infusion study, which showed localization of the IFN- to these same regions of the surface epithelium and not to any of the other tissues that express the receptor subunits, including the caruncular stroma. It is possible that IFN- reaches other cells, but this was not detectable in our studies.
The data are consistent with the hypothesis originally proposed by Godkin et al. [31] that the antiluteolytic factor secreted by the sheep conceptus exerts its effects primarily via the uterine epithelium. It is curious that up-regulation of several interferon-stimulated genes (ISGs), including ISG 17 (ubiquitin cross-reactive protein), 2'5'-oligoadenylate synthetase, signal transducer and activator of transcription 1 (STAT1), STAT2, interferon regulatory factor 1 (IRF-1) and IRF-9, have been observed to occur primarily in the intercaruncular stromal tissue and deeper glandular epithelium, and not in superficial and upper glandular epithelial cells [46–49], which carry the highest concentration of receptor. Choi et al. [49] hypothesized that the failure of the conceptus and IFN- to induce these ISGs in the LE and GE cells was because these cells express the transcription factor IRF-2, a repressor, in response to IFN-, whereas stromal and glandular epithelial cells do not. There seem to be two possible explanations for the up-regulation of ISGs in the stroma. Either they possess low amounts of receptor, undetectable by our localization procedures, and respond to small amounts of interferon that passes across the uterine epithelium, or the induction of the ISGs in stromal cells is secondary to a primary response in the epithelium.
Western blotting revealed that both IFNAR1 and IFNAR2 existed in at least two molecular weight forms (Fig. 3). The molecular weights of the slower migrating bands for both subunits were calculated to be around 110 000, values similar to those described by others [43, 44, 50]. The precise nature of the lower molecular weight forms of both subunits, which have been observed previously in cross-linking experiments [41, 51], remains mysterious. These forms probably did not arise as a result of proteolysis during processing of the membrane fractions [41]. They may represent novel, alternatively transcribed forms of the receptor subunits or incompletely glycosylated precursor forms. Each must have at least a partial cytoplasmic domain, because the antisera were raised against these regions of the proteins. The presence of these smaller forms of the receptor might explain why binding studies [31, 33, 51] have always revealed two populations of receptors in the ovine endometrium, one with a binding affinity in the 10-10 M range, the other with a dissociation constant about 10-fold higher. In bovine uterus, where affinity cross-linking has revealed only a single band of 110 kDa, only the high-affinity population of receptors can be detected [41]. It seems likely therefore that one or both of the 75–80 kDa IFNAR-related polypeptides found in ovine uterus is the low-affinity form of the receptor detected in binding studies.
In summary, these studies demonstrate the high expression and colocalization of IFNAR1 and IFNAR2c in cells of the LE and GE of the intact uterus of ewes, and that these cells are the ones targeted by IFN- during pregnancy. The experiments also confirm that expression of the receptor subunits in the uterine endometrium are present both during pregnancy and the estrous cycle, although they may be slightly up-regulated when a conceptus is present [20]. Although apparently novel forms of IFNAR1 and IFNAR2 have been identified in these experiments, its relevance to IFN- action on the uterus remains unclear.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 R.M.R. was supported by grant HD 21896 from the National Institutes of Health. Part of this work was presented at the 34th Annual SSR meeting in Ottawa, ON, Canada, in 2001. 
2 Correspondence: R. Michael Roberts, University of Missouri-Columbia, 158 ASRC, 920 E. Campus Dr., Columbia, MO 65211. FAX: 573 882 6827; robertsrm{at}missouri.edu
3 Current address: Department of Bioinformatics, Lexicon Genetics Inc., The Woodlands, TX 77381
Accepted: April 11, 2002.
Received: February 6, 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 binding site, and observed ligand-induced tightening. Biochemistry 2002 41:3575-3585[CrossRef][Medline]. Proc Natl Acad Sci U S A 1989 86:558-562 with the type I interferon receptor on Daudi cells. J Interferon Cytokine Res 1995 15:(suppl 1):S97. J Clin Endocrinol Metab 1970 30::544-547 and localized to glandular epithelial and stromal cells from pregnant cows. Endocrine 1999 10:243-252[Medline]This article has been cited by other articles:
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G. Song, F. W Bazer, and T. E Spencer Pregnancy and interferon tau regulate RSAD2 and IFIH1 expression in the ovine uterus Reproduction, January 1, 2007; 133(1): 285 - 295. [Abstract] [Full Text] [PDF]
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V. Emond, L. A. MacLaren, S. Kimmins, J. A. Arosh, M. A. Fortier, and R. D. Lambert Expression of Cyclooxygenase-2 and Granulocyte-Macrophage Colony-Stimulating Factor in the Endometrial Epithelium of the Cow Is Up-Regulated During Early Pregnancy and in Response to Intrauterine Infusions of Interferon-{tau} Biol Reprod, January 1, 2004; 70(1): 54 - 64. [Abstract] [Full Text] [PDF]
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