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Regular Article |
a Center for Animal Biotechnology and Genomics, Department of Animal Science,
b Department of Veterinary Anatomy and Public Health, and
c Departments of Poultry Science and Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843
ABSTRACT
Interferon tau (IFN
) is the signal for maternal recognition of pregnancy in ruminants. The positive effects of IFN on IFN-stimulated gene (ISG) expression are mediated by ISG factor 3 (ISGF3), which is composed of signal transducer and activator of transcription (Stat) 1, Stat 2, and IFN regulatory factor-9 (IRF-9), and by gamma-activated factor (GAF), which is a Stat 1 homodimer. Induction of ISGs, such as ISG17 and 2',5'-oligoadenylate synthetase, by IFN during pregnancy is limited to the endometrial stroma (S) and glandular epithelium (GE) of the ovine uterus. The IRF-2, a potent transcriptional repressor of ISG expression, is expressed in the luminal epithelium (LE). This study determined effects of the estrous cycle, pregnancy, and IFN on expression of Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 genes in the ovine endometrium. In cyclic ewes, Stat 1, Stat 2, IRF-1, and IRF-9 mRNA and protein were detected at low levels in the S and GE. During pregnancy, expression of these genes increased only in the S and GE. Expression of IRF-2 was detected only in the LE and superficial GE (sGE) of both cyclic and pregnant ewes. In cyclic ewes, intrauterine administration of IFN stimulated Stat 1, Stat 2, IRF-9, and IRF-1 expression in the endometrium. Ovine IRF-2 repressed transcriptional activity driven by IFN-stimulated response elements that bind ISGF3, but not by gamma-activation sequences that bind GAF. These results suggest that IRF-2 in the LE and sGE restricts IFN induction of ISGs to the S and GE. In the S and GE, IFN hyperactivation of ISG expression likely involves formation and actions of the transcription factors ISGF3 and, perhaps, IRF-1.
conceptus, gene regulation, hormone action, interferon, IRF, signal transduction, Stat, uterus
INTRODUCTION
Tau interferons (IFN) are a unique subclass of the 172-amino acid type I omega (
) IFNs produced exclusively by the trophectoderm of ruminant (i.e., sheep, cattle, and goat) conceptuses [1]. The mononuclear cells of the trophectoderm synthesize and secrete IFN between Day 11 and Days 20–24 of pregnancy in sheep [2, 3], which then acts in a paracrine manner on the endometrial luminal epithelium (LE) and superficial glandular epithelium (sGE) to suppress transcription of estrogen receptor alpha (ER
) and oxytocin receptor (OTR) genes [4–6]. These actions prevent development of the luteolytic mechanism by abrogating oxytocin-induced luteolytic pulses of prostaglandin F2 [6–8]. In addition to suppressing or silencing transcription of ER and OTR genes, IFN induces or increases expression of a number of IFN-stimulated genes (ISGs) in the ruminant endometrium as well as in endometrial and fibroblast cell lines. These ISGs include signal transducer and activator of transcription (Stat) 1 and 2 [9–11], ß2-microglobulin [12], IFN regulatory factor one (IRF-1) [6, 9, 10, 13], ISG17/ubiquitin cross-reactive protein [10, 14–16], Mx protein [17], granulocyte chemotactic protein-2 [18], and 2',5'-oligoadenylate synthetase (OAS) [19–22]. In the endometrium of early pregnant ewes as well as cyclic ewes receiving intrauterine injections of recombinant ovine IFN, ISG17 and OAS genes are up-regulated only in the stroma (S) and middle- to deep uterine glands in the stratum spongiosum [15, 22, 23].
Induction and increases in ISG expression in response to IFN are mediated by an intracellular signal transduction system involving Stats and IRFs [6, 9, 10, 11, 13, 24]. The IFN activates a signal transduction pathway similar to that of IFN/ß in ovine endometrial cell lines [9, 10] and human fibroblast cell lines [11]. In ovine endometrial LE cells, IFN stimulates tyrosine phosphorylation and nuclear translocation of Stats 1, 2, 3, 5a/b, and 6 within 30 min [10]. However, exposure to IFN for more than 30 min extends Stat 1 and 2 tyrosine phosphorylation, whereas Stats 3, 5a/b, and 6 are rapidly dephosphorylated. The IFN induces homodimerization of Stat 1 to form the transcription factor gamma-activated factor (GAF) as well as heterodimerization of Stat 1 and Stat 2, which complex with IRF-9 (also known as ISGF3
/p48) to form the transcription factor complex IFN-stimulated gene factor 3 (ISGF3). The GAF regulates transcription through -activated sequence (GAS) elements in the promoter region of selected ISGs such as IRF-1 [25, 26]. The ISGF3 binds to IFN-stimulated response elements (ISREs) to drive transcription of ISGs such as ISG17 [27], Mx [28], and OAS [29]. Similarly, expression of Stat1, Stat2, IRF-9, and IRF-1 genes is induced or increased by IFN and other type I IFNs via the ISGF3 and GAF transcription factor complexes [10, 11, 26, 30, 31]. The simultaneous induction of Stat 2 and IRF-9 gene expression by IFN appears to shift transcription factor formation from GAF toward predominantly ISGF3 [10, 11].
In the pregnant ovine uterus, ISGs including OAS and ISG17 are induced or up-regulated in the endometrial S and glandular epithelium (GE), but expression of these genes is not detectable in the endometrial LE or sGE [15, 22]. The lack of induction or increases in ISG expression in either LE or sGE during pregnancy and in response to IFN may be due either to the lack of Stat 1, Stat 2, IRF-9, or, perhaps, IRF-1 gene expression in the LE and sGE or the presence of IRF-2, a transcriptional repressor of ISGs as well as type I IFNs themselves [32–35]. In pregnant ewes, IRF-2 protein is present in the LE and sGE on Days 13, 15, 17, and 20 [6]. Therefore, the objectives of the present study were to determine effects of the estrous cycle, pregnancy, and intrauterine administration of recombinant ovine IFN on expression of the Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 genes in endometrium of the ovine uterus and to determine effects of ovine IRF-2 on transactivation of ISRE- and GAS-driven promoters.
MATERIALS AND METHODS
Animals and Experimental Design
Mature ewes of primarily Rambouillet breeding were observed daily for estrus using vasectomized rams. All ewes exhibited at least two estrous cycles of normal duration (
16–18 days). Experimental and surgical procedures involving animals were approved by the Agricultural Animal Care and Use Committee of Texas A&M University.
Study 1
At estrus, ewes were assigned randomly to cyclic or pregnant status. Ewes assigned to pregnant status were bred to intact rams during estrus. Ewes were then ovariohysterectomized (n = 4 ewes/day) on Days 11, 13, or 15 of the estrous cycle and Days 11, 13, 15, or 17 of pregnancy (Day 0 = estrus/mating). Pregnancy was confirmed by presence of an apparently normal conceptus in uterine flushings. At hysterectomy, several sections (0.5 cm) from the middle of each uterine horn were fixed in fresh, 4% paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% (v/v) ethanol for 24 h and then dehydrated and embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). Several sections (1–1.5 cm) from the middle of each uterine horn were also embedded in Tissue-Tek Optimal Cutting Temperature (OCT) compound (Miles, Inc., Oneonta, NY), 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 for this study.
Study 2
Eight cyclic ewes were fitted with uterine catheters on Day 5 of the estrous cycle (Day 0 = estrus) as described previously [23]. Ewes (n = 4 ewes/treatment) were then allotted randomly to receive intrauterine injections of either control serum proteins (6 mg/day) or recombinant ovine IFN (roIFN; 2 x 107 antiviral units/day) [36] on Days 11–15 after estrus. For intrauterine administration, the uterine horns of each ewe received injections of either roIFN (5 x 106 antiviral units/horn per injection) or control ovine serum proteins (CX; equal amount of total protein/horn per injection) twice daily. This regimen of roIFN treatment has been shown to mimic the antiluteolytic effects of the conceptus on the endometrium during the pregnancy-recognition period [23, 37]. All ewes were ovariohysterectomized on Day 16. The endometrium was dissected from the myometrium, frozen in liquid nitrogen, and stored at -80°C for RNA extraction.
Cloning of a Partial cDNA for Ovine IRF-9 and Full-Length cDNA for Ovine IRF-1 and IRF-2
Partial cDNAs for ovine IRF-1 and IRF-9 were amplified by reverse transcription-polymerase chain reaction (PCR) of total RNA from Day 15 pregnant ovine endometrium using primers based on the human IRF-1 sequence (GenBank accession no. NM_002198; forward: 5'-TCC ACC CAC CAA GAA CC-3', reverse: 5'-TTC TGG CTC CTC CTT ACA GC-3') or human IRF-9 sequence (NM_006084; forward: 5'-TGA ATT TAA GGA GGT TCC-3', reverse, 5'-GCA GCA GTG AGT AGT CTG-3'). Reverse transcription of endometrial total RNA was performed as described previously [37]. The PCR was conducted as follows for IRF-1 using PCR Optimized Buffer A (Invitrogen, Carlsbad, CA): 1) 95°C for 2 min; 2) 30 sec at 95°C, 30 sec at 57°C, and 30 sec at 72°C for 30 cycles; and 3) 72°C for 10 min. For IRF-9, PCR was performed using PCR Optimized Buffer C (Invitrogen) as follows: 1) 95°C for 2 min; 2) 30 sec at 95°C, 1 min at 42°C, 1 min at 72°C for four cycles; 3) 30 sec at 95°C, 1 min at 53°C, and 1 min at 72°C for 35 cycles; and 3) 72°C for 10 min. The amplified IRF-1 (502 base pairs [bp]) and IRF-9 (392 bp) PCR products were subcloned into pCRII cloning vector using a T/A Cloning Kit (Invitrogen) and sequenced in both directions using an ABI PRISM Dye Terminator Cycle Sequencing Kit and ABI PRISM automated DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA) to confirm identity.
Full-length cDNAs for ovine IRF-1 and ovine IRF-2 were obtained by conventional screening of a Lambda ZAP II cDNA library of Day 15 pregnant ovine endometrium (Stratagene, La Jolla, CA) at low stringency using radiolabeled cDNA probes of an ovine IRF-1 partial cDNA and murine IRF-2 cDNA. Positive clones containing entire ovine IRF-1 and IRF-2 coding sequences were sequenced in both directions and deposited in GenBank (GenBank accession no. AF331970 and AF228445, respectively). Mammalian expression vectors for ovine IRF-1 and IRF-2 were constructed by subcloning the EcoRV/NotI and BamHI/NotI fragments containing full-length ovine IRF-1 and IRF-2 cDNA sequences into BamHI (blunt end)/NotI-digested and BamHI/NotI-digested pEF1-Myc/His-LacZ mammalian expression vector (Invitrogen), respectively.
RNA Isolation and Analyses
RNA isolation Total cellular RNA was isolated from endometrium using Trizol reagent (Gibco-BRL, Bethesda, MD). The quantity of RNA was assessed spectrophotometrically and the integrity of RNA examined by gel electrophoresis in a denaturing 1% (w/v) agarose gel.
Slot blot hybridization analysis Steady-state levels of Stat and IRF mRNAs were assessed by slot blot hybridization using methods described previously [37, 38]. Denatured total endometrial RNA (20 µg) from each ewe was analyzed using a radiolabeled antisense human Stat 1 (M97935) [39], human Stat 2 (NM005419) [40], ovine IRF-9, ovine IRF-1, or ovine IRF-2 cRNA. To correct for variation in total RNA loading, a duplicate RNA slot membrane was hybridized with radiolabeled antisense 18S rRNA cRNA (pT718S; Ambion, Austin, TX). Following washing, nonspecific hybridization was removed by RNase A digestion [41]. The radioactivity associated with each slot was quantitated by electronic autoradiography using an Instant Imager (Packard Instrument Company, Meridian, CT) and is expressed as total counts (TC).
In Situ Hybridization Analysis
The mRNAs for Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 were localized in uterine tissue sections (5 µm) by in situ hybridization analysis as described previously [38]. Deparaffinized, rehydrated, and deproteinated uterine tissue sections were hybridized with radiolabeled antisense or sense cRNA probes generated from linearized human Stat 1, human Stat 2, ovine IRF-9, ovine IRF-1, or ovine IRF-2 cRNA cDNAs using in vitro transcription with -35S-uridine triphosphate. After hybridization, washing, and ribonuclease A digestion, slides were dipped in NTB-2 liquid photographic emulsion (Eastman Kodak, Rochester, NY), stored at 4°C for 1 wk (Stat 1, Stat 2, IRF-9, and IRF-1) or 3 wk (IRF-2), and developed in Kodak D-19 developer. Slides were then counterstained with Harris modified hematoxylin (Fisher Scientific, Fairlawn, NJ), dehydrated through a graded series of alcohol to xylene, and protected with a coverslip.
Generation of Polyclonal Antibodies to Ovine IRF-2 in Chickens
Single Comb White Leghorn hens (13 wk old) were used for production of polyclonal antibodies against ovine IRF-2 protein. An IRF-2-specific peptide segment was selected based on the predicted amino acid sequence of ovine IRF-2 (AAF34781.1) by use of the EMBL PredictProtein server. The selected antigenic sequence was NH2-CITQARVKSY-COOH (IRF-2 CT) corresponding to the C-terminus of the native protein. An N-terminal cysteine residue was added for ease of coupling to a carrier protein. The peptide was synthesized by Sigma Genosys (Woodlands, TX) and coupled to thyroglobulin (Sigma, St. Louis, MO) with m-maleimidobezoyl-N-hydroysuccinimide ester (Pierce, Rockford, IL) using an approximately 40-fold molar excess of peptide over protein (2 mg peptide and 15 mg of thyroglobulin). The conjugates (400 mg) were solubilized in 2 ml of PBS buffer (0.15 M), emulsified with 2 ml of Freunds Complete Adjuvant (Sigma), and used to immunize four birds in the breast muscle, with each bird receiving 100 mg of the conjugate. The chickens were boosted at 2-wk intervals using the same dose and volume of the respective conjugates emulsified in Freunds Incomplete Adjuvant (Sigma). Eggs were collected for purification of IgY from the yolks. Briefly, yolk lipids were removed from the emulsified yolks by euglobulin precipitation, and chicken IgY was then further purified by ammonium sulfate precipitation [42]. Peptide-specific antibodies were purified by immunoaffinity chromatography over a 1-ml SulfoLink Coupling gel (Pierce) derivatized with 950 mg of the respective peptides. Peptide-specific antibodies were eluted with 50 mM glycine (pH 2.5). The purified fractions were collected in Tris buffer (2.0 M, pH 8.0) for immediate neutralization and stored at 4°C.
Immunocytochemical Analyses
Frozen sections (4–8 µm) of uterine tissues embedded in OCT compound in study 1 were cut with a cryostat and mounted on Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). 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 (two parts 0.02 M PBS, 1.0% BSA, and 0.3% Tween-20 [pH 8.0] to one 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 antibodies. Antibodies were mouse anti-Stat 1 (#S21120) at 30 µg/ml and mouse anti-IRF-9/ISGF3 (#I29320) at 20 µg/ml from Transduction Laboratories (Lexington, KY); rabbit anti-Stat 2 (#sc-476) at 20 µg/ml and rabbit anti-IRF-1 (#sc-497) at 20 µg/ml from Santa Cruz Biotechnology (Santa Cruz, CA); chicken anti-IRF-2 at 20 µg/ml; and normal rabbit IgG (#I5006), normal mouse IgG (#I5381), or normal chicken IgY (Sigma) at the same concentrations as primary antibodies. Following three rinses in PBS for 10 min each, sections were incubated with fluorescein-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA) for 1 h at room temperature and again washed in PBS three times for 10 min each. Sections were then overlaid with a coverslip and Prolong Antifade mounting reagent (Molecular Probes, Eugene, OR).
Photomicroscopy and Digital Imaging
Images of representative fields of in situ hybridization and immunofluorescence slides were recorded using a Zeiss Axioplan2 microscope (Carl Zeiss, Thornwood, NY) fitted with a Hamamatsu C-5810 chilled three-color CCD camera (Hamamatsu Corporation, Bridgewater, NJ). Digital images were captured and/or assembled using Adobe Photoshop 4.0 (Adobe Systems, Seattle, WA) and a MacIntosh PowerMac G3 computer (Apple Computer, Cupertino, CA). Black-and-white prints were electronically printed using a Kodak DS8650 color printer.
Transient Transfection Assays
Constructs The 5xISRE-TK-LUC (pISRE-LUC) and TK-LUC (pTAL-LUC) constructs were purchased from Clontech (Palo Alto, CA). The 3xGAS-TK-LUC constructs contain three copies of the rat IRF-1 GAS element as described previously [10].
Transfection procedure
Immortalized ovine endometrial stromal cells [9] were subcultured into 12-well plates (70–80% confluent) and transiently cotransfected (n = 4 wells per construct and treatment) with the indicated LUC reporter construct (0.5 µg/well) and pEF1-Myc/His-LacZ (0.05 µg/well; Invitrogen) as a marker for transfection efficiency using the GenePORTER transfection reagent (Gene Therapy Systems, Inc., San Diego, CA) according to the manufacturer's recommendations. After transfection, the cells were left untreated as a control or were treated with roIFN (10 000 antiviral units/ml) for 24 h. Cells were then harvested using Cell Culture Lysis Buffer (Promega, Madison, WI).
Luciferase and ß-galactosidase assays
Luciferase expression assays were conducted using the Promega Luciferase Assay System according to the manufacturer's recommendations and quantitated using a Packard Luminometer. ß-Galactosidase assays were performed using a Galacto-Light Plus Kit (Tropix, Bedford, MA) according to the manufacturer's recommendations. For each experiment, luciferase expression was analyzed by least-squares ANOVA procedures using the ß-galactosidase values as a covariate to correct for differences in transfection efficiency between wells and plates within time points. Data are reported as fold-induction or fold-suppression of IFN based on least-squares mean relative light units. Each transient transfection experiment was repeated a minimum of four times.
Statistical Analyses
Data from slot blot hybridization analyses were subjected to least-squares ANOVA using the General Linear Models procedures of the Statistical Analysis System [43]. Slot blot hybridization data for Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 mRNAs (TC) were normalized for differences in sample loading using the 18S rRNA data as a covariate in least-squares ANOVA. Data from study 1 were analyzed for effects of day, pregnancy status (cyclic or pregnant), and their interaction. Within pregnancy status, least-squares regression analyses were used to determine effects of day on endometrial mRNA levels. Data from study 2 were subjected to one-way least-squares ANOVA. All tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error [44]. Fold-induction or fold-suppression data derived from each transfection experiment were analyzed by one-way ANOVA to determine effects of ovine IRF-1 and IRF-2 expression on activity of the promoter-reporter constructs. A P value of 0.05 or less was considered to be statistically significant. Data are presented as least-square means (TC) with SEM.
RESULTS
Effects of the Estrous Cycle and Pregnancy on Stat and IRF Expression in the Ovine Endometrium (Study 1)
Slot blot hybridization analyses Effects of day and pregnancy status on steady-state levels of Stat and IRF mRNAs in the ovine endometrium are summarized in Table 1. In cyclic ewes, Stat 1, IRF-9, and IRF-2 mRNA levels decreased (P < 0.05, linear) between Days 11 and 15 of the cycle. Endometrial levels of Stat 2 and IRF-1 mRNA did not decline in cyclic ewes (P > 0.10). Pregnancy affected expression of Stat 1 (P = 0.003), Stat 2 (P = 0.03), IRF-9 (P = 0.05), IRF-1 (P = 0.0001), and IRF-2 (P = 0.05) mRNAs in the ovine endometrium (day x status interaction). In pregnant ewes, steady-state levels of Stat 1, Stat 2, IRF-9, and IRF-1 mRNAs increased in the endometrium (P < 0.05; quadratic for Stat 1, Stat 2, and IRF-9; cubic for IRF-1).
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In situ hybridization analyses The overall pattern of expression of Stat and IRF mRNAs during the cycle and early pregnancy paralleled that found by slot blot hybridization analysis of endometrial total RNA. During the estrous cycle, Stat 1, Stat 2, IRF-9, and IRF-1 mRNA expression was detected at relatively low levels in all cell types (Figs. 1–4). In the intercaruncular endometrium, expression of these mRNAs was higher in the stratum compactum S than in the stratum spongiosum S, LE, or GE. In cyclic ewes, IRF-1 mRNA increased in the LE between Days 11 and 15 (Fig. 4). In contrast, IRF-2 mRNA was expressed in the LE and sGE to middle GE during the estrous cycle but was not detected in S as compared to the background present in the sense control (Fig. 5).
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Expression of Stat 1, Stat 2, IRF-9, and IRF-1 mRNAs increased in the endometrium during early pregnancy (Figs. 1–4). Relative levels of expression of these mRNAs were not different in the endometrium of cyclic and pregnant ewes on Day 11, except for IRF-1 mRNA, which was more abundant in focal areas of S in the caruncular areas of the uterus. On Day 13 of pregnancy, an increase in Stat 1, Stat 2, and IRF-9 mRNA expression was detected in the stratum compactum S and middle GE. The IRF-1 mRNA increased in stratum compactum S but not in GE on Day 13. By Day 15 of pregnancy, strong expression of Stat 1, Stat 2, IRF-9, and IRF-1 mRNAs was detected throughout the S and GE of the intercaruncular endometrium. No increase in expression of these Stat and IRF mRNAs was detected in the LE or sGE. By Day 17 of pregnancy, relative levels of Stat 1, Stat 2, IRF-9, and IRF-1 mRNA expression declined in the GE but remained higher in the S than observed in endometrium from Day 11 of pregnancy.
During early pregnancy, IRF-2 mRNA was expressed in LE and sGE (Fig. 5). No difference in endometrial expression of IRF-2 mRNA was detected in these epithelia between cyclic and pregnant ewes on Days 11 and 13. However, IRF-2 mRNA levels were greater in the LE and sGE on Day 15 of pregnancy, and they remained elevated on Day 17 of pregnancy.
Immunofluorescence Localization of Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 Protein in Ovine Endometrium (Study 1)
Immunoflourescence localization of Stat and IRF proteins was conducted to confirm Stat and IRF mRNA expression results obtained by in situ hybridization. As compared to the IgG controls, immunoreactive Stat 1, Stat 2, and IRF-9 proteins were not detected in either LE or GE in endometrium from cyclic or early pregnant ewes (Fig. 6). However, a notable increase in Stat 1, Stat 2, and IRF-9 protein expression in S and GE was detected between Days 11 and 15 of early pregnancy (Fig. 6). These proteins were detected in both the nucleus and cytoplasm. The rabbit polyclonal IRF-9 antibody nonspecifically cross-reacted with an unidentified antigen in the basal lamina of the endometrial LE in all uterine samples regardless of day. All antibodies used in this study have been used previously for Western blot analyses of ovine endometrial cell lines as well as human fibroblasts [10, 11].
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The IRF-1 protein was present only in the S of Day 11 cyclic and pregnant ewes (Fig. 7). On Day 15 of the estrous cycle, IRF-1 protein was detected in LE and sGE as well as in S. Between Days 11 and 15 of pregnancy, IRF-1 protein increased in the S and the middle to deep GE. However, no increase in IRF-1 protein expression was detected in the LE and sGE of pregnant ewes. In the middle to deep uterine glands, a punctate pattern of immunoreactive IRF-1 protein was observed in the uterine glands. Although IRF-1 protein was present in the GE cells, expression was predominantly located in the nuclei of intraepithelial cells, which are presumed to be immune cells.
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Chicken anti-ovine IRF-2 IgY detected only recombinant ovine IRF-2, not recombinant ovine IRF-1 or IRF-6 proteins, in Western blot assays (data not shown). In addition, the purified chicken anti-ovine IRF-2 IgY detected the appropriately sized protein in extracts of immortalized ovine LE cells as well as Cos-7 cells overexpressing ovine IRF-2, but in not ovine IRF-1 or ovine IRF-6 (data not shown). Immunoreactive IRF-2 protein was detected in the endometrial LE and sGE of cyclic and pregnant ewes (Fig. 7). As for immunoreactive IRF-1, abundant IRF-2 protein was detected in intraepithelial immune cells in the middle to deep GE regardless of day. On Day 15 of pregnancy, levels of IRF-2 protein were higher in the nuclei of LE and sGE cells on Day 15 of pregnancy compared to Day 11 of pregnancy or Day 15 of the cycle. Preadsorption of the antipeptide IRF-2 IgY with recombinant ovine IRF-2 eliminated L, GE, and immune cell staining, but it also revealed low levels of nonspecific binding of the chicken anti-ovine IRF-2 IgY to the basal lamina of the endometrial epithelia as well as blood vessels in a manner similar to IgY alone (data not shown).
Effects of Intrauterine Administration of Recombinant Ovine IFN on Stat and IRF mRNA Expression in Ovine Endometrium (Study 2)
To determine if the effects of pregnancy on endometrial Stat and IRF gene expression in vivo were due to IFN from the conceptus, cyclic ewes were fitted with uterine catheters on Day 5 and given daily intrauterine injections of either control proteins or roIFN from Days 11 to 15. As illustrated in Figure 8, intrauterine administration of roIFN into cyclic ewes induced an approximately 3-fold increase (P < 0.01, CX vs. IFN) in steady-state levels of endometrial Stat 1 mRNA. Similarly, roIFN increased steady-state levels of endometrial Stat 2 and IRF-9 mRNA by approximately 2-fold (P < 0.01) and of IRF-1 mRNA by approximately 1.4-fold (P < 0.10, CX vs. IFN). However, IRF-2 mRNA levels were not affected by roIFN treatment (P > 0.10).
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Effects of Ovine IRF-1 and Ovine IRF-2 on Activity of Promoters Containing ISRE or GAS Sequences in Transient Transfection Assays
An immortalized ovine endometrial S cell line was transiently transfected with mammalian overexpression vectors for ovine IRF-1, ovine IRF-2, or the combination of ovine IRF-1 and IRF-2 to determine their effects on transactivation of a promoter-reporter construct containing five consensus ISRE-binding sites (Fig. 9). Ovine IRF-1 alone transactivated (2 ± 0.2-fold) the 5xISRE-TK-LUC reporter. In contrast, ovine IRF-2 alone strongly repressed (24 ± 3-fold) transcriptional activity of the 5xISRE-TK-LUC reporter. Cotransfection of ovine IRF-1 with ovine IRF-2 also resulted in repression (17 ± 3-fold) of the 5xISRE-TK-LUC reporter. Transcriptional activity of a reporter containing three IRF-1 GAS elements (3xGAS-TK-LUC) was not affected by overexpression of either ovine IRF-1 or ovine IRF-2 in the ovine LE cells (data not shown). Activity of the minimal TK-LUC reporter was also not affected by overexpression of either ovine IRF-1 or ovine IRF-2 (data not shown).
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DISCUSSION
To our knowledge, this is the first report of temporal and spatial alterations in Stat 1, Stat 2, and IRF-9 gene expression in the ovine uterus. Results presented here confirm and extend those of previous reports that these classical ISGs are directly regulated by IFN in the ovine endometrium [9–11, 15, 22, 23]. Similar to expression of OAS, Mx, and ISG17 [15, 17, 22], expression of Stat 1, Stat 2, IRF-9, and IRF-1 in the ovine endometrial S and GE during early pregnancy was coordinate with temporal production of IFN by the conceptus [2, 3]. The increases in expression of these ISGs during pregnancy can be attributed to IFN from the conceptus, because intrauterine infusion of cyclic ewes with roIFN alone increased Stat 1, Stat 2, IRF-9, and IRF-1 expression in the endometrium. Originally, Stats 1 and 2 and IRF-9 were discovered as ISGs and termed ISG factors 1 (p91), 2 (p113), and 3 (p48), respectively [45, 46]. Expression of these genes is increased by type I IFNs [31] via multiple ISREs in their promoter regions [47, 48]. Likewise, the 5' promoter/enhancer region of other ISGs in the ovine uterus, including ISG17, Mx, and OAS, contain multiple ISREs that bind ISGF3 and induce or increase their expression in response to type I IFNs [13, 27–30].
The relative abundance of Stat 1, Stat 2, IRF-9, and IRF-1 is hypothesized to dictate the temporal and spatial influences of conceptus IFN on ISG expression in the endometrial S and GE as determined from studies in ovine endometrial cell lines [9, 10] as well as human fibroblast cell lines lacking specific IFN signaling components [11]. Collectively, available results support the concept that the continual, elevated expression of ISGs in the endometrial S and GE during pregnancy can be attributed to IFN stimulation of janus kinase (JAK), JAK1 and Tyk2, activation of Stats 1 and 2 by phosphorylation, formation of the transcription factor complexes GAF and ISGF3, and increased expression of IRF-1 and then Stat 1, Stat 2, and IRF-9 (Fig. 10). Similar to in vitro observations on IFN signal transduction pathways [9–11], IFN activation of Stats 1 and 2 is proposed to result in ISGF3 formation and hyperactivated expression of ISRE-containing target genes, such as Stat 1, Stat 2, IRF-9, ISG17, and OAS, in the S and GE. Similarly, activation of Stat 1 elicits formation of Stat 1 homodimers or GAF that activates GAS-containing target genes, such as IRF-1 [10, 25, 49]. The IRF-1 can bind to ISREs [50] and may assist in maintaining activation of ISRE-containing target genes by type I IFNs [13, 27], because it contains a transactivation domain [51]. The simultaneous induction of Stat 2 and IRF-9 gene expression by IFN appears to shift transcription factor formation from GAF toward predominantly ISGF3 [10, 11]. The persistent formation of ISGF3 would favor the expression of ISGs in the S and GE.
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In this study, low levels of IRF-2 gene expression were detected in the LE and sGE from ewes on Days 11, 13, and 15 of the estrous cycle. These findings conflict with those of a previous report [6], perhaps due to differences in sensitivities of antibodies or methods for immunohistochemistry. In the present study, a peptide-specific antibody generated to ovine IRF-2 was used in immunofluorescence analyses of frozen ovine uterine tissue. Results indicated that IRF-2 mRNA and protein expression increased in the endometrial LE and sGE between Days 11 and 15 of pregnancy, as reported previously [6]. The increase in IRF-2 gene expression in the LE and sGE likely involves other factors besides IFN, because IFN does not induce or increase IRF-2 expression in the endometrium of cyclic ewes (Fig. 8) or in bovine and ovine endometrial epithelial cell lines [13, 52]. The decline in IRF-2 mRNA expression in the whole endometrium between Days 15 and 17 of pregnancy is likely due to loss of some LE during initial synepithelial placentation [53].
Expression of Stat 1, Stat 2, and IRF-9 mRNA and protein was not detected in the LE and sGE on Days 11–19 of early pregnancy in this study. These results agree with those reported for ISG17 and OAS expression in the ovine uterus [15, 16, 22, 23]. The absence of these ISGs in the LE and sGE during early pregnancy is hypothesized to result from expression of IRF-2, a potent repressor and attenuator of IFN/ß-induced gene expression [33, 34, 54–57]. In the present and a previous study [6], IRF-2 protein was detected only in the LE and sGE on Days 13, 15, 17, and 20 of pregnancy. Results from the transient transfection studies presented here further support the hypothesis that IRF-2 expression in the LE and sGE restricts and specifies increases in ISG expression to the S and GE in response to pregnancy and intrauterine IFN. Transient transfection assays revealed that ovine IRF-2 strongly inhibited transcriptional activity of an ISRE-driven reporter construct, which supports the results of Fleming et al. [52], who found that ovine IRF-2 inhibited reporter constructs driven by closely related IRF-binding elements or IRF-Es in transient transfection assays.
Collectively, available in vitro and in vivo results support the hypothesis that IRF-2 prevents the induction of ISG expression in ovine LE and sGE in response to IFN (Fig. 10). The IRF-2, a unique member of the IRF family of transcription factors, is a potent repressor of IFN/ß-induced gene transcription [33, 34, 54–57]. Both IRF-2 and ISGF3 bind to ISREs [58]. Unlike ISGF3 and IRF-1, IRF-2 is a stable nuclear protein and is expressed in many cell types [55]. Recently, it was demonstrated that IRF-2 competes with IRF-1 for ISRE binding, and that IRF-1 and IRF-2 formed a heterocomplex wherein IRF-2 decreased transactivation by IRF-1 [35]. The IRF-2 contains a repression domain [59] and has a rapid on-off rate for ISRE binding [34]. Senger et al. [35] provided strong evidence that IRF-2 represses transcription by inhibiting recruitment of coactivators into enhanceosomes by transcription factors. Hida et al. [34] proposed that IRF-2 continuously interacts with ISRE elements in promoter regions of ISGs to inhibit ISGF3 transcription factor complex induction of ISGs. Although the IRF-1 gene is not directly attenuated by IRF-2, IRF-1 gene expression did not increase in the endometrial LE and sGE during early pregnancy in the present study. Increases in IRF-1 gene transcription in several cell lines requires induction of the Stat 1 gene and formation of Stat 1 dimers, that is, Stat 1 homodimers or GAF, or Stat 1–2 heterodimers [10, 11, 24, 25]. Therefore, the lack of IRF-1 induction by IFN in the ovine uterine endometrial LE may be explained by the lack of detectable increases in Stat 1 and Stat 2 gene expression in these cells. Another possibility is that IFN activates other signal transduction pathways that are unrelated to the JAK-Stat pathway, such as nuclear factor-kappa B by stimulation of phosphatidylinositol 3-kinase and Akt [60], but that are involved in IFN regulation of LE gene expression. These signaling pathways are involved in other type I IFN effects on gene expression and warrant further investigation. A plausible hypothesis is that activation of non-JAK-Stat pathways by IFN and the consequent lack of ISG induction in the LE relate to the higher levels of basal prostaglandin F2 secretion in early pregnant ewes [7, 8].
In the present study, an increase in IRF-1 expression was detected in the endometrium from ewes on Days 11–15 of the estrous cycle. The precise cellular and molecular mechanism regulating this event is not known, but it correlates with a decline in overall endometrial IRF-2 gene expression in the present study as well as with increases in ER and OTR gene expression that are specific to the endometrial LE and sGE [4–6]. The ovine ER and bovine OTR genes contain several IRF-Es [52, 61]. Recently, Fleming et al. [52] found that the promoter/enhancer region of the ovine ER gene contains two functional IRF-Es and one ISRE. Furthermore, overexpression of ovine IRF-2 inhibited transcriptional activity of several regions of the ovine ER promoter containing a functional IRF-E or ISRE. Therefore, the overall ratio of IRF-1 to IRF-2 protein in endometrial LE and sGE may regulate ER and, perhaps, OTR expression during development of the endometrial luteolytic mechanism in cyclic ewes and during pregnancy recognition in pregnant ewes.
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ACKNOWLEDGMENTS
The authors thank Mr. Todd Taylor and Dr. Shawn W. Ramsey of the Texas A&M University Sheep and Goat Center for assistance with animal husbandry and Drs. Curt Horvath and James Darnell (Rockefeller University) for provision of human Stat 1 and Stat 2 cDNAs.
FOOTNOTES
1 Supported by NIH grant HD32534 (to F.W.B. and T.E.S.) and, in part, by NIH grants HD08501 (to G.A.J.) and P30 ES09106. 
2 Correspondence: Fuller W. Bazer, Center for Animal Biotechnology and Genomics, 442D Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. FAX: 979 862 2662; fbazer{at}cvm.tamu.edu
3 Current address: Animal and Veterinary Science Department, Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-2330.
4 These authors contributed equally to this work and should be considered first coauthors.
Accepted: May 15, 2001.
Received: March 29, 2001.
REFERENCES
) regulation of IFN-stimulated gene expression in cell lines lacking specific IFN-signaling components. Endocrinology 2001; 142::1786-1794/ß-signaling. Immunity 2000; 13:643-655[CrossRef][Medline] gene regulatory elements. EMBO J 1988; 7:3397-3405[Medline]This article has been cited by other articles:
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|
|
|
|
 |
|
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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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|
|
|
|
|
|
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|
|
|
|
|
|
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|
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Y. Chen, E. Antoniou, Z. Liu, L. B Hearne, and R M. Roberts A microarray analysis for genes regulated by interferon-{tau} in ovine luminal epithelial cells Reproduction, July 1, 2007; 134(1): 123 - 135. [Abstract] [Full Text] [PDF]
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|
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T. E Spencer, G. A Johnson, F. W Bazer, and R. C Burghardt Implantation mechanisms: insights from the sheep Reproduction, December 1, 2004; 128(6): 657 - 668. [Abstract] [Full Text] [PDF]
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G. A. Johnson, R. C. Burghardt, M. M. Joyce, T. E. Spencer, F. W. Bazer, C. Pfarrer, and C. A. Gray Osteopontin Expression in Uterine Stroma Indicates a Decidualization-Like Differentiation During Ovine Pregnancy Biol Reprod, June 1, 2003; 68(6): 1951 - 1958. [Abstract] [Full Text] [PDF]
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Y. Choi, G. A. Johnson, T. E. Spencer, and F. W. Bazer Pregnancy and Interferon Tau Regulate Major Histocompatibility Complex Class I and {beta}2-Microglobulin Expression in the Ovine Uterus Biol Reprod, May 1, 2003; 68(5): 1703 - 1710. [Abstract] [Full Text] [PDF]
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R. Telgmann, R. A.D. Bathgate, S. Jaeger, G. Tillmann, and R. Ivell Transcriptional Regulation of the Bovine Oxytocin Receptor Gene Biol Reprod, March 1, 2003; 68(3): 1015 - 1026. [Abstract] [Full Text] [PDF]
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K. D. Carpenter, C. A. Gray, S. Noel, A. Gertler, F. W. Bazer, and T. E. Spencer Prolactin Regulation of Neonatal Ovine Uterine Gland Morphogenesis Endocrinology, January 1, 2003; 144(1): 110 - 120. [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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