Pregnancy and interferon tau regulate RSAD2 and IFIH1 expression in the ovine uterus

in Reproduction

Radical S-adenosyl methionine domain containing 2 (RSAD2) encodes a cytoplasmic antiviral protein induced by interferons (IFN). Interferon-induced with helicase C domain 1 (IFIH1) is a RNA helicase involved in innate immune defense against viruses, growth suppression, and apoptosis. Interferon tau (IFNT), a Type I IFN produced by the peri-implantation ruminant conceptus, acts on the uterine endometrium to signal pregnancy recognition and promote receptivity to implantation. Transcriptional profiling identified RSAD2 and IFIH1 as IFNT regulated genes in the ovine uterine endometrium. This study tested the hypothesis that RSAD2 and IFIH1 were induced in the endometrium in a cell type-specific manner by IFNT from the conceptus during early pregnancy. Endometrial RSAD2 and IFIH1 mRNA increased between days 12 and 16 of pregnancy, but not of the estrous cycle. In pregnant ewes, RSAD2 and IFIH1 mRNAs increased in endometrial glands, and stroma and immune cells, but not in the luminal epithelium. Neither gene was expressed in the trophectoderm of day 18 or 20 conceptuses. Progesterone (P4) treatment of ovariectomized ewes did not induce expression RSAD2 or IFIH1 mRNA in the endometrium; however, intrauterine injections of IFNT induced expression of RSAD2 and IFIH1 mRNA in endometria of ewes treated with P4, as well as in ewes treated with P4 and the progesterone receptor antagonist, ZK 136,317. These results indicate that conceptus IFNT induces both RSAD2 and IFIH1 in a P4-independent manner in the ovine uterine endometrium. These two IFNT-stimulated genes are proposed to have biological roles in the establishment of uterine receptivity to the conceptus during implantation through induction of an antiviral state and modulation of local immune cells in the endometrium.

Abstract

Radical S-adenosyl methionine domain containing 2 (RSAD2) encodes a cytoplasmic antiviral protein induced by interferons (IFN). Interferon-induced with helicase C domain 1 (IFIH1) is a RNA helicase involved in innate immune defense against viruses, growth suppression, and apoptosis. Interferon tau (IFNT), a Type I IFN produced by the peri-implantation ruminant conceptus, acts on the uterine endometrium to signal pregnancy recognition and promote receptivity to implantation. Transcriptional profiling identified RSAD2 and IFIH1 as IFNT regulated genes in the ovine uterine endometrium. This study tested the hypothesis that RSAD2 and IFIH1 were induced in the endometrium in a cell type-specific manner by IFNT from the conceptus during early pregnancy. Endometrial RSAD2 and IFIH1 mRNA increased between days 12 and 16 of pregnancy, but not of the estrous cycle. In pregnant ewes, RSAD2 and IFIH1 mRNAs increased in endometrial glands, and stroma and immune cells, but not in the luminal epithelium. Neither gene was expressed in the trophectoderm of day 18 or 20 conceptuses. Progesterone (P4) treatment of ovariectomized ewes did not induce expression RSAD2 or IFIH1 mRNA in the endometrium; however, intrauterine injections of IFNT induced expression of RSAD2 and IFIH1 mRNA in endometria of ewes treated with P4, as well as in ewes treated with P4 and the progesterone receptor antagonist, ZK 136,317. These results indicate that conceptus IFNT induces both RSAD2 and IFIH1 in a P4-independent manner in the ovine uterine endometrium. These two IFNT-stimulated genes are proposed to have biological roles in the establishment of uterine receptivity to the conceptus during implantation through induction of an antiviral state and modulation of local immune cells in the endometrium.

Introduction

Interferon tau (IFNT), the maternal recognition of pregnancy signal in ruminants (sheep, cattle, goats), is secreted by the elongating peri-implantation conceptus (embryo–fetus and associated membranes) and inhibits development of the endometrial luteolytic mechanism (Spencer & Bazer 2002, 2004). IFNT is produced by sheep conceptuses between days 10 and 21 of gestation with maximal production on days 14 to 16 (Farin et al. 1989, Guillomot et al. 1990). During pregnancy recognition, IFNT acts in a paracrine fashion on endometrial luminal epithelium (LE) and superficial ductal glandular epithelium (sGE) of the ovine uterus to repress transcription of the estrogen receptor alpha gene (Spencer et al. 1996, Fleming et al. 2001), thereby preventing estrogen induction of expression of the oxytocin receptor gene (Fleming et al. 2006) which precludes oxytocin-induced endometrial release of luteolytic pulses of prostaglandin F2 alpha (Spencer & Bazer 2004). The antiluteolytic actions of IFNT allow maintenance of a functional corpus luteum and secretion of progesterone (P4), which is the hormone of pregnancy necessary for successful implantation and development of the conceptus to term (Spencer & Bazer 2004). In addition, to antiluteolytic effects on the endometrium, IFNT induces a number of IFN-stimulated genes (ISGs) in a cell-specific manner within the endometrium, and ISGs are hypothesized to play important roles in uterine receptivity and conceptus implantation during establishment of pregnancy (Hansen et al. 1999a, Spencer & Bazer 2004, Gray et al. 2006, Klein et al. 2006). Several ISGs are first induced by P4 and stimulated by IFNT, whereas other genes are stimulated by IFNT from the conceptus in a P4-independent manner (Gray et al. 2006).

Recent transcriptional profiling experiments identified RSAD2 and IFIH1 as genes induced by IFNT from the conceptus in ovine and bovine endometria during early pregnancy (Gray et al. 2006, Klein et al. 2006). Radical S-adenosyl methionine domain containing 2 (RSAD2; alias viperin) is a cytoplasmic antiviral protein induced by Type I IFNs that can inhibit infection of cells with human cytomegalovirus (Chin & Cresswell 2001). Interferon-induced with helicase C domain 1 (IFIH1; alias MDA5) is a RNA helicase that through its ATP-dependent unwinding of RNA, promotes mRNA degradation by specific RNases and is involved in innate immune defense against viruses as well as cellular growth suppression (Kang et al. 2002, 2004). IFIH1 senses intracellular viral infection and triggers innate antiviral responses including the production of Type I IFNs (Yoneyama et al. 2005). Both RSAD2 and IFIH1 are produced during a viral infection in response to IFNs to limit viral replication and modulate subsequent adaptive immunity (Katze et al. 2002, Helbig et al. 2005). Similar to other Type I IFNs, IFNT elicits antiviral, antiproliferative, and immunomodulatory activities in homologous and heterologous cells (Pontzer et al. 1991, Alexenko et al. 1997, Khan et al. 1998, Johnson et al. 1999a, 1999b, 1999c, 1999d, 1999e). Induction of an antiviral state in the endometrium during early pregnancy may be beneficial by inhibiting sexually transmitted viruses as well as modulating local immune cells to promote tolerance of the allogeneic conceptus and stimulating production of cytokines beneficial for conceptus survival and growth (Hansen 1995, Tekin & Hansen 2002, Croy et al. 2003b).

Although RSAD2 and IFIH1 have been identified as pregnancy- and IFNT-stimulated genes in the ovine uterine endometrium, the temporal and spatial alterations in their expression in the endometrium during early pregnancy and in response to P4 and IFNThave not been investigated. Our working hypothesis that RSAD2 and IFIH1 are induced in the endometrium in a cell-type specific manner by IFNT from the conceptus during early pregnancy and have biological roles in establishing uterine receptivity to implantation by the conceptus. As first step in testing this hypothesis, studies were conducted to determine effects of: (1) stage of the estrous cycle and early pregnancy on RSAD2 and IFIH1 expression in the ovine uterus; (2) P4 and IFNT on RSAD2 and IFIH1 expression in the ovine uterus; and (3) IFNT on RSAD2 and IFIH1 expression in ruminant endometrial cell lines.

Materials and Methods

Animals

Mature crossbred Suffolk sheep (Ovis aries) were observed daily for estrus in the presence of vasectomized rams and used in the experiment after they exhibited at least two estrous cycles of normal duration (16–18 days). At estrus, ewes were assigned randomly to cyclic or pregnant status. All experimental and surgical procedures were in compliance with the Guide for the Care and Use of Agriculture Animals in Teaching and Research, and were approved by the Institutional Animal Care and Use Committee of Texas A&M University.

Experimental design

Study 1

At estrus (day 0), ewes were mated to either an intact or vasectomized ram as described previously (Spencer et al. 1999a) and then hysterectomized (n=5 ewes/day) on day 10, 12, 14, or 16 of the estrous cycle or day 10, 12, 14, 16, 18, or 20 of pregnancy. To confirm the pregnancy status, the uterine lumen was flushed with saline on days 10–16 of pregnancy and examined for the presence of a morphologically normal conceptus(es). At hysterectomy, several sections (~0.5 cm) from the mid-portion of each uterine horn ipsilateral to the corpus luteum were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% ethanol for 24 h and then dehydrated and embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO, USA). Several sections (1–1.5 cm) from the middle of each uterine horn were embedded in Tissue-Tek OCT compound (Miles, Oneonta, NY, USA), 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 subsequent RNA extraction. In monovulatory pregnant ewes, uterine tissue samples were marked as either contralateral or ipsilateral to the ovary bearing the corpus luteum; no tissues from the contralateral uterine horn were used for this study.

Study 2

Sixteen cyclic ewes were ovariectomized and fitted with intrauterine (IU) catheters on day 5 post-estrus as described previously (Gray et al. 2006) and injected daily i.m. with 75 mg P4 between days 5 and 16. Ewes were then assigned randomly (n=5 ewes/treatment) to receive one of the following treatment regimens between days 11 and 16: (1) P4 and daily IU infusions of control serum proteins (P4 + CX); (2) P4 and 75 mg of ZK136,317 (Schering, Berlin, Germany), a progesterone receptor (PGR) antagonist and CX proteins (P4 + ZK + CX); (3) P4 and IU IFNT (2 × 107 antiviral units) (P4 + IFN); or (4) P4 and ZK and IU IFNT (P4 + ZK + IFN). The P4 and ZK were administered daily in corn oil vehicle. Both uterine horns of each ewe received twice daily injections of either CX proteins (50 μg/horn per injection) or recombinant ovine IFNT (5 × 106 antiviral units/horn per injection with CX proteins). Recombinant ovine IFNTwas produced in Pichia pastoris and purified as described previously (Van Heeke et al. 1996). Proteins were prepared for IU injection as described previously (Spencer et al. 1999b). This regimen of P4 and IFNT mimics the effects of P4 and the conceptus on endometrial expression of hormone receptors and IFNT-stimulated genes during early pregnancy in ewes (Spencer et al. 1995, Johnson et al. 2001, Kim et al. 2003). All ewes were hysterectomized on day 17. The uterus was processed for histology and the endometrium obtained for RNA extraction as described in Study 1.

Cell culture

Immortalized ovine uterine endometrial LE cells were cultured as described previously (Johnson et al. 1999c). Bovine endometrial (BEND) cells (Johnson et al. 1999a) were kindly provided by Dr Thomas R Hansen (Colorado State University, Fort Collins, CO, USA). Ovine LE and BEND cells were maintained in 150 mm culture dishes containing DMEM (Dulbecco’s modified essential medium) with F-12 salts (DMEM-F12; Sigma-Aldrich Corp.) supplemented with 5% serum and antibiotics. When cells reached 70–80% confluency, they were treated with either IFNT (2 × 107 antiviral unit (AVU)/ml) or left untreated as a control for 24 h in serum-free medium. The experiment was independently repeated three times in each cell type.

RNA isolation

Total cellular RNA was isolated from frozen endometrium or cultured cells using the Trizol reagent (Gibco-BRL) according to manufacturer’s recommendations. The quantity and quality of total RNA was determined by spectrometry and denaturing agarose gel electrophoresis respectively.

Cloning of partial cDNAs for ovine RSAD2 and IFIH1

Partial cDNAs for ovine RSAD2 and IFIH1 mRNAs were amplified by RT-PCR using total RNA endometrial tissues from day 18 of pregnancy using specific primers based on human RSAD2 mRNA (Genbank NM_080657; forward, 5′-GAG GCC AAG AAA GGT CTG C-3′; reverse, 5′-CCAAGA ACG CTT CAA ACT CC-3′) and human IFIH1 mRNA (Genbank AF095844; forward, 5′-TTC CGC AAA GAG TTC AAA CC-3′; reverse, 5′-AAT GTG TTC TTC GGG TTT GG-3′). The RT of cellular total RNA into cDNA was performed, as described previously (Stewart et al. 2000). The PCR amplification was conducted as follows for RSAD2 and IFIH1: (1) 95 °C for 5 min; (2) 95 °C for 30 s, 56.5 °C for 40 s (for RSAD2), 57 °C for 40 s (for IFIH1), and 72 °C for 1 min for 35 cycles; and (3) 72 °C for 10 min. The partial cDNAs for ovine RSAD2 and IFIH1 PCR products were cloned into pCRII using a T/A Cloning Kit (Invitrogen) and their sequences verified using an ABI PRISM Dye Terminator Cycle Sequencing Kit and ABI PRISM automated DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA).

Slot blot hybridization analyses

Steady-state levels of mRNA in ovine endometrium were assessed by slot blot hybridization as described previously (Spencer et al. 1999c, Choi et al. 2001a). For RSAD2 and IFIH1 antisense cRNA probes, the plasmids were linearized with XbaI and in vitro transcription was conducted with SP6 RNA polymerase. Sense cRNA probes were generated using BamHI and T7 RNA polymerase. Radiolabeled antisense and sense cRNA probes were then generated by in vitro transcription with [α-32P]-UTP. Denatured total endometrial RNA (20 μg) from each ewe was hybridized with radiolabeled cRNA probes. To correct for variation in total RNA loading, a duplicate RNA slot membrane was hybridized with radiolabeled antisense 18S cRNA (pT718S; Ambion, Austin, TX, USA). Following washing, the blots were digested with RNase A and radioactivity associated with slots quantified using a Typhoon 8600 MultiImager (Molecular Dynamics, Piscataway, NJ, USA).

Semiquantitative RT-PCR analysis

RSAD2 and IFIH1 mRNA levels in immortalized ovine endometrial LE and BEND cells were assessed using semi-quantitative RT-PCR as described previously (Stewart et al. 2000). Briefly, isolated total cellular RNA was treated with RQ1 RNase Free-DNase1 (Promega) and then ethanol-precipitated. The cDNA was synthesized from total cellular RNA (5 μg) isolated from both cell-lines using random and oligo (dT) primers and SuperScript II Reverse Transcriptase (Life Technologies). Newly synthesized cDNA was acid-ethanol precipitated, resuspended in 20 μl sterile water, and stored at −20 °C. The cDNAs were diluted (1:10) in sterile water before use in PCR. The primers, PCR amplification and verification of their sequences were conducted as described in the section on cloning partial cDNAs. Housekeeping β-actin (ACTB) primers were forward (5′-ATGAAGATCCTCACGGAACG-3′) and reverse (5′-GAAGGTGGTCTCGTGAATGC-3′), which amplified a 270-bp product. PCR amplification was conducted as follows for ACTB: (1) 95 °C for 5 min; (2) 95 °C for 30 s, 57°C for 30 s, and 72 °C for 1 min for 25 cycles; and (3) 72 °C for 10 min. After PCR, equal amounts of reaction product were analyzed using a 1.5% agarose gel, and PCR products were visualized using ethidium bromide staining. The amount of DNA present was quantified by measuring the intensity of light emitted from correctly sized bands under u.v. light using a ChemiDoc EQ system and Quantity One software (Bio-Rad).

In situ hybridization analyses

Location of mRNA expression in uterine sections (5 μm) was determined by radioactive in situ hybridization analysis as described previously (Spencer et al. 1999c, Choi et al. 2001a). Briefly, deparaffinized, rehydrated and deproteinated uterine tissue sections were hybridized with radiolabeled antisense or sense cRNA probes generated from linearized RSAD2 and IFIH1 partial cDNAs using in vitro transcription with [α-35S]-UTP. After hybridization, washing, and RNase A digestion slides were dipped in NTB-2 liquid photographic emulsion (Kodak), and exposed at 4 °C for 1–2 weeks. Slides were developed in Kodak D-19 developer, counterstained with Gill’s hematoxylin (Fisher Scientific, Fairlawn, NJ, USA), and then dehydrated through a graded series of alcohol to xylene. Coverslips were then affixed with Permount (Fisher). Images of representative fields were recorded under brightfield or darkfield illumination using a Nikon Eclipse 1000 photomicroscope (Nikon Instruments Inc., Lewisville, TX, USA) fitted with a Nikon DXM1200 digital camera.

Statistical analyses

All quantitative data were subjected to least-squares analyses of variance (ANOVA) using the Statistical Analysis System (SAS Institute, Cary, NC, USA). Slot blot hybridization data were corrected for differences in sample loading using the 18S rRNA data as a covariate. Data from Study 1 were analyzed for effects of day, pregnancy status (cyclic or pregnant), and their interaction. Data from Study 2 were analyzed using orthogonal contrasts (P4 + CX vs P4 + IFN; P4 + ZK + CX vs P4 + ZK + IFN; and P4 + CX vs P4 + ZK + CX) to elucidate effects of treatment. Semi-quantitative RT-PCR data was analyzed using the ACTB data as a covariate. All tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. A P-value of 0.05 or less was considered significant. Data are presented as least-square means (LSM) with standard errors (s.e.).

Results

RSAD2 and IFIH1 expression increases in the endometrium by a cell type-specific manner

Expression levels of RSAD2 and IFIH1 mRNAs in the endometrium of cyclic ewes were low and not affected (P>0.10) by day (Fig. 1). In contrast, RSAD2 mRNA increased (P<0.01, quadratic) about sixfold between days 12 and 16 and was maintained through day 20 in pregnant ewes. Similarly, IFIH1 mRNA increased (P<0.01, quadratic) about 2.5-fold between days 12 and 16. The presence of a conceptus increased endometrial RSAD2 and IFIH1 mRNA between days 10 and 16 (P<0.01, day x status; Fig. 1).

In situ hybridization analyses determined the location of RSAD2 (Fig. 2) and IFIH1 (Fig. 3) mRNAs in uteri of cyclic and pregnant ewes. RSAD2 mRNA was low and not different between uteri from day 10 cyclic and pregnant ewes (Fig. 2). Between days 10 and 12 of pregnancy, RSAD2 mRNA increased in the middle glands and to a lower extent in the stratum compactum stroma. Between days 14 and 20 of pregnancy, RSAD2 mRNA was present predominantly in the endometrial glands, and stroma and immune cells, but not in LE, sGE, myometrium, or conceptus trophectoderm. Interestingly, RSAD2 mRNA declined in the endometrial glands after day 16 of pregnancy. Similar to RSAD2, IFIH1 mRNA was low and not different between uteri from day 10 cyclic and pregnant ewes (Fig. 3). Between days 10 and 12 of pregnancy, IFIH1 mRNA increased slightly in the middle glands and stratum compactum stroma. Between days 14 and 18 of pregnancy, IFIH1 mRNA was present predominantly in the stratum compactum stroma of the endometrium, middle glands, and immune cells, and not observed in the endometrial LE, sGE, myometrium, or conceptus trophectoderm. The melanocytes underneath the LE do not have IFIH1 mRNA, but appear white in darkfield photomicrographs. Between days 18 and 20, IFIH1 mRNA abundance declined in the endometrial stroma. The presence of RSAD2 and IFIH1 mRNAs in immune cells within the endometrium was based on visual observations of cell morphology.

Intrauterine administration of recombinant ovine IFNT induces RSAD2 and IFIH1 mRNA in the ovine endometrium

In order to determine if differences in expression of the selected genes in endometrium of pregnant compared with cyclic ewes was due to IFNT from the conceptus, cyclic ewes were ovariectomized and fitted with IU catheters on day 5 and hysterectomized on day 17 (see Fig. 4A). Treatment of ewes with the ZK 136,317 PGR antagonist did not affect (P>0.10, P4 + CX vs P4 + ZK + CX) endometrial RSAD2 or IFIH1 mRNA abundance (Fig. 4B). For ewes receiving P4 alone, IU recombinant ovine IFNT increased (P<0.001) steady-state levels of RSAD2 and IFIH1 mRNAs 10-fold and 8.3-fold respectively, in the endometria (P<0.001, P4 + CX vs P4 + IFN; Figs 4B and 5A). Similarly, for ewes receiving P4 + ZK treatment, IU recombinant ovine IFNT increased RSAD2 and IFIH1 mRNAs in the endometrium about 9.3-fold and 5.6-fold respectively (P<0.001, P4 + ZK + CX vs P4 + ZK + IFN).

In situ hybridization analyses verified that IFNT increased RSAD2 and IFIH1 mRNA abundance in the endometrium (Figs 4C and 5B). Similar to day 16–18 pregnant ewes, RSAD2 and IFIH1 mRNA were increased by IFNT primarily in the endometrial stroma and immune cells and, to a lower extent, in the endometrial glands of uteri from ewes receiving P4 + IFNT treatment. In P4 + ZK ewes, IFNT increased RSAD2 and IFIH1 mRNA in the endometrial stroma and immune cells. Further, IFNT increased IFIH1 mRNA in endometrial LE of ewes receiving P4 + ZK treatment (Fig. 5B).

Effects of IFNTon RSAD2 and IFIH1 in endometrial cells

In untreated ovine endometrial LE (oLE) and BEND cells maintained in serum-free medium, IFIH1 but not RSAD2 mRNA was detected (Fig. 6). Treatment of both oLE and BEND cells with recombinant ovine IFNT increased (P<0.0001) RSAD2 and IFIH1 mRNA levels.

Discussion

During the peri-implantation period of pregnancy, gene expression in endometria of ruminants is programmed primarily by P4 from the ovarian corpus luteum and IFNT from the conceptus (Spencer & Bazer 2002, Spencer et al. 2004c). In the present study, we identified two antiviral-related genes, RSAD2 and IFIH1, as being induced in the ovine endometrium in response to IFNT from the conceptus in a P4-independent manner. These genes were selected for analysis based on transcriptional profiling studies of ruminant endometria (Gray et al. 2006, Klein et al. 2006) as well as knowledge that both RSAD2 and IFIH1 are produced during a viral infection in response to IFNs to limit viral replication and modulate subsequent adaptive immunity (Katze et al. 2002, Helbig et al. 2005). In the present study, the ontogeny of RSAD2 and IFIH1 in the ovine endometrium correlates directly with increasing amounts of IFNT produced by the rapidly elongating conceptus, which is maximum between days 14 and 16 and declines thereafter, as the trophectoderm begins implantation and trophoblast giant binucleate cells begin to differentiate (Guillomot et al. 1990). Clearly, P4 and IFNT have complex, independent, and complementary effects on expression of a number of genes in the ovine endometrium during early pregnancy (see Gray et al. 2006, Klein et al. 2006). In the present study, P4 was found to be not required for IFNT induction of RSAD2 and IFIH1 in the endometrium, which is similar to findings for other IFNT-stimulated genes in the ovine endometrium including CXCL10 (chemokine (C-X-C motif) ligand 10), IFITM3 (interferon induced trans-membrane protein 3 (1-8U)), B2M (beta-2-microglobin), MIC (MHC class I polypeptide-related alpha chain), and STAT1 (Gray et al. 2006). Further, treatment of ovine endometrial LE and BEND cells with recombinant ovine IFNT induced RSAD2 and IFIH1 expression without a requirement for serum or P4 in the medium. In contrast, IFNT induction of several non-classical IFN-stimulated genes, such as LGALS15 (galectin 15), CTSL (cathespin L), and CST3 (cystatin C), in endometrial LE and sGE is dependent on P4 (Gray et al. 2004, Song et al. 2005, 2006), which is hypothesized to involve P4 down-regulation of the PGR in those epithelia (Spencer et al. 2004c, Gray et al. 2006).

In the ovine uterus, induction of RSAD2 and IFIH1 mRNA by the presence of the conceptus during pregnancy and by IFNT was limited to endometrial stroma and middle to deep glands, as well as resident immune cells based on visual observations of cell morphology. The majority of ISGs induced by IFNT without a requirement for P4 in the ovine uterus are restricted to endometrial stroma and middle to deep glands as well as immune cells (see (Spencer et al. 2004a, 2004c) for review). A variety of ruminant and human cell lines have been used to determine that IFNT activates the classical JAK-STAT-IFN regulatory factor (IRF) signaling pathway utilized by other Type I IFNs that involves ISGF3 (STAT1, STAT2, ISGF3G complex), GAF (gamma activated unit) (STAT1 dimer), and IRF one (IRF1; see Stark et al. 1998, Spencer et al. 2004c). ISGF3 transactivates genes through binding an IFN-stimulated response element (ISRE), whereas GAF binds to a gamma activation sequence element in genes such as IRF1. Further, IRF1 transactivates genes through an IRF element (IRFE). Similar to findings for RSAD2 and IFIH1 in the present study, results of in vivo studies indicate that many classical IFN-stimulated genes (STAT1, STAT2, IRF1, ISGF3G, GBP2, IFI6, IFI56, ISG15, MIC, B2M, OAS) are not induced or increased by IFNT in LE and sGE of the sheep uterus (Johnson et al. 1999d, 2001, Choi et al. 2001b, 2003, Kim et al. 2003). This finding was initially surprising because all ovine endometrial cell types express IFNAR1 and IFNAR2 subunits of the common Type I IFN receptor (Rosenfeld et al. 2002). However, available results also indicate that IRF2, a potent transcriptional repressor of IFN-stimulated genes (Mamane et al. 1999), is expressed specifically in endometrial LE and sGE and represses transcriptional activity of promoters containing ISRE or IRFE (Choi et al. 2001b). Thus, IRF2 in LE and sGE is proposed to restrict IFNT induction of many IFN-stimulated genes to endometrial stroma and glandular epithelium. In fact, all components of ISGF3 (STAT1, STAT2, ISGF3G) and other studied IFN-stimulated genes (B2M, GBP2, G1P2, ISG15, G1P3, IFI56, MIC, OAS) contain ISREs in their promoters. Further, the promoter regions of the human and fish RSAD2 genes contain multiple IRFEs (Chin & Cresswell 2001, Sun & Nie 2004). Similarly, the promoter region of the human IFIH1 gene has predicted ISRE and IRFE (unpublished results). Thus, the constitutive presence and pregnancy-specific increase in IRF2 in ovine endometrial LE/sGE in vivo is proposed to prevent IFNT induction of RSAD2 and IFIH1 in those epithelia. P4 appears to be involved in this cell-type specification of IFNT actions, because IFNT induced IFIH1 mRNA in the LE of the endometrium in P + ZK-treated ewes in the present study. Immortalized ovine endometrial LE and BEND cells lack or have very low levels of IRF2 mRNA (Song & Spencer, unpublished results); thus, they are fully responsive to IFNT in vitro (Johnson et al. 1999b, Perry et al. 1999, Stewart et al. 2001). In human 2fTGH cells, Type I IFNB (interferon beta) can induce IFIH1 expression, but this is not the case for STAT1 null U3A cells derived from 2fTGH cells (Kang et al. 2004). Thus, the classical JAK-STAT-IRF signaling pathway active in endometrial stroma and glands, and perhaps resident immune cells, is likely responsible for IFNT induction of IFIH1 via activation and formation of the ISGF3 complex (Kang et al. 2004). One interesting finding of the present studies was the loss of RSAD2 mRNA in the middle to deep endometrial glands between days 16 and 18 of pregnancy. This loss correlates with a reduction in IFNT production by the conceptus as well as a decline in IRF1 abundance in those glands (Choi et al. 2001b). Available evidence supports the concept that distinct cell-type specific differences exist in the ruminant endometrium with respect to responses to IFNT from the conceptus between the endometrial glands, stroma and resident immune cells.

The IFNT-stimulated genes in endometria of ruminants are hypothesized to be important for conceptus implantation (Hansen et al. 1999b, Spencer et al. 2004b, Klein et al. 2006). RSAD2 contains a radical S-adenosylmethionine (SAM) domain that catalyzes diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation, and protein radical formation. Radical SAM proteins function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis, and biodegradation pathways (Sofia et al. 2001) which could be important in endometrial cells during the peri-implantation period to support conceptus development and implantation. IFIH1 (alias melanoma differentiation associated gene 5) is a RNA helicase induced during differentiation, cancer reversion, and programmed cell death (Kang et al. 2002, 2004). IFIH1 acts to sense intracellular viral infection and mediate a signal for innate antiviral responses including production of Type I IFNs (Kang et al. 2002, Yoneyama et al. 2005). Other Type I IFNs (IFNA and IFNB) are not induced in the endometrium in response to IFNT (Spencer & Bazer, unpublished results).

One biological role of RSAD2 and IFIH1 could be to prevent viral infection of the uterus during the critical peri-implantation period of pregnancy, particularly when the conceptus does not have a developed immune system or antiviral defenses. RSAD2 and IFIH1 are implicated in establishing an antiviral state by modulation of innate immune responses. For example, stable expression of RSAD2 in fibroblasts inhibits human cytomegalovirus infection (Chin & Cresswell 2001). Given that IFIH1 also has growth suppressive properties, IFNT induction may suppress the activation of cells within the endometrium, which could be beneficial for pregnancy. In other species such as rodents and humans, resident and recruited immune cells within the endometrium play important roles in placentation and the success of pregnancy (Croy et al. 2003a, 2003b). Unfortunately, knowledge of which immune cells are present in the ovine uterus during pregnancy and their biological functions is sparse. During the estrous cycle, the density of macrophages and T lymphocytes in the ovine and bovine uteri do not change (Hansen 1998). However, during early pregnancy, the number of CD45R+ lymphocytes increases in both endometrium (Segerson et al. 1991) and uterine and jugular venous blood (Lee et al. 1988, Alders & Shelton 1990). It has been postulated that these are NK (natural killer) cells that produce factors to enhance establishment of pregnancy. In the present study, the number of IFIH1-and, in particular, RSAD2-positive immune cells markedly increased in the endometria during pregnancy and in response to IFNT, but it is not clear whether these cells were recruited in response to IFNT or already present and stimulated by IFNT. The IFNT stimulated resident immune cells in the endometrium may migrate from the uterus, because IFNT-stimulated genes are higher in the peripheral blood leukocytes isolated from pregnant as compared with non-pregnant ewes and cows (Yankey et al. 2001). Eosinophils are also present in the endometrium of early pregnant ewes, and their numbers increase between days 11 and 19 of early pregnancy, perhaps due to actions of both P4 and perhaps IFNT (Asselin et al. 2001). In fact, IFNT possesses immunoregulatory activity and can inhibit mitogen-induced lymphocyte proliferation (Newton et al. 1989, Tekin et al. 2000) as well as modulate activity of NK cells (Tuo et al. 1993, Tekin & Hansen 2002). These effects of IFNT may prevent immune cell-mediated destruction of the conceptus (Hansen 1995). Finally, some IFNT-stimulated genes, such as CXCL10, from immune cells may have direct effects on conceptus implantation (Nagaoka et al. 2003, Imakawa et al. 2006). Collectively, available evidence supports the hypothesis that RSAD2 and IFIH1 modulate uterine receptivity to conceptus implantation by induction of an antiviral state and modulation of immune cell functions.

Figure 1
Figure 1

Steady-state levels of RSAD2 and IFIH1 mRNAs in endometria from cyclic and early pregnant ewes as determined by slot blot hybridization analysis. In cyclic ewes, RSAD2 mRNA level was low between days 10 and 16. In contrast, RSAD2 mRNA increased (P<0.01) sixfold between days 12 and 16 and was maintained to day 20. Similarly, IFIH1 mRNAwas very low in the endometria of cyclic ewes and increased (P<0.01) about 2.5-fold between days 12 and 16 of pregnancy. Data are expressed as LSM relative units (RU) with standard error (s.e.).

Citation: Reproduction 133, 1; 10.1530/REP-06-0092

Figure 2
Figure 2

In situ hybridization analyses of RSAD2 mRNA in uteri of cyclic and pregnant ewes. Cross-sections of the uterine wall from cyclic (C) and pregnant (P) ewes were hybridized with radiolabeled antisense or sense ovine RSAD2 cRNA probes. RSAD2 mRNA is expressed detected in endometrial stroma, glands, and resident immune cells. Legend: LE, luminal epithelium; GE, glandular epithelium; M, myometrium; S, stroma; Tr, trophectoderm. Scale bar represents 10 μm.

Citation: Reproduction 133, 1; 10.1530/REP-06-0092

Figure 3
Figure 3

In situ hybridization analyses of IFIH1 mRNAs in uteri of cyclic and pregnant ewes. Cross-sections of the uterine wall from cyclic (C) and pregnant (P) ewes were hybridized with radiolabeled antisense or sense ovine IFIH1 cRNA probes. IFIH1 mRNA was detected only in endometrial stroma and glands. Legend: LE, luminal epithelium; GE, glandular epithelium; S, stroma; Tr, trophectoderm. Scale bar represents 10 μm.

Citation: Reproduction 133, 1; 10.1530/REP-06-0092

Figure 4
Figure 4

Effects of progesterone and IFNT on RSAD2 mRNA in the ovine uterus. (A) Experimental design. See Materials and Methods for complete description. Legend: CX, control serum proteins; Hystx, hysterectomy; Ovx/Cath, ovariectomy and uterine catheterization; P4, progesterone; IFNT, recombinant ovine interferon tau; ZK, ZK137,316 anti-progestin. (B) Steady-state levels of RSAD2 mRNA in endometria as determined by slot blot hybridization analysis. Intrauterine infusion of IFNT increased RSAD2 mRNA by 10-fold in the endometrium (P4 + CX vs P4 + IFN, P<0.001), but not in ewes receiving the ZK anti-progestin (P4 + IFN vs P4 + ZK + IFN, P>0.10). Similarly, IFNT increased RSAD2 mRNA 9.3-fold in ewes receiving ZK anti-progestin (P4 + ZK + CX vs P4 + ZK + IFN, P<0.001), but not in ewes receiving the ZK anti-progestin (P4 + IFN vs P4 + ZK + IFN, P>0.10). The asterisk (*) denotes an effect of treatment. (C) In situ hybridization analysis of RSAD2 mRNA expression. In situ hybridization analyses verified that roIFNT increased RSAD2 mRNA expression in a cell-type specific manner in P4-treated ewes consistent with increased expression in uteri from day 16 and 18 pregnant ewes. Intrauterine injections of IFNT increased RSAD2 and IFIH1 mRNAs in endometrial stroma and glands, but not LE, blood vessels or myometrium. Further, IFNT increased RSAD2 mRNA in endometria of P4 + ZK-treated ewes. Scale bar represents 10 μm.

Citation: Reproduction 133, 1; 10.1530/REP-06-0092

Figure 5
Figure 5

Effects of progesterone and IFNT on IFIH1 mRNA in the ovine uterus. (A) Steady-state levels of IFIH1 mRNA in endometria were determined by slot blot hybridization analysis. Intrauterine infusion of IFNT increased IFIH1 mRNA about 8.3-fold in endometria (P4 + CX vs P4 + IFN, P<0.001), but not in ewes receiving the ZK anti-progestin (P4 + IFN vs P4 + ZK + IFN, P>0.10). The asterisk (*) denotes an effect of treatment. (B) In situ hybridization analysis of IFIH1 mRNA expression. Intrauterine injections of IFNT increased IFIH1 mRNA expression in a cell-type specific manner in P4-treated ewes consistent with the increased in expression in uteri from day 16 and day 18 pregnant ewes. Infusion of roIFNT increased expression of IFIH1 mRNA in endometrial stroma and GE, but not LE, blood vessels or myometrium. Further, IFNT increased IFIH1 mRNA in endometria from ewes treated with P4 + ZK. Cross-sections of the uterine wall from treated-ewes were hybridized with radiolabeled antisense or sense ovine IFIH1 cRNA probes. Scale bar represents 10 μm.

Citation: Reproduction 133, 1; 10.1530/REP-06-0092

Figure 6
Figure 6

Semi-quantitative RT-PCR analyses of RSAD2 and IFIH1 mRNAs in total cellular RNA isolated from immortalized ovine endometrial LE (oLE) and bovine endometrial (BEND) cells. All PCR products were separated in a 1.5% agarose gel and stainedwith ethidium bromide. Positions of the 100-bp (bp) DNA marker (M) ladder are shown. Total cellular RNA from endometria of a day 18 pregnant ewes and sterile water (no template) were positive and negative controls respectively. A graph illustrating the effect of IFNT on relative mRNA levels for RSAD2 and IFIH1 is presented below each gel, and the asterisk (*) denotes a significant (P<0.001) effect of IFNT treatment.

Citation: Reproduction 133, 1; 10.1530/REP-06-0092

Received 1 July 2006
 Revised manuscript received 12 September 2006
 Accepted 16 October 2006

The authors thank all members of the Bazer/Spencer Laboratory for assistance and management of animals, as well as Dr Thomas R Hansen (Colorado State University) for the BEND cells. This research was funded by National Institutes of Health Grants 5 R01 HD32534 and 5 P30 ES09106.

References

  • Alders RG & Shelton JN1990 Lymphocyte subpopulations in lymph and blood draining from the uterus and ovary in sheep. Journal of Reproductive Immunology1727–40.

    • Search Google Scholar
    • Export Citation
  • Alexenko AP Leaman DW Li J & Roberts RM1997 The antiproliferative and antiviral activities of IFN-tau variants in human cells. Journal of Interferon and Cytokine Research17769–779.

    • Search Google Scholar
    • Export Citation
  • Asselin E Johnson GA Spencer TE & Bazer FW2001 Monocyte chemotactic protein-1 and -2 messenger ribonucleic acids in the ovine uterus: regulation by pregnancy progesterone and interferon-tau. Biology of Reproduction64992–1000.

    • Search Google Scholar
    • Export Citation
  • Chin KC & Cresswell P2001 Viperin (cig5) an IFN-inducible antiviral protein directly induced by human cytomegalovirus. PNAS9815125–15130.

    • Search Google Scholar
    • Export Citation
  • Choi Y Johnson GA Burghardt RC Berghman LR Joyce MM Taylor KM Stewart MD Bazer FW & Spencer TE2001a Interferon regulatory factor-two restricts expression of interferon-stimulated genes to the endometrial stroma and glandular epithelium of the ovine uterus. Biology of Reproduction651038–1049.

    • Search Google Scholar
    • Export Citation
  • Choi Y Johnson GA Burghardt RC Berghman LR Joyce MM Taylor KM Stewart MD Bazer FW & Spencer TE2001b Interferon regulatory factor-two restricts expression of interferon-stimulated genes to the endometrial stroma and glandular epithelium of the ovine uterus. Biology of Reproduction651038–1049.

    • Search Google Scholar
    • Export Citation
  • Choi Y Johnson GA Spencer TE & Bazer FW2003 Pregnancy and interferon tau regulate MHC class I and beta-2-microglobulin expression in the ovine uterus. Biology of Reproduction681703–1710.

    • Search Google Scholar
    • Export Citation
  • Croy B He H Esadeg S Wei Q McCartney D Zhang J Borzychowski A Ashkar A Black G Evans Set al.2003a Uterine natural killer cells: insights into their cellular and molecular biology from mouse modelling. Reproduction126149–160.

    • Search Google Scholar
    • Export Citation
  • Croy BA Esadeg S Chantakru S van den Heuvel M Paffaro VA He H Black GP Ashkar AA Kiso Y & Zhang J2003b Update on pathways regulating the activation of uterine NK cells their interactions with decidual spiral arteries and homing of their precursors to the uterus. Journal of Reproductive Immunology59175–191.

    • Search Google Scholar
    • Export Citation
  • Farin CE Imakawa K & Roberts RM1989In situ localization of mRNA for the interferon ovine trophoblast protein-1 during early embryonic development of the sheep. Molecular Endocrinology31099–1107.

    • Search Google Scholar
    • Export Citation
  • Fleming JA Choi Y Johnson GA Spencer TE & Bazer FW2001 Cloning of the ovine estrogen receptor-alpha promoter and functional regulation by ovine interferon-tau. Endocrinology1422879–2887.

    • Search Google Scholar
    • Export Citation
  • Fleming JG Spencer TE Safe SH & Bazer FW2006 Estrogen regulates transcription of the ovine oxytocin receptor gene through GC-rich SP1 promoter elements. Endocrinology147899–911.

    • Search Google Scholar
    • Export Citation
  • Gray CA Adelson DL Bazer FW Burghardt RC Meeusen EN & Spencer TE2004 Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in the trophectoderm. PNAS1017982–7987.

    • Search Google Scholar
    • Export Citation
  • Gray CA Abbey CA Beremand PD Choi Y Farmer JL Adelson DL Thomas TL Bazer FW & Spencer TE2006 Identification of endometrial genes regulated by early pregnancy progesterone and interferon tau in the ovine uterus. Biology of Reproduction74383–394.

    • Search Google Scholar
    • Export Citation
  • Guillomot M Michel C Gaye P Charlier N Trojan J & Martal J1990 Cellular localization of an embryonic interferon ovine trophoblastin and its mRNA in sheep embryos during early pregnancy. Biology of the Cell68205–211.

    • Search Google Scholar
    • Export Citation
  • Hansen PJ1995 Interactions between the immune system and the ruminant conceptus. Journal of Reproduciton Fertility. Supplement4969–82.

    • Search Google Scholar
    • Export Citation
  • Hansen PJ1998 Regulation of uterine immune function by progesterone–lessons from the sheep. Journal of Reproductive Immunology4063–79.

    • Search Google Scholar
    • Export Citation
  • Hansen TR Austin KJ Perry DJ Pru JK Teixeira MG & Johnson GA1999a Mechanism of action of interferon-tau in the uterus during early pregnancy. Journal of Reproduciton Fertility. Supplement54329–339.

    • Search Google Scholar
    • Export Citation
  • Hansen TR Austin KJ Perry DJ Pru JK Teixeira MG & Johnson GA1999b Mechanism of action of interferon-tau in the uterus during early pregnancy. Journal of Reproduciton Fertility54329–339.

    • Search Google Scholar
    • Export Citation
  • Helbig KJ Lau DT Semendric L Harley HA & Beard MR2005 Analysis of ISG expression in chronic hepatitis C identifies viperin as a potential antiviral effector. Hepatology42702–710.

    • Search Google Scholar
    • Export Citation
  • Imakawa K Imai M Sakai A Suzuki M Nagaoka K Sakai S Lee SR Chang KT Echternkamp SE & Christenson RK2006 Regulation of conceptus adhesion by endometrial CXC chemokines during the implantation period in sheep. Molecular Reproduction and Development73850–858.

    • Search Google Scholar
    • Export Citation
  • Johnson GA Austin KJ Collins AM Murdoch WJ & Hansen TR1999a Endometrial ISG17 mRNA and a related mRNA are induced by interferon-tau and localized to glandular epithelial and stromal cells from pregnant cows. Endocrine10243–252.

    • Search Google Scholar
    • Export Citation
  • Johnson GA Burghardt RC Newton GR Bazer FW & Spencer TE1999b Development and characterization of immortalized ovine endometrial cell lines. Biology of Reproduction611324–1330.

    • Search Google Scholar
    • Export Citation
  • Johnson GA Burghardt RC Newton GR Bazer FW & Spencer TE1999c Development and characterization of immortalized ovine endometrial cell lines. Biology of Reproduction611324–1330.

    • Search Google Scholar
    • Export Citation
  • Johnson GA Spencer TE Hansen TR Austin KJ Burghardt RC & Bazer FW1999d Expression of the interferon tau inducible ubiquitin cross-reactive protein in the ovine uterus. Biology of Reproduction61312–318.

    • Search Google Scholar
    • Export Citation
  • Johnson JA Hochkeppel HK & Gangemi JD1999e IFN-tau exhibits potent suppression of human papillomavirus E6/E7 oncoprotein expression. Journal of Interferon and Cytokine Research191107–1116.

    • Search Google Scholar
    • Export Citation
  • Johnson GA Stewart MD Gray CA Choi Y Burghardt RC Yu-Lee LY Bazer FW & Spencer TE2001 Effects of the estrous cycle pregnancy and interferon tau on 2′ 5′- oligoadenylate synthetase expression in the ovine uterus. Biology of Reproduction641392–1399.

    • Search Google Scholar
    • Export Citation
  • Kang DC Gopalkrishnan RV Wu Q Jankowsky E Pyle AM & Fisher PB2002 mda-5: an interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties. PNAS99637–642.

    • Search Google Scholar
    • Export Citation
  • Kang DC Gopalkrishnan RV Lin L Randolph A Valerie K Pestka S & Fisher PB2004 Expression analysis and genomic characterization of human melanoma differentiation associated gene-5 mda-5: a novel type I interferon-responsive apoptosis-inducing gene. Oncogene231789–1800.

    • Search Google Scholar
    • Export Citation
  • Katze MG He Y & Gale M Jr2002 Viruses and interferon: a fight for supremacy. Nature Reviews. Immunology2675–687.

  • Khan OA Jiang H Subramaniam PS Johnson HM & Dhib-Jalbut SS1998 Immunomodulating functions of recombinant ovine interferon tau: potential for therapy in multiple sclerosis and autoimmune disorders. Multiple Sclerosis463–69.

    • Search Google Scholar
    • Export Citation
  • Kim S Choi Y Bazer FW & Spencer TE2003 Identification of genes in the ovine endometrium regulated by interferon tau independent of signal transducer and activator of transcription 1. Endocrinology1445203–5214.

    • Search Google Scholar
    • Export Citation
  • Klein C Bauersachs S Ulbrich SE Einspanier R Meyer HHD Schmidt SEM Reichenbach H-D Vermehren M Sinowatz F Blum Het al.2006 Monozygotic twin model reveals novel embryo-induced transcriptome changes of bovine endometrium in the pre-attachment period. Biology of Reproduction74253–264.

    • Search Google Scholar
    • Export Citation
  • Lee CS Gogolin-Ewens K & Brandon MR1988 Identification of a unique lymphocyte subpopulation in the sheep uterus. Immunology63157–164.

    • Search Google Scholar
    • Export Citation
  • Mamane Y Heylbroeck C Genin P Algarte M Servant MJ LePage C DeLuca C Kwon H Lin R & Hiscott J1999 Interferon regulatory factors: the next generation. Gene2371–14.

    • Search Google Scholar
    • Export Citation
  • Nagaoka K Sakai A Nojima H Suda Y Yokomizo Y Imakawa K Sakai S & Christenson RK2003 A chemokine interferon (IFN)-gamma-inducible protein 10 kDa is stimulated by IFN-tau and recruits immune cells in the ovine endometrium. Biology of Reproduction681413–1421.

    • Search Google Scholar
    • Export Citation
  • Newton GR Vallet JL Hansen PJ & Bazer FW1989 Inhibition of lymphocyte proliferation by ovine trophoblast protein-1 and a high molecular weight glycoprotein produced by the peri-implantation sheep conceptus. American Journal of Reproductive Immunology1999–107.

    • Search Google Scholar
    • Export Citation
  • Perry DJ Austin KJ & Hansen TR1999 Cloning of interferon-stimulated gene 17: the promoter and nuclear proteins that regulate transcription. Molecular Endocrinology131197–1206.

    • Search Google Scholar
    • Export Citation
  • Pontzer CH Bazer FW & Johnson HM1991 Antiproliferative activity of a pregnancy recognition hormone ovine trophoblast protein-1. Cancer Research515304–5307.

    • Search Google Scholar
    • Export Citation
  • Rosenfeld CS Han CS Alexenko AP Spencer TE & Roberts RM2002 Expression of interferon receptor subunits IFNAR1 and IFNAR2 in the ovine uterus. Biology of Reproduction67847–853.

    • Search Google Scholar
    • Export Citation
  • Segerson EC Matterson PM & Gunsett FC1991 Endometrial T-lymphocyte subset infiltration during the ovine estrous cycle and early pregnancy. Journal of Reproductive Immunology20221–236.

    • Search Google Scholar
    • Export Citation
  • Sofia HJ Chen G Hetzler BG Reyes-Spindola JF & Miller NE2001 Radical SAM a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Research291097–1106.

    • Search Google Scholar
    • Export Citation
  • Song G Spencer TE & Bazer FW2005 Cathepsins in the ovine uterus: regulation by pregnancy progesterone and interferon tau. Endocrinology1464825–4833.

    • Search Google Scholar
    • Export Citation
  • Song G Spencer TE & Bazer FW2006 Progesterone and interferon tau regulate cystatin C (CST3) in the endometrium. Endocrinology1473478–3483.

    • Search Google Scholar
    • Export Citation
  • Spencer TE & Bazer FW2002 Biology of progesterone action during pregnancy recognition and maintenance of pregnancy. Frontiers in Bioscience7d1879–d1898.

    • Search Google Scholar
    • Export Citation
  • Spencer TE & Bazer FW2004 Conceptus signals for establishment and maintenance of pregnancy. Reproductive Biology and Endocrinology249.

  • Spencer TE Becker WC George P Mirando MA Ogle TF & Bazer FW1995 Ovine interferon-tau regulates expression of endometrial receptors for estrogen and oxytocin but not progesterone. Biology of Reproduction53732–745.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Ott TL & Bazer FW1996 tau-Interferon: pregnancy recognition signal in ruminants. Proceedings of the Society for Experimental Biology and Medicine213215–229.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Bartol FF Bazer FW Johnson GA & Joyce MM1999a Identification and characterization of glycosylation-dependent cell adhesion molecule 1-like protein expression in the ovine uterus. Biology of Reproduction60241–250.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Gray A Johnson GA Taylor KM Gertler A Gootwine E Ott TL & Bazer FW1999b Effects of recombinant ovine interferon tau placental lactogen and growth hormone on the ovine uterus. Biology of Reproduction611409–1418.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Stagg AG Ott TL Johnson GA Ramsey WS & Bazer FW1999c Differential effects of intrauterine and subcutaneous administration of recombinant ovine interferon tau on the endometrium of cyclic ewes. Biology of Reproduction61464–470.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Burghardt RC Johnson GA & Bazer FW2004a Conceptus signals for establishment and maintenance of pregnancy. Animal Reproduction Science82–83537–550.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Johnson GA Bazer FW & Burghardt RC2004b Implantation mechanisms: insights from the sheep. Reproduction128657–668.

  • Spencer TE Johnson GA Burghardt RC & Bazer FW2004c Progesterone and placental hormone actions on the uterus: insights from domestic animals. Biology of Reproduction712–10.

    • Search Google Scholar
    • Export Citation
  • Stark GR Kerr IM Williams BR Silverman RH & Schreiber RD1998 How cells respond to interferons. Annual Review of Biochemistry67227–264.

    • Search Google Scholar
    • Export Citation
  • Stewart MD Johnson GA Gray CA Burghardt RC Schuler LA Joyce MM Bazer FW & Spencer TE2000 Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy. Biology of Reproduction621779–1789.

    • Search Google Scholar
    • Export Citation
  • Stewart DM Johnson GA Vyhlidal CA Burghardt RC Safe SH YuLee LY Bazer FW & Spencer TE2001 Interferon-tau activates multiple signal transducer and activator of transcription proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells. Endocrinology14298–107.

    • Search Google Scholar
    • Export Citation
  • Sun BJ & Nie P2004 Molecular cloning of the viperin gene and its promoter region from the mandarin fish Siniperca chuatsi. Veterinary Immunology and Immunopathology101161–170.

    • Search Google Scholar
    • Export Citation
  • Tekin S & Hansen PJ2002 Natural killer-like cells in the sheep: functional characterization and regulation by pregnancy-associated proteins. Experimental Biology and Medicine (Maywood)227803–811.

    • Search Google Scholar
    • Export Citation
  • Tekin S Ealy AD Wang SZ & Hansen PJ2000 Differences in lymphocyte-regulatory activity among variants of ovine IFN-tau. Journal of Interferon & Cytokine Research201001–1005.

    • Search Google Scholar
    • Export Citation
  • Tuo W Ott TL & Bazer FW1993 Natural killer cell activity of lymphocytes exposed to ovine type I trophoblast interferon. American Journal of Reproductive Immunology2926–34.

    • Search Google Scholar
    • Export Citation
  • Van Heeke G Ott TL Strauss A Ammaturo D & Bazer FW1996 High yield expression and secretion of the ovine pregnancy recognition hormone interferon-tau by Pichia pastoris. Journal of Interferon & Cytokine Research16119–126.

    • Search Google Scholar
    • Export Citation
  • Yankey SJ Hicks BA Carnahan KG Assiri AM Sinor SJ Kodali K Stellflug JN & Ott TL2001 Expression of the antiviral protein Mx in peripheral blood mononuclear cells of pregnant and bred non-pregnant ewes. Journal of Endocrinology170R7–11.

    • Search Google Scholar
    • Export Citation
  • Yoneyama M Kikuchi M Matsumoto K Imaizumi T Miyagishi M Taira K Foy E Loo YM Gale M Jr Akira Set al.2005 Shared and unique functions of the DExD/H-box helicases RIG-I MDA5 and LGP2 in antiviral innate immunity. Journal of Immunology1752851–2858.

    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

 

     An official journal of

    Society for Reproduction and Fertility

 

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 485 159 6
PDF Downloads 243 80 5
  • View in gallery

    Steady-state levels of RSAD2 and IFIH1 mRNAs in endometria from cyclic and early pregnant ewes as determined by slot blot hybridization analysis. In cyclic ewes, RSAD2 mRNA level was low between days 10 and 16. In contrast, RSAD2 mRNA increased (P<0.01) sixfold between days 12 and 16 and was maintained to day 20. Similarly, IFIH1 mRNAwas very low in the endometria of cyclic ewes and increased (P<0.01) about 2.5-fold between days 12 and 16 of pregnancy. Data are expressed as LSM relative units (RU) with standard error (s.e.).

  • View in gallery

    In situ hybridization analyses of RSAD2 mRNA in uteri of cyclic and pregnant ewes. Cross-sections of the uterine wall from cyclic (C) and pregnant (P) ewes were hybridized with radiolabeled antisense or sense ovine RSAD2 cRNA probes. RSAD2 mRNA is expressed detected in endometrial stroma, glands, and resident immune cells. Legend: LE, luminal epithelium; GE, glandular epithelium; M, myometrium; S, stroma; Tr, trophectoderm. Scale bar represents 10 μm.

  • View in gallery

    In situ hybridization analyses of IFIH1 mRNAs in uteri of cyclic and pregnant ewes. Cross-sections of the uterine wall from cyclic (C) and pregnant (P) ewes were hybridized with radiolabeled antisense or sense ovine IFIH1 cRNA probes. IFIH1 mRNA was detected only in endometrial stroma and glands. Legend: LE, luminal epithelium; GE, glandular epithelium; S, stroma; Tr, trophectoderm. Scale bar represents 10 μm.

  • View in gallery

    Effects of progesterone and IFNT on RSAD2 mRNA in the ovine uterus. (A) Experimental design. See Materials and Methods for complete description. Legend: CX, control serum proteins; Hystx, hysterectomy; Ovx/Cath, ovariectomy and uterine catheterization; P4, progesterone; IFNT, recombinant ovine interferon tau; ZK, ZK137,316 anti-progestin. (B) Steady-state levels of RSAD2 mRNA in endometria as determined by slot blot hybridization analysis. Intrauterine infusion of IFNT increased RSAD2 mRNA by 10-fold in the endometrium (P4 + CX vs P4 + IFN, P<0.001), but not in ewes receiving the ZK anti-progestin (P4 + IFN vs P4 + ZK + IFN, P>0.10). Similarly, IFNT increased RSAD2 mRNA 9.3-fold in ewes receiving ZK anti-progestin (P4 + ZK + CX vs P4 + ZK + IFN, P<0.001), but not in ewes receiving the ZK anti-progestin (P4 + IFN vs P4 + ZK + IFN, P>0.10). The asterisk (*) denotes an effect of treatment. (C) In situ hybridization analysis of RSAD2 mRNA expression. In situ hybridization analyses verified that roIFNT increased RSAD2 mRNA expression in a cell-type specific manner in P4-treated ewes consistent with increased expression in uteri from day 16 and 18 pregnant ewes. Intrauterine injections of IFNT increased RSAD2 and IFIH1 mRNAs in endometrial stroma and glands, but not LE, blood vessels or myometrium. Further, IFNT increased RSAD2 mRNA in endometria of P4 + ZK-treated ewes. Scale bar represents 10 μm.

  • View in gallery

    Effects of progesterone and IFNT on IFIH1 mRNA in the ovine uterus. (A) Steady-state levels of IFIH1 mRNA in endometria were determined by slot blot hybridization analysis. Intrauterine infusion of IFNT increased IFIH1 mRNA about 8.3-fold in endometria (P4 + CX vs P4 + IFN, P<0.001), but not in ewes receiving the ZK anti-progestin (P4 + IFN vs P4 + ZK + IFN, P>0.10). The asterisk (*) denotes an effect of treatment. (B) In situ hybridization analysis of IFIH1 mRNA expression. Intrauterine injections of IFNT increased IFIH1 mRNA expression in a cell-type specific manner in P4-treated ewes consistent with the increased in expression in uteri from day 16 and day 18 pregnant ewes. Infusion of roIFNT increased expression of IFIH1 mRNA in endometrial stroma and GE, but not LE, blood vessels or myometrium. Further, IFNT increased IFIH1 mRNA in endometria from ewes treated with P4 + ZK. Cross-sections of the uterine wall from treated-ewes were hybridized with radiolabeled antisense or sense ovine IFIH1 cRNA probes. Scale bar represents 10 μm.

  • View in gallery

    Semi-quantitative RT-PCR analyses of RSAD2 and IFIH1 mRNAs in total cellular RNA isolated from immortalized ovine endometrial LE (oLE) and bovine endometrial (BEND) cells. All PCR products were separated in a 1.5% agarose gel and stainedwith ethidium bromide. Positions of the 100-bp (bp) DNA marker (M) ladder are shown. Total cellular RNA from endometria of a day 18 pregnant ewes and sterile water (no template) were positive and negative controls respectively. A graph illustrating the effect of IFNT on relative mRNA levels for RSAD2 and IFIH1 is presented below each gel, and the asterisk (*) denotes a significant (P<0.001) effect of IFNT treatment.

  • Alders RG & Shelton JN1990 Lymphocyte subpopulations in lymph and blood draining from the uterus and ovary in sheep. Journal of Reproductive Immunology1727–40.

    • Search Google Scholar
    • Export Citation
  • Alexenko AP Leaman DW Li J & Roberts RM1997 The antiproliferative and antiviral activities of IFN-tau variants in human cells. Journal of Interferon and Cytokine Research17769–779.

    • Search Google Scholar
    • Export Citation
  • Asselin E Johnson GA Spencer TE & Bazer FW2001 Monocyte chemotactic protein-1 and -2 messenger ribonucleic acids in the ovine uterus: regulation by pregnancy progesterone and interferon-tau. Biology of Reproduction64992–1000.

    • Search Google Scholar
    • Export Citation
  • Chin KC & Cresswell P2001 Viperin (cig5) an IFN-inducible antiviral protein directly induced by human cytomegalovirus. PNAS9815125–15130.

    • Search Google Scholar
    • Export Citation
  • Choi Y Johnson GA Burghardt RC Berghman LR Joyce MM Taylor KM Stewart MD Bazer FW & Spencer TE2001a Interferon regulatory factor-two restricts expression of interferon-stimulated genes to the endometrial stroma and glandular epithelium of the ovine uterus. Biology of Reproduction651038–1049.

    • Search Google Scholar
    • Export Citation
  • Choi Y Johnson GA Burghardt RC Berghman LR Joyce MM Taylor KM Stewart MD Bazer FW & Spencer TE2001b Interferon regulatory factor-two restricts expression of interferon-stimulated genes to the endometrial stroma and glandular epithelium of the ovine uterus. Biology of Reproduction651038–1049.

    • Search Google Scholar
    • Export Citation
  • Choi Y Johnson GA Spencer TE & Bazer FW2003 Pregnancy and interferon tau regulate MHC class I and beta-2-microglobulin expression in the ovine uterus. Biology of Reproduction681703–1710.

    • Search Google Scholar
    • Export Citation
  • Croy B He H Esadeg S Wei Q McCartney D Zhang J Borzychowski A Ashkar A Black G Evans Set al.2003a Uterine natural killer cells: insights into their cellular and molecular biology from mouse modelling. Reproduction126149–160.

    • Search Google Scholar
    • Export Citation
  • Croy BA Esadeg S Chantakru S van den Heuvel M Paffaro VA He H Black GP Ashkar AA Kiso Y & Zhang J2003b Update on pathways regulating the activation of uterine NK cells their interactions with decidual spiral arteries and homing of their precursors to the uterus. Journal of Reproductive Immunology59175–191.

    • Search Google Scholar
    • Export Citation
  • Farin CE Imakawa K & Roberts RM1989In situ localization of mRNA for the interferon ovine trophoblast protein-1 during early embryonic development of the sheep. Molecular Endocrinology31099–1107.

    • Search Google Scholar
    • Export Citation
  • Fleming JA Choi Y Johnson GA Spencer TE & Bazer FW2001 Cloning of the ovine estrogen receptor-alpha promoter and functional regulation by ovine interferon-tau. Endocrinology1422879–2887.

    • Search Google Scholar
    • Export Citation
  • Fleming JG Spencer TE Safe SH & Bazer FW2006 Estrogen regulates transcription of the ovine oxytocin receptor gene through GC-rich SP1 promoter elements. Endocrinology147899–911.

    • Search Google Scholar
    • Export Citation
  • Gray CA Adelson DL Bazer FW Burghardt RC Meeusen EN & Spencer TE2004 Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in the trophectoderm. PNAS1017982–7987.

    • Search Google Scholar
    • Export Citation
  • Gray CA Abbey CA Beremand PD Choi Y Farmer JL Adelson DL Thomas TL Bazer FW & Spencer TE2006 Identification of endometrial genes regulated by early pregnancy progesterone and interferon tau in the ovine uterus. Biology of Reproduction74383–394.

    • Search Google Scholar
    • Export Citation
  • Guillomot M Michel C Gaye P Charlier N Trojan J & Martal J1990 Cellular localization of an embryonic interferon ovine trophoblastin and its mRNA in sheep embryos during early pregnancy. Biology of the Cell68205–211.

    • Search Google Scholar
    • Export Citation
  • Hansen PJ1995 Interactions between the immune system and the ruminant conceptus. Journal of Reproduciton Fertility. Supplement4969–82.

    • Search Google Scholar
    • Export Citation
  • Hansen PJ1998 Regulation of uterine immune function by progesterone–lessons from the sheep. Journal of Reproductive Immunology4063–79.

    • Search Google Scholar
    • Export Citation
  • Hansen TR Austin KJ Perry DJ Pru JK Teixeira MG & Johnson GA1999a Mechanism of action of interferon-tau in the uterus during early pregnancy. Journal of Reproduciton Fertility. Supplement54329–339.

    • Search Google Scholar
    • Export Citation
  • Hansen TR Austin KJ Perry DJ Pru JK Teixeira MG & Johnson GA1999b Mechanism of action of interferon-tau in the uterus during early pregnancy. Journal of Reproduciton Fertility54329–339.

    • Search Google Scholar
    • Export Citation
  • Helbig KJ Lau DT Semendric L Harley HA & Beard MR2005 Analysis of ISG expression in chronic hepatitis C identifies viperin as a potential antiviral effector. Hepatology42702–710.

    • Search Google Scholar
    • Export Citation
  • Imakawa K Imai M Sakai A Suzuki M Nagaoka K Sakai S Lee SR Chang KT Echternkamp SE & Christenson RK2006 Regulation of conceptus adhesion by endometrial CXC chemokines during the implantation period in sheep. Molecular Reproduction and Development73850–858.

    • Search Google Scholar
    • Export Citation
  • Johnson GA Austin KJ Collins AM Murdoch WJ & Hansen TR1999a Endometrial ISG17 mRNA and a related mRNA are induced by interferon-tau and localized to glandular epithelial and stromal cells from pregnant cows. Endocrine10243–252.

    • Search Google Scholar
    • Export Citation
  • Johnson GA Burghardt RC Newton GR Bazer FW & Spencer TE1999b Development and characterization of immortalized ovine endometrial cell lines. Biology of Reproduction611324–1330.

    • Search Google Scholar
    • Export Citation
  • Johnson GA Burghardt RC Newton GR Bazer FW & Spencer TE1999c Development and characterization of immortalized ovine endometrial cell lines. Biology of Reproduction611324–1330.

    • Search Google Scholar
    • Export Citation
  • Johnson GA Spencer TE Hansen TR Austin KJ Burghardt RC & Bazer FW1999d Expression of the interferon tau inducible ubiquitin cross-reactive protein in the ovine uterus. Biology of Reproduction61312–318.

    • Search Google Scholar
    • Export Citation
  • Johnson JA Hochkeppel HK & Gangemi JD1999e IFN-tau exhibits potent suppression of human papillomavirus E6/E7 oncoprotein expression. Journal of Interferon and Cytokine Research191107–1116.

    • Search Google Scholar
    • Export Citation
  • Johnson GA Stewart MD Gray CA Choi Y Burghardt RC Yu-Lee LY Bazer FW & Spencer TE2001 Effects of the estrous cycle pregnancy and interferon tau on 2′ 5′- oligoadenylate synthetase expression in the ovine uterus. Biology of Reproduction641392–1399.

    • Search Google Scholar
    • Export Citation
  • Kang DC Gopalkrishnan RV Wu Q Jankowsky E Pyle AM & Fisher PB2002 mda-5: an interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties. PNAS99637–642.

    • Search Google Scholar
    • Export Citation
  • Kang DC Gopalkrishnan RV Lin L Randolph A Valerie K Pestka S & Fisher PB2004 Expression analysis and genomic characterization of human melanoma differentiation associated gene-5 mda-5: a novel type I interferon-responsive apoptosis-inducing gene. Oncogene231789–1800.

    • Search Google Scholar
    • Export Citation
  • Katze MG He Y & Gale M Jr2002 Viruses and interferon: a fight for supremacy. Nature Reviews. Immunology2675–687.

  • Khan OA Jiang H Subramaniam PS Johnson HM & Dhib-Jalbut SS1998 Immunomodulating functions of recombinant ovine interferon tau: potential for therapy in multiple sclerosis and autoimmune disorders. Multiple Sclerosis463–69.

    • Search Google Scholar
    • Export Citation
  • Kim S Choi Y Bazer FW & Spencer TE2003 Identification of genes in the ovine endometrium regulated by interferon tau independent of signal transducer and activator of transcription 1. Endocrinology1445203–5214.

    • Search Google Scholar
    • Export Citation
  • Klein C Bauersachs S Ulbrich SE Einspanier R Meyer HHD Schmidt SEM Reichenbach H-D Vermehren M Sinowatz F Blum Het al.2006 Monozygotic twin model reveals novel embryo-induced transcriptome changes of bovine endometrium in the pre-attachment period. Biology of Reproduction74253–264.

    • Search Google Scholar
    • Export Citation
  • Lee CS Gogolin-Ewens K & Brandon MR1988 Identification of a unique lymphocyte subpopulation in the sheep uterus. Immunology63157–164.

    • Search Google Scholar
    • Export Citation
  • Mamane Y Heylbroeck C Genin P Algarte M Servant MJ LePage C DeLuca C Kwon H Lin R & Hiscott J1999 Interferon regulatory factors: the next generation. Gene2371–14.

    • Search Google Scholar
    • Export Citation
  • Nagaoka K Sakai A Nojima H Suda Y Yokomizo Y Imakawa K Sakai S & Christenson RK2003 A chemokine interferon (IFN)-gamma-inducible protein 10 kDa is stimulated by IFN-tau and recruits immune cells in the ovine endometrium. Biology of Reproduction681413–1421.

    • Search Google Scholar
    • Export Citation
  • Newton GR Vallet JL Hansen PJ & Bazer FW1989 Inhibition of lymphocyte proliferation by ovine trophoblast protein-1 and a high molecular weight glycoprotein produced by the peri-implantation sheep conceptus. American Journal of Reproductive Immunology1999–107.

    • Search Google Scholar
    • Export Citation
  • Perry DJ Austin KJ & Hansen TR1999 Cloning of interferon-stimulated gene 17: the promoter and nuclear proteins that regulate transcription. Molecular Endocrinology131197–1206.

    • Search Google Scholar
    • Export Citation
  • Pontzer CH Bazer FW & Johnson HM1991 Antiproliferative activity of a pregnancy recognition hormone ovine trophoblast protein-1. Cancer Research515304–5307.

    • Search Google Scholar
    • Export Citation
  • Rosenfeld CS Han CS Alexenko AP Spencer TE & Roberts RM2002 Expression of interferon receptor subunits IFNAR1 and IFNAR2 in the ovine uterus. Biology of Reproduction67847–853.

    • Search Google Scholar
    • Export Citation
  • Segerson EC Matterson PM & Gunsett FC1991 Endometrial T-lymphocyte subset infiltration during the ovine estrous cycle and early pregnancy. Journal of Reproductive Immunology20221–236.

    • Search Google Scholar
    • Export Citation
  • Sofia HJ Chen G Hetzler BG Reyes-Spindola JF & Miller NE2001 Radical SAM a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Research291097–1106.

    • Search Google Scholar
    • Export Citation
  • Song G Spencer TE & Bazer FW2005 Cathepsins in the ovine uterus: regulation by pregnancy progesterone and interferon tau. Endocrinology1464825–4833.

    • Search Google Scholar
    • Export Citation
  • Song G Spencer TE & Bazer FW2006 Progesterone and interferon tau regulate cystatin C (CST3) in the endometrium. Endocrinology1473478–3483.

    • Search Google Scholar
    • Export Citation
  • Spencer TE & Bazer FW2002 Biology of progesterone action during pregnancy recognition and maintenance of pregnancy. Frontiers in Bioscience7d1879–d1898.

    • Search Google Scholar
    • Export Citation
  • Spencer TE & Bazer FW2004 Conceptus signals for establishment and maintenance of pregnancy. Reproductive Biology and Endocrinology249.

  • Spencer TE Becker WC George P Mirando MA Ogle TF & Bazer FW1995 Ovine interferon-tau regulates expression of endometrial receptors for estrogen and oxytocin but not progesterone. Biology of Reproduction53732–745.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Ott TL & Bazer FW1996 tau-Interferon: pregnancy recognition signal in ruminants. Proceedings of the Society for Experimental Biology and Medicine213215–229.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Bartol FF Bazer FW Johnson GA & Joyce MM1999a Identification and characterization of glycosylation-dependent cell adhesion molecule 1-like protein expression in the ovine uterus. Biology of Reproduction60241–250.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Gray A Johnson GA Taylor KM Gertler A Gootwine E Ott TL & Bazer FW1999b Effects of recombinant ovine interferon tau placental lactogen and growth hormone on the ovine uterus. Biology of Reproduction611409–1418.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Stagg AG Ott TL Johnson GA Ramsey WS & Bazer FW1999c Differential effects of intrauterine and subcutaneous administration of recombinant ovine interferon tau on the endometrium of cyclic ewes. Biology of Reproduction61464–470.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Burghardt RC Johnson GA & Bazer FW2004a Conceptus signals for establishment and maintenance of pregnancy. Animal Reproduction Science82–83537–550.

    • Search Google Scholar
    • Export Citation
  • Spencer TE Johnson GA Bazer FW & Burghardt RC2004b Implantation mechanisms: insights from the sheep. Reproduction128657–668.

  • Spencer TE Johnson GA Burghardt RC & Bazer FW2004c Progesterone and placental hormone actions on the uterus: insights from domestic animals. Biology of Reproduction712–10.

    • Search Google Scholar
    • Export Citation
  • Stark GR Kerr IM Williams BR Silverman RH & Schreiber RD1998 How cells respond to interferons. Annual Review of Biochemistry67227–264.

    • Search Google Scholar
    • Export Citation
  • Stewart MD Johnson GA Gray CA Burghardt RC Schuler LA Joyce MM Bazer FW & Spencer TE2000 Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy. Biology of Reproduction621779–1789.

    • Search Google Scholar
    • Export Citation
  • Stewart DM Johnson GA Vyhlidal CA Burghardt RC Safe SH YuLee LY Bazer FW & Spencer TE2001 Interferon-tau activates multiple signal transducer and activator of transcription proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells. Endocrinology14298–107.

    • Search Google Scholar
    • Export Citation
  • Sun BJ & Nie P2004 Molecular cloning of the viperin gene and its promoter region from the mandarin fish Siniperca chuatsi. Veterinary Immunology and Immunopathology101161–170.

    • Search Google Scholar
    • Export Citation
  • Tekin S & Hansen PJ2002 Natural killer-like cells in the sheep: functional characterization and regulation by pregnancy-associated proteins. Experimental Biology and Medicine (Maywood)227803–811.

    • Search Google Scholar
    • Export Citation
  • Tekin S Ealy AD Wang SZ & Hansen PJ2000 Differences in lymphocyte-regulatory activity among variants of ovine IFN-tau. Journal of Interferon & Cytokine Research201001–1005.

    • Search Google Scholar
    • Export Citation
  • Tuo W Ott TL & Bazer FW1993 Natural killer cell activity of lymphocytes exposed to ovine type I trophoblast interferon. American Journal of Reproductive Immunology2926–34.

    • Search Google Scholar
    • Export Citation
  • Van Heeke G Ott TL Strauss A Ammaturo D & Bazer FW1996 High yield expression and secretion of the ovine pregnancy recognition hormone interferon-tau by Pichia pastoris. Journal of Interferon & Cytokine Research16119–126.

    • Search Google Scholar
    • Export Citation
  • Yankey SJ Hicks BA Carnahan KG Assiri AM Sinor SJ Kodali K Stellflug JN & Ott TL2001 Expression of the antiviral protein Mx in peripheral blood mononuclear cells of pregnant and bred non-pregnant ewes. Journal of Endocrinology170R7–11.

    • Search Google Scholar
    • Export Citation
  • Yoneyama M Kikuchi M Matsumoto K Imaizumi T Miyagishi M Taira K Foy E Loo YM Gale M Jr Akira Set al.2005 Shared and unique functions of the DExD/H-box helicases RIG-I MDA5 and LGP2 in antiviral innate immunity. Journal of Immunology1752851–2858.

    • Search Google Scholar
    • Export Citation