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Endocrinology, doi:10.1210/en.2007-0863
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Endocrinology Vol. 149, No. 3 1252-1259
Copyright © 2008 by The Endocrine Society

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

João F. Oliveira1, Luiz E. Henkes1, Ryan L. Ashley, Scott H. Purcell, Natalia P. Smirnova, D. N. Rao Veeramachaneni, Russell V. Anthony and Thomas R. Hansen

Animal Reproduction and Biotechnology Laboratory (J.F.O., L.E.H., R.L.A., S.H.P., N.P.S., D.N.R.V., R.V.A., T.R.H.), Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523; and BioRep (J.F.O.), Departamento de Clínica de Grandes Animais, Centro de Ciências Rurais, Universidade Federal de Santa Maria 97105-900, Santa Maria, Rio Grande do Sul, Brazil

Address all correspondence and requests for reprints to: Dr. Thomas R. Hansen, Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Campus Delivery 1683, Fort Collins, Colorado 80523. E-mail: thomas.hansen{at}colostate.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ruminant conceptus synthesizes and secretes interferon (IFN)-{tau}, which presumably acts via an intrauterine paracrine mechanism to signal maternal recognition of pregnancy. The aims of this study were to determine whether IFN-stimulated genes (ISG) such as ISG15 and OAS-1 are differentially expressed in blood cells circulating in the uterus of ewes; whether extrauterine components of the reproductive tract such as the corpus luteum (CL) also express mRNA for these ISG, and whether antiviral activity is greater in uterine vein than in uterine artery during early pregnancy. The concentrations of mRNA for both ISG were significantly greater (P < 0.0001) in endometrium and jugular blood of 15-d pregnant ewes than in nonpregnant ewes. ISG15 and OAS-1 mRNA concentrations were also greater (P < 0.05) in CL from 15-d pregnant ewes than in nonpregnant ewes. Immunohistochemistry revealed intense staining for ISG15 in large luteal cells on d 15 of pregnancy. Blood cells from uterine artery and vein of 15-d pregnant ewes had similar ISG15 and OAS-1 mRNA concentrations, suggesting that these cells were not conditioned by IFN-{tau} within the uterus. By using an antiviral assay, uterine venous blood was found to contain 500- to 1000-fold higher concentrations of bioactive IFN-{tau} than in uterine arterial blood on d 15 of pregnancy. It is concluded that uterine vein releases IFN-{tau}, which induces ISG in extrauterine tissues such as the CL during the time of maternal recognition of pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EXPANDING RUMINANT blastocyst releases interferon (IFN)-{tau}, which acts locally in the uterus as a paracrine signal for maternal recognition of pregnancy (1, 2). IFN-{tau} interacts with its receptor in the uterus and alters secretion of prostaglandin (PG)-F2{alpha}, thus preventing lysis of the corpus luteum (CL) (3, 4). IFN-{tau} regulates endometrial PG production through reducing oxytocin receptors as an outcome of the down-regulation of estrogen receptor (2). Furthermore, an alteration in the PGE2 to PGF2{alpha} ratio in favor of PGE2 during pregnancy (5) and in response to IFN-{tau} treatment of endometrial cells in bovine endometrial primary cultures (6) may also be important in maintaining the CL during early pregnancy.

In addition to its antiluteolytic effects, IFN-{tau} increases expression of several IFN-stimulated genes (ISG) in the uterus, such as 2',5'-oligoadenylate synthetase (OAS-1) (7, 8), signal transducer and activator of transcription (Stat)-1 and Stat2 (9, 10, 11, 12), major histocompatibility complex class I and β2-microglobulin (13), IFN regulatory factor (IRF)-1 and IRF-9 (9), myxovirus (influenza virus) resistance 2 Mx (14), OAS-1 (15), and ubiquitin-like IFN-stimulated gene 15-kDa protein (ISG15) (16). More recently, several other ISG that are either induced by pregnancy (17, 18, 19) or via culture of endometrial cells with recombinant ovine (ro)IFN-{tau} (20) have been identified by microarray analysis.

The functions of many of these IFN-induced proteins in the antiviral response are well characterized, but their roles during early pregnancy have not been described. Austin et al. (21) linked ISG15 to early pregnancy by identifying a 17-kDa protein that was secreted by the bovine endometrium in response to pregnancy and conceptus-derived IFN-{tau}. ISG15 is up-regulated in the uterine endometrium of pregnant sheep and cows in response to IFN-{tau} (16, 21, 22). Johnson et al. (23) found that bovine ISG15 conjugates to intracellular proteins during pregnancy, suggesting that ISG15 may be involved in the regulation of proteins critical to the establishment and maintenance of pregnancy.

The expression of IFN-{tau}-induced proteins during early pregnancy, such as Mx and ISG15, was recently shown to occur in other tissues. Yankey et al. (24) have shown that Mx gene expression increases 4- to -5-fold in peripheral blood mononuclear cells (PBMC) within 24–48 h of initial IFN-{tau} signaling in pregnant sheep, whereas Chen et al. (25) demonstrated expression of ISG in the CL after either intrauterine infusion or im injection with roIFN-{tau}. Han et al. (26) predicted the pregnancy status with an accuracy ranging from 89–100% based on lower levels of ISG15 mRNA in blood cells from nonpregnant cows. Gifford et al. (27) also demonstrated that ISG15, Mx, and other ISG were down-regulated in peripheral blood from d-18 nonpregnant dairy cows when compared with pregnant dairy cows. However, exactly how the blood cells were conditioned by pregnancy or IFN-{tau} to express these genes remains to be established. Also, pregnancy-induced expression of ISG in the CL has not been investigated until the present work.

Based on the results described above and historical reports of conceptus-derived IFN-{tau}, working exclusively through an intrauterine-paracrine action, we hypothesized that the blood cells are conditioned during trafficking through the uterus. The aim of this study was to determine whether ISG in blood cells are differentially expressed as they enter and then exit the uterus and to determine whether peripheral tissues such as the CL contain elevated levels of mRNA for these ISG via trafficking of uterine PBMC from the uterus to the CL on d 15 of pregnancy. Given that expression of ISG15 and OAS-1 mRNA in PBMC were similar in the uterine vein and artery from d 15 pregnant ewes in the current study, we next hypothesized that IFN-{tau} was actually released into the uterine vein, which would result in a massive peripheral response to IFN-{tau} and may explain the high levels of ISG15 and OAS-1 in the CL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
All experimental procedures using animals were reviewed and approved by the Colorado State University Animal Care and Use Committee. Mature, crossbred, white-faced ewes were synchronized by two injections of PGF2{alpha} (Lutalyse; Pfizer, New York, NY; 10 mg, im) given 14 d apart. After observation of standing estrus with a vasectomized ram, all ewes were bred by one of two intact rams. Pregnancy was confirmed by the presence of a conceptus. Some ewes were not exposed to rams to generate blood samples and tissues representing the estrous cycle.

Endometrium, CL, and jugular blood samples were collected on d 15 (d 0 = day of mating) from nonpregnant and pregnant ewes (n = 3 ewes per group). The neck of each ewe was shaved, and jugular blood samples were collected using Vacutainer tubes (Becton Dickinson and Co., Franklin Lakes, NJ). Immediately after collection of jugular samples, ewes were anesthetized using sodium pentobarbital (20 mg/kg, iv). Hysterectomy was then performed, and CL and endometrial samples were collected. In d-15 pregnant ewes, uterine vein and uterine artery blood was collected after sedation. Jugular vein blood and CL also were collected on d 21 and 30 of pregnancy as described above.

Isolation of large luteal cells
Corpora lutea were decapsulated, sliced, and dissociated into single-cell suspensions with 0.25% collagenase and 0.05% deoxyribonuclease. The cells were separated by centrifugal elutriation into small (l0–20 µm) and large (20–35 µm) luteal cell fractions (28). The large luteal cells were cultured overnight in six-well plates (5 x 105 cells per well) in MEM supplemented with 5% fetal bovine serum (FBS). On the next morning, the medium was changed and cells were placed in serum-free conditions for 1 h and treated with or without 200 ng/ml roIFN-{tau} for 24 h. Cells were next washed with cold PBS and lysed directly in the culture plate by adding 1 ml/well of TRI reagent (Sigma Chemical Co., St. Louis, MO).

Isolation of RNA
Tissue RNA extraction was performed using TRI reagent as previously described (29). Blood (200 µl) was mixed with 750 µl TRI reagent (Sigma) and stored at –80 C until analysis as previously described by Han et al. (26). The remaining blood sample was chilled, and plasma was separated via centrifugation at 1000 x g for 10 min, aliquoted, and stored at –20 C.

After extraction of RNA by using phenol/chloroform/isopropyl alcohol, RNA was treated with DNase I. The total RNA was purified using RNeasy MinElute Cleanup Kit (QIAGEN, Valencia, CA). Single-stranded cDNA was synthesized from total RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) following the manufacturer’s instructions. Pregnancy status was determined based on the presence or absence of one or more embryos. A sample of endometrium and CL was collected for protein extraction and for preparation of histological sections (CL only).

Semiquantitative real-time PCR (qRT-PCR)
ISG15 and OAS-1 are two of the most highly up-regulated genes in blood cells from d-18 pregnant cows compared with nonpregnant cows (our unpublished results). For this reason, these ISG were the focus of the present study. The primers for ISG15 (forward, 5'-GGTATCCGAGCTGAAGCAGTT-3'; reverse, 5'-ACCTCCCTGCTGTCAAGGT-3') and GAPDH (forward, 5'-GATTGTCAGCAATGCCTCCT-3'; reverse, 5'-GGTCATAAGTCCCTCCACGA-3') were based on Han et al. (26). OAS-1 primers (forward, 5'-TAGGCCTGGAACATCAGGTC-3'; reverse, 5'-TTTGGTCTGGCTGGATTACC-3') were designed based on sequences retrieved from the NCBI (accession no. NM_001040606). qRT-PCR amplification was performed at 95 C for 30 sec, 62 C for 30 sec, and 72 C for 15 sec for 40 cycles. qRT-PCR was performed to determine expression of the ISG. cDNA (5 µl) was used as a template for qRT-PCR amplification using iQ SYBR Green Supermix (Bio-Rad). GAPDH was used as an internal reference for normalization of mRNA expression.

Western blot
CL tissue (100 mg) was homogenized in 1 ml Laemmli buffer. Lysates (100 µg/lane) were separated by SDS-PAGE using 12% polyacrylamide gels followed by transfer to nitrocellulose membrane. Membranes were blocked overnight in 5% nonfat milk made in Tris-buffered saline plus Tween 20 at 4 C. Samples were analyzed for ISG15 by Western analysis using a monoclonal anti-ISG15 (5F10) antibody (generated in the Hansen laboratory) at 1:50,000 dilution (22). Secondary goat antimouse IgG-horseradish peroxidase was used at 1:200 dilution (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were detected by chemiluminescence using the Amersham ECL Plus Western Blotting Detection System (GE Healthcare, Piscataway, NJ). An anti-actin antibody (sc-47778; Santa Cruz Biotechnology) was used at 1:1000 dilution to further demonstrate equal loading.

Immunohistochemical localization of ISG15 in the CL
Transverse pieces of CL were fixed in 4% buffered paraformaldehyde for 24 h, followed by paraffin embedding and sectioning. Antigen retrieval was accomplished by microwaving two times for 5 min each in 0.01 M citrate buffer (pH 6.0). ISG15 and its conjugates were localized using monoclonal 5F10 antibody (1:10,000) described by Austin et al. (22).

IFN protection assay
IFN concentrations were determined based on a modified antiviral assay (30) by using Madin-Darby bovine kidney cells (MDBK). MDBK cells were plated at 30 x 103 cells per well in 96-well plates in regular media (MEM supplemented with 5% FBS). After reaching confluence, cells were exposed to serial dilutions of ovine plasma (1:4 to 1:32) and cultured for 24 h at 37 C. The medium was changed, and the cells were challenged with vesicular stomatitis virus in MEM with 1% FBS for an additional 42 h and stained with 0.5% crystal violet solution in 70% methanol. OD of the remaining monolayers was measured by absorbance at 540 nm. IFN protection index was calculated by the following formula: protection index = (OD exp/OD cell control) x 100%, where OD exp is OD of the experimental well (plasma-treated) and OD cell control is the average OD of cell control wells. Universal type I IFN (Biomedical Laboratories, Piscataway, NJ) at starting concentration of 100,000 U/ml with serial dilutions from 1:100 to 1:25,600 was used as the IFN standard. Estimation of IFN U/ml in tested plasma samples was made by comparison of IFN protection indices for tested samples with IFN protection indices of appropriate dilution of Universal IFN standard.

Statistical analysis
Statistical analyses were performed using Statistical Analysis System Package version 9.1 for Windows (SAS Institute, Cary, NC). Data for mRNA expression were analyzed using a one-way ANOVA based on {Delta}{Delta}Ct values. For analysis of IFN levels using the antiviral assay, the data were first transformed using the arcsin function and then submitted to ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mRNA profiles of ISG15 and OAS-1 in endometrium and blood during early pregnancy in sheep
Concentrations of mRNA encoding both ISG15 and OAS-1 were significantly (P < 0.05) up-regulated in the endometrium of pregnant ewes on d 15 compared with nonpregnant females (Fig. 1Go). The jugular blood mRNA profiles of ISG15 and OAS-1 on d 15, 20, and 30 of pregnancy in sheep are shown in Fig. 2Go. ISG15 and OAS-1 jugular blood cell mRNAs exhibited a 102- and 47-fold increase, respectively, on d 15 of pregnancy when compared with the estrous cycle. Jugular blood ISG15 mRNA was significantly up-regulated (P < 0.001) on d 15, 20, and 30 of pregnancy, whereas OAS-1 mRNA concentrations were up-regulated (P < 0.05) on d 15 and 20 but not on d 30 of pregnancy compared with d 15 of the estrous cycle. However, on d 15 of pregnancy, jugular vein, uterine vein, and uterine arterial blood mRNA profiles of ISG15 or OAS-1 did not differ (P > 0.05) (Fig. 3Go).


Figure 1
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  		FIG. 1. Endometrial expression of mRNA for ISG15 (A) and OAS-1 (B) on d 15 of the estrous cycle (NP) and pregnancy (P) in ewes. Values represent the mean ± SE. *, Means are different (P < 0.05).

 

Figure 2
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  		FIG. 2. Peripheral blood profiles (jugular vein) of mRNA for ISG15 and OAS-1 on d 15 of the estrous cycle (NP) and on d 15, 20, and 30 of pregnancy in ewes. Comparisons were made within mRNA target across the days of pregnancy. Values represent the mean ± SE. Significant differences (P < 0.05) are indicated by different letters (uppercase letters, ISG15; lowercase letters, OAS-1).

 

Figure 3
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  		FIG. 3. ISG15 (A) and OAS-1 (B) mRNA profiles in jugular vein (JV), uterine vein (UV) and uterine artery (UA) blood samples from d-15 pregnant ewes. Jugular vein blood cell mRNAs from d-15 nonpregnant (NP) ewes are provided as a negative control. Values represent the mean ± SE. Significant differences (P < 0.05) are indicated by different letters.

 
Expression of ISG15 and OAS-1 in the CL
The relative expression of mRNA for ISG15 and OAS-1 was significantly higher (P < 0.05) in CL on d 15 of pregnancy when compared with d-15 nonpregnant ewes (Fig. 4Go). In pregnant ewes, ISG15 mRNA concentration was lower in the CL when compared with the jugular vein. However, jugular vein ISG15 mRNA concentrations were significantly higher (P < 0.05) than jugular vein and endometrial (not shown) concentrations from nonpregnant ewes. Expression of OAS-1 mRNA followed a similar pattern to ISG15 mRNA in that jugular and CL OAS-1 levels were higher (P < 0.05) on d 15 of pregnancy when compared with d 15 of the estrous cycle.


Figure 4
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  		FIG. 4. ISG15 (A) and OAS-1 (B) mRNA profiles in jugular vein blood samples and CL in d-15 nonpregnant (NP) or pregnant (P) sheep. CL15NP, CL d-15 nonpregnant; CL15P, CL, d-15 pregnant; JNP, jugular vein nonpregnant; JP, jugular vein pregnant. Values represent the mean ± SE. Significant differences (P < 0.05) are indicated by different letters.

 
Western blot detection (Fig. 5Go) of ISG15 also confirmed that both free and conjugated ISG15 were induced in the CL on d 15 of pregnancy when compared with the estrous cycle.


Figure 5
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  		FIG. 5. Western blot detection of ISG15 protein in CL collected on d 15 of the estrous cycle or pregnancy in sheep. A, Protein extracts (100 µg/lane; each lane represents a different ewe) were loaded on Western blots from d 15 either nonpregnant (NP) or pregnant (P) ewes. Free ISG15 migrates at about 15 kDa on the blots. ISG15, when covalently attached as a conjugated protein, forms higher molecular weight complexes. The same protein samples were also probed for actin to further demonstrate equal loading of protein. B, Quantitation of these data (means ± SE). *, Means differ at P < 0.05.

 
Immunohistochemical localization revealed intense staining for ISG15 in CL collected on d 15 of pregnancy, with a diminution of staining by d 20 and 30 of pregnancy (Fig. 6Go). On d 15 of pregnancy, staining for ISG15 was predominantly localized to the cytoplasm of large luteal cells, with variable staining for ISG15 in the small luteal cells (Fig. 7Go). Although large luteal cells were conspicuous by their size, the more numerous small luteal cells were identified by their morphological features such as irregular shape with tapering cytoplasmic processes and sometimes discernible irregular nuclear contours (31). It was evident that the immunostaining was limited only to cytoplasm when sections without nuclear counterstaining were examined using differential interference contrast microscopy. There was no staining for ISG15 in ovarian stroma (Fig. 6Go) and vascular endothelial cells or in circulating erythrocytes and neutrophils (Fig. 7Go). Other negative controls, which included deletion of primary antibodies, revealed no background staining.


Figure 6
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  		FIG. 6. Representative immunohistochemical localization (x100 magnification) of ISG15 protein in ovine CL on d 10, 15, 21, and 30 of pregnancy. The left panel represents deletion of the primary antibody against ISG15. The right panel represents immunohistochemical localization of ISG15.

 

Figure 7
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  		FIG. 7. Differential interference contrast photomicrograph of ovine CL on d 15 of pregnancy stained for ISG15. Note immunostaining is predominantly localized to large luteal cells (white arrows), whereas the staining is variable in small luteal cells (black arrows). Immunostaining is not observed in vascular endothelial cells (arrowheads) or circulating red blood cells (RBC) and neutrophils (WBC). Scale bar, 25 µm.

 
Ovine large luteal cells were enriched from CL collected on d 10 of the estrous cycle and were cultured with 200 ng/ml roIFN-{tau} for 24 h to determine whether these cells responded. These cells responded to culture with roIFN-{tau} by massive induction (P < 0.001) of ISG15 mRNA (Fig. 8Go).


Figure 8
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  		FIG. 8. ISG15 mRNA profiles in ovine large luteal cells untreated (CTRL) and treated with roIFN-{tau} (TAU). Values represent the mean ± SE. Significant differences (P < 0.05) are indicated by different letters.

 
Type I IFN bioactivity in uterine and peripheral blood
According to the background limits and standardization of the antiviral assay, antiviral levels less than 20% are representative of nonspecific factors that are present in sheep plasma. Antiviral activity (Fig. 9Go) in jugular vein blood on d 15 of pregnancy and the estrous cycle and uterine artery blood on d 15 of pregnancy represented background limits for the assay. On d 15 of pregnancy, antiviral activity in the uterine vein blood was significantly higher (P < 0.05) than those observed in uterine artery and jugular vein. The levels of IFN in the uterine vein in pregnant ewes on d 15 ranged from 500–1000 U/ml of uterine vein plasma.


Figure 9
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  		FIG. 9. Antiviral indices (nontransformed data) detected in the jugular vein (JV), uterine artery (UA), and uterine vein (UV) plasma on d 15 of pregnancy and the estrous cycle (NP). Values less than 20% represent nonspecific protection from plasma factors. The levels of IFN in the uterine vein correspond to 500-1000 U/ml (5–10 ng/ml using 108 U/mg) of plasma. Values represent the mean ± SE. Significant differences (P < 0.05) are indicated by different letters.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because the ovine conceptus is free-floating in the uterus until d 16–17, signals such as IFN-{tau} are released in a paracrine fashion that regulate endometrial gene expression and indirectly induce antiluteolytic responses that are required for maintenance of pregnancy (32, 33). Early studies suggested that IFN-{tau}, originally called ovine trophoblast protein-1 (34), is sequestered within the uterine lumen and does not enter the peripheral circulation in high enough concentrations to be detected. However, more recently, ISG such as ISG15 (16, 35) and OAS-1 (15) have been shown to be up-regulated in uterine cross-sections as deep as the myometrium. For this reason, IFN-{tau} was suspected to induce a secondary mediator of ISG in the myometrium, or limited amounts of IFN-{tau} were suspected to pass through the subluminal epithelial basement membrane to elicit local induction of genes in the endometrium and the myometrium. An interferonomedin mechanism was proposed, which favored the former mechanism (36).

Intrauterine delivery of roIFN-{tau} (paracrine) suppressed expression of estrogen receptor and oxytocin receptor, whereas sc delivery of roIFN-{tau} (endocrine) had no effect, suggesting that roIFN-{tau} acts exclusively within the uterus to elicit the antiluteolytic response (37). Oddly, only intrauterine roIFN-{tau} induced endometrial ISG15 expression, whereas both modes of roIFN-{tau} delivery induced endometrial Mx expression. Also, both modes of roIFN-{tau} treatment induced ISG15 and Mx mRNA expression in the CL (37). This group concluded that the antiluteolytic effects of IFN-{tau} were mediated through an intrauterine paracrine response, whereas other peripheral responses were mediated through signals other than conceptus-derived IFN-{tau}.

PBMC from pregnant sheep were first reported to contain increased transcription of Mx mRNA (24), which was also reported for ISG15 and Mx in cattle (26, 27). One theory on how this might be regulated was that PBMC were conditioned through exposure to IFN-{tau} or an interferonomedin while residing in the uterus. This hypothesis was tested in the present study by examining two ISG mRNAs, ISG15 and OAS-1, in blood cells collected from jugular, uterine artery, and uterine vein from d-15 pregnant sheep. These ISG were selected for study because they had the greatest fold up-regulation of known genes in blood cells from d-18 pregnant cows that were screened by using the Affymetrix bovine microarray (Hansen, T. R., J. K. Pru, H. Hans, and K. J. Austin, manuscript in preparation).

ISG15 (38) and OAS-1 (15) mRNA were also confirmed to be up-regulated in the ovine endometrium on d 15 of pregnancy as a positive control for the present experiments, even though this response had already been reported by others (15, 25). Because OAS-1 has not been examined in blood cell mRNA, we compared the expression of OAS-1 to ISG15 and found that ISG15 was up-regulated by d 15 and remained up-regulated through d 30 of pregnancy. A similar up-regulation of blood cell OAS-1 mRNA was noted by d 15. However, by d 30, blood cell OAS-1 mRNA concentrations were low and did not differ from concentrations found in blood from d 15 of the estrous cycle. IFN-{tau} mRNA was originally described to be expressed by the conceptus from d 14–22 of pregnancy by Northern blot analysis (39). More sensitive qRT-PCR revealed that IFN-{tau} mRNA also could be detected as late as d 30–45, but concentrations were about 4-fold lower than on d 20 (40). The reason for the loss of OAS-1 mRNA in blood by d 30 when compared with relatively greater amounts of ISG15 mRNA might be related to stability of the mRNAs or differences in sensitivity of the promoters on these genes to low levels of IFN-{tau}.

We hypothesized that ISG15 mRNA would be lower in blood from the uterine artery compared with uterine vein blood on d 15 of pregnancy. Thus, we were surprised to observe no difference in either ISG15 or OAS-1 mRNA when comparing blood from jugular vein, uterine vein, and uterine artery. Because of this result, we concluded that PBMC are not conditioned in response to IFN-{tau} or an interferonomedin while residing in the uterus. Rather, we suspected that these blood cells might exit the uterus via lymphatic drainage. Lymph nodes (mesenteric and submandibular) were also collected in a different experiment on d 15 of pregnancy and also contained levels of ISG15 mRNA that did not differ when comparing drainage from the uterus (mesenteric) with drainage from the head (submandibular) (our unpublished data). For this reason, we suspected that IFN-{tau} was released from the uterine vein and consequently induced a peripheral response such as increased expression of ISG15 mRNA in PBMC and possibly other extrauterine reproductive tissues such as the CL.

Using sensitive qRT-PCR approaches, we demonstrate that ISG15 mRNA is up-regulated in CL from ewes on d 15 of pregnancy when compared with the estrous cycle. Western blot analysis also revealed massive induction of free and conjugated ISG15 in CL collected on d 15 of pregnancy when compared with the estrous cycle.

Using immunohistochemistry, we were able to confirm the up-regulation of ISG15 in the CL on d 15 of pregnancy. Although ISG-15 was localized to a variable extent in small luteal cells, localization was predominantly in large luteal cells. Interestingly, no immunostaining was observed in circulating neutrophils, indicating that the increase in mRNA expression for ISG15 in blood cells was owing to expression in leukocytes other than neutrophils. It is also possible that translational events, such as expression of ISG15 protein, do not occur at levels that are significant enough to be detected by immunohistochemical approaches in neutrophils.

Treatment of isolated ovine large luteal cells with roIFN-{tau} caused a dramatic increase in ISG15 mRNA expression. These data demonstrated that IFN-{tau} receptors exist on ovine large luteal cells and the receptors are functionally coupled through signal transduction pathways that lead to the transcription of the ISG15 gene.

In the present experiments, it was not clear whether up-regulation of ISG15 in PBMC and CL was regulated by release of IFN-{tau} or an interferonomedin from the uterus. Recently, we standardized an antiviral assay using bovine type I IFN to study maternal and fetal responses to viral infection in cattle (47). This bioassay for type I IFN was used to analyze blood collected on d 15 of pregnancy in the present study. Bioactive IFN was significantly greater in blood from the uterine vein when compared with uterine artery and jugular vein blood. These data provide the first evidence that a type I IFN is actually released from the uterus during maternal recognition of pregnancy in sheep and has an endocrine action. Type I IFN other than IFN-{tau} are not thought to be released in significant amounts by the embryo or the endometrium. Because IFN-{tau} is produced at levels approaching 100–200 µg in a 24-h period by the ovine conceptus (reviewed in Ref. 41), we suspect that this antiviral activity reflects IFN-{tau} rather than other type I IFN. The levels of IFN-{tau} in uterine vein blood are significant at 500–1000 U/ml (~5–10 ng/ml; using 108 U/mg), especially when considering that uterine blood flow ipsilateral to the CL is 13.5 ml/min (~1.35 µg IFN-{tau}/10 min) (42). The IFN-{tau} released via the uterine vein would return to the heart and lungs and then would be delivered from the heart to targeted tissues via arteriole blood flow. Or, IFN-{tau} might be delivered to the CL in a countercurrent exchange from the uterine vein to the ovarian artery as described for PGF2{alpha} (43), although molecules similar in size to IFN-{tau} have not yet been shown to be delivered to the CL through this pathway. Based on 500–1000 U IFN/ml plasma, there would be enough IFN-{tau} released by the uterine vein to activate the IFN receptor and ISG in targeted tissues. However, very little IFN-{tau} recirculates after activating target receptors. This interpretation is based on very low antiviral activity in jugular vein and uterine artery blood on d 15 of pregnancy that did not differ from jugular vein blood collected on d 15 of the estrous cycle. This finding, in addition to the detection of ISG mRNA and proteins in the CL, is interpreted to mean that the CL might be rescued during pregnancy not only by endocrine factors released by the uterus, such as IFN-{tau} but also by expression of specific ISG in the ovary.

Two uterine mechanisms have been proposed to explain how luteolysis is prevented in ruminants: the alteration in the proportion of PGE2/PGF2{alpha} in favor of PGE2 (5, 44) and the down-regulation of oxytocin receptors as a consequence of down-regulation of estrogen receptor by IFN-{tau} (36, 45). Arosh et al. (44) proposed a model integrating actions of PGE2 at the time of establishment of pregnancy in cattle where PGE2 produced in the uterus through an IFN-{tau} stimulus is transported to the CL by PG transporters. Our findings demonstrating that ISG are expressed in CL, more predominantly in large luteal cells, suggest that IFN-{tau} release from uterus can directly regulate synthesis of PG by the CL and potentially the intraluteal PGE2/PGF2 ratio.

In the present study, we demonstrated expression of ISG in the CL by qRT-PCR, Western blots, and immunohistochemistry. We also described release of significant amounts of IFN-{tau} into the uterine vein in d-15 pregnant ewes. Because ISG15 was shown to be specifically localized to large luteal cells, we suspect that conceptus-derived IFN-{tau} is released into the uterine vein and through an endocrine mechanism directly impacts CL function and survival at the level of the large luteal cell. It is tempting to speculate that IFN-{tau} directly disrupts large luteal cell production of PGF and oxytocin, redirects PG synthesis toward PGE2, or enhances degradation of intraluteal PGF. These concepts will be the focus of future experiments in our laboratories.

Pregnancy losses during early pregnancy significantly impact return to estrus in dairy and beef cattle. About 40% of early embryonic death occurs from d 15–17 of the estrous cycle (46). Results of the present study indicate that at least two ISG expressed in endometrium during early pregnancy are also expressed in other tissues, specifically blood and luteal cells. The expression of proteins related to the IFN pathway like Mx and ISG15 have previously been reported in sheep (24) and cattle (26). Additionally, Chen et al. (25) detected up-regulation of genes related to the IFN pathway in the CL after intrauterine infusion of IFN-{tau} or im injection of high IFN doses. Thus, use of ISG15 and OAS-1 as well as other ISG can be a valuable tool for the assessment of embryonic mortality as well as distinguishing pregnancy losses caused by death of embryos from failure of fertilization in cattle and sheep. They also might be used to identify nonpregnant ruminants so that they could be immediately resynchronized and mated.

However, the use of IFN-{tau} as an indicator of pregnancy status by collecting jugular or tail vein blood would be counterindicated based on the results in this study. The expression of ISG in extrauterine tissues and the demonstration of release of IFN-{tau} through the uterine vein suggest a novel endocrine mechanism for maternal recognition of pregnancy in ruminants.


   Acknowledgments
 
We thank Dr. Torrance Nett (Colorado State University) for providing some of the d 15 ovine CL tissues and Dr. Gordon Niswender (Colorado State University) for providing isolated large luteal cells for these experiments.


   Footnotes
 
This study was supported by National Research Initiative Competitive Grants 2006-35203-17258 (T.R.H.) and 2005-35203-15885 (R.V.A.) from the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service. Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil, supported J.F.O. during his sabbatical research at Colorado State University.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 6, 2007

1 J.F.O. and L.E.H. are co-first authors and contributed equally to the research. Back

Abbreviations: CL, Corpus luteum; FBS, fetal bovine serum; IFN, interferon; ISG, IFN-stimulated genes; OAS-1, 2',5'-oligoadenylate synthetase; PBMC, peripheral blood mononuclear cells; PG, prostaglandin; qRT-PCR, semiquantitative real-time PCR; ro, recombinant ovine.

Received June 27, 2007.

Accepted for publication November 29, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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