Kathy J. Austin

Endometrial ISG17 mRNA and a related mRNA are induced by interferon-tau and localized to glandular epithelial and stromal cells from pregnant cows.

The interferon stimulated gene product, ISG17, conjugates to bovine uterine proteins in response to conceptus-derived interferon (IFN)-τ. The objectives of the present experiments were to examine induction of ISG17 (0.65 kb) and a related 2.5 kb mRNA in response to IFN-τ and pregnancy using Northern blotting procedures, and to determine cell types in the endometrium that expressed ISG17 mRNA usingin situ hybridization. RNA was isolated from endometrial explants or from bovine endometrial (BEND) cells cultured in the absence (control) or presence of 25 nM recombinant (r) bolFN-τ for 0, 3, 6, 12, 24, or 48 h. The major ISG17 0.65 kb mRNA and a minor 2.5 kb mRNA were induced (p<0.05) after 6 h (explants) or 3 h (BEND cells) treatment with rbolFN-τ. Both mRNAs were present in endometrium from day 18 pregnant cows, but were absent in endometrium from nonpregnant cows. The ISG17 mRNA was localized to stromal and glandular epithelial cells on d 18 of pregnancy. The 2.5 kb mRNA may encode a novel ISG17 homolog, or a unique polyISG17 repeat that is similar in structure to the polyubiquitin genes. Because ISG17 mRNA is induced in stromal and glandular epithelial cells, it could be assumed that ISG17 has a role in regulating intracellular proteins in both cell types.

Pregnancy and Interferon-τ Upregulate Gene Expression of Members of the 1-8 Family in the Bovine Uterus

Abstract The 1-8 family (1-8U, 1-8D, Leu-13/9-27) of interferon (IFN)-inducible genes encodes proteins that are components of multimeric complexes involved with transduction of antiproliferative and homotypic adhesion signals. Human 1-8 family members are highly similar and are regulated by type 1 and type 2 IFNs. Because the bovine uterus is bathed in conceptus-derived IFNτ during early pregnancy, it was hypothesized that members of the 1-8 family were upregulated in the bovine uterus during early pregnancy. Oligonucleotide primers were designed based on human and rat 1-8U and Leu-13 cDNAs and used in reverse transcription polymerase chain reactions to amplify bovine cDNAs from endometrial RNA. The bovine 1-8U cDNA was sequenced, found to be 84% identical to the human 1-8U, and used to screen a bovine endometrial cDNA library to isolate the full-length 1-8U and Leu-13 cDNAs. The inferred amino acid sequences of bovine 1-8U and Leu-13 were 72% and 73% identical to their respective human counterparts. Bovine 1-8U and Leu-13 retain an amino acid motif that is conserved in other 1-8 family members and in some ubiquitin-conjugating enzymes (E2s). This motif is critical for function of E2s in covalently linking ubiquitin to targeted proteins. Northern blotting revealed that bovine endometrial 1-8U and Leu-13 mRNAs were upregulated on Day 15 of pregnancy (P < 0.0001) and continued to accumulate through Day 18 of pregnancy (P < 0.05) when compared with endometrium from nonpregnant cows. The bovine 1-8U and Leu-13 mRNAs were also upregulated (P < 0.05) by IFNτ (25 nM) within 3 h, continued to accumulate through 12 h, and reached a plateau at 12–24 h in cultured bovine endometrial cells. In situ hybridization revealed that mRNAs encoding 1-8 family members were heavily localized to glandular epithelium but also were present to a lesser extent in the luminal epithelium and stroma. The temporal upregulation of 1-8U and Leu-13 mRNAs by pregnancy and IFNτ and tissue distribution of these mRNAs paralleled closely that of the ubiquitin homolog, IFN-stimulated gene product 17. These IFN-induced proteins probably work together to prepare the endometrium for adhesion of the developing conceptus.

Bovine granulocyte chemotactic protein-2 is secreted by the endometrium in response to interferon-tau (IFN-τ).

Interferon-tau (IFN-τ) is secreted by the bovine conceptus and may regulate synthesis of uterine endometrial cytokines to provide an environment that is conducive to embryo development and implantation. Interferon-τ stimulates secretion of an 8-kDa uterine protein (P8) in the cow. P8 was purified, digested to yield internal peptides, and partially sequenced to determine identity. Two internal peptides had 100% (13-mer) and 92% (12-mer) amino acid sequence identity with bovine granulocyte chemotactic protein-2 (bGCP-2). Bovine GCP-2 is an α-chemokine that acts primarily as a potent chemoattractant for granulocyte cells of the immune system. A peptide was synthesized based on a region of bGCP-2 that overlapped with a P8 peptide amino acid sequence, coupled to keyhole limpet hemocyanin, and used to generate high titer polyclonal antiserum in sheep. Western blots revealed that bGCP-2 was not released by endometrium from day 14 nonpregnant cows, but was released in response to 25 nM IFN-τ (p<0.05). Uterine GCP-2 exhibited high affinity to heparin agarose, a characteristic shared by all α chemokines. This is the first report describing presence of GCP-2 in the uterine endometrium and regulation by IFN-τ. The regulation of bGCP-2 by IFN-τ may have important implications for cytokine networking in the uterus during pregnancy. Also, the regulation of inflammation and angiogenesis by bGCP-2 working together with other cytokines may be integral to establishing early pregnancy and implantation in the cow.

Effect of protein supplementation on expression and distribution of urea transporter-B in lambs fed low-quality forage

Two experiments were conducted to determine the effects of ruminal protein degradability, supplementation frequency, and increasing dietary protein on the expression and distribution of urea transporter-B (UT-B) in lambs fed low-quality forage (mature crested wheatgrass hay; 4.2 to 4.7% CP). In Exp. 1, 15 Dorset wether lambs (initial BW=45.8+/-1.3 kg) were blocked by initial BW and assigned to 1 of 3 treatments within a randomized complete block design for 28 d, with supplements fed to achieve 7, 10, or 13% total dietary CP. In Exp. 2, 13 Dorset wether lambs (initial BW=34+/-4 kg) were used in a completely randomized design and given 1 of 4 isonitrogenous supplements: 1) ruminally degradable protein (RDP) fed daily (n=3), 2) RDP fed on alternate days (n=3), 3) ruminally undegradable protein (RUP) fed on alternate days (n=3), or 4) a 50:50 mixture of RDP and RUP fed on alternate days (n=4) for 18 d. Alternate-day treatments were fed at twice that of daily supplementation. On the last day of both experiments, lambs were killed and samples taken for Western blot analyses for UT-B. Immunoblotting using a rabbit polyclonal antibody to UT-B confirmed the presence of distinct 32-kDa (consistent with a nonglycosylated UT-B protein) and 47-kDa (probable N-glycosylated form of UT-B) protein bands in all 9 tissues analyzed. In both experiments, the liver, dorsal rumen, reticulum, and ventral rumen displayed strong bands at 32 kDa and lighter bands at 47 kDa, whereas the cecum, large colon, spiral colon, and parotid salivary gland displayed slight 32-kDa bands and stronger, more visible bands at 47 kDa. Both protein bands were apparent in the kidney at similar visual intensities in Exp. 1, whereas the relative intensities of the 2 UT-B bands in the kidney were variable, and appeared somewhat reciprocal among animals in Exp. 2. Although the abundance of the 47-kDa UT-B band in the ventral rumen was greater (P=0.03) in lambs fed RDP daily in Exp. 2, no other treatment differences (P >or= 0.15 to 0.99) in the abundance of the 32- or 47-kDa UT-B proteins within tissues were observed in either experiment. Although protein supplementation strategy had little effect on UT-B expression in tissues other than the ventral rumen, differences in the degree of glycosylation of UT-B across tissues may provide insight into its regulation.

Effects of dietary aflatoxin on the hepatic expression of apoptosis genes in growing barrows.

The most common and toxic form of aflatoxin, aflatoxin B(1) (AFB(1)), is produced by molds growing on crops. Use of moldy corn can result in high concentrations of AFB(1) in swine diets, which could potentially lead to an increased incidence of aflatoxicosis, a disease associated with decreased health and performance through reduced feed intake, reduced BW gain, and impaired liver function. The objective of this study was to determine the effects of AFB(1) on the hepatic gene expression of growing barrows. Ninety Duroc × Yorkshire crossbred barrows (age = 35 ± 5 d; initial BW = 14.2 ± 3.0 kg) were allocated to 9 pens with 10 pigs per pen, and randomly assigned in a 3 × 3 factorial arrangements of treatments to receive diets containing 0 µg/kg of AFB(1), 250 µg/kg of AFB(1), or 500 µg/kg of AFB(1) for 7, 28, or 70 d. Because performance was most affected in animals administered AFB(1) for an extended period, liver samples from d 70 animals were used for RNA-sequencing analysis. Of 82,744 sequences probed, 179 had transcripts that were highly correlated (r ≥ |0.8|; P < 0.0001) with treatment. Of the 179 significant transcripts, 46 sequences were negatively and 133 sequences positively related to treatment. Forty-three unique functional groups were identified. Genes within the apoptosis regulation functional group were selected for 1) confirmation of d 70 gene expression differences using real-time reverse-transcription (RT)-PCR (n = 4 genes), and 2) investigation of d 7 expression to identify early responses to AFB(1) (n = 15 genes) using real-time RT-PCR. Expression of the 4 apoptosis genes selected for confirmation, cyclin-dependent kinase inhibitor 1A, zinc finger matrin type 3, kininogen 1, and pim-1 oncogene, was confirmed with real-time RT-PCR. Of the 15 genes tested in d 7 liver samples, 4 were differentially expressed: cyclin-dependent kinase inhibitor 1A; zinc finger matrin type 3; tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; and apoptosis enhancing nuclease. Results from this study demonstrate that administration of an AFB(1)-contaminated diet to growing barrows alters hepatic gene expression, and in particular apoptosis genes.

Interferon-Tau Suppresses Prostaglandin F2α Secretion Independently of the Mitogen-Activated Protein Kinase and Nuclear Factor κ B Pathways

Abstract Pregnancy is established in ruminants through inhibitory actions of interferon (IFN)-τ on the release of prostaglandin F2α (PGF), which allows the corpus luteum to survive and continue to produce progesterone. Experiments were designed to 1) delineate the signal transduction pathway coordinating the synthesis of PGF, 2) determine how rapidly recombinant bovine (rb) IFN-τ attenuated phorbol ester (PDBu)-induced secretion of PGF, and 3) establish the site at which rbIFN-τ attenuates the secretion of PGF in cultured bovine endometrial (BEND) cells. BEND cells were untreated (control) or treated for 5, 10, 60, 180, or 300 min with PDBu (100 ng/ml), rbIFN-τ (50 or 500 ng/ml), PDBu + rbIFN-τ, or PDBu + PD98059 (MEK-1 inhibitor; 50 μM). Secretion of PGF was induced (P < 0.0001) by PDBu within 180 min, but induction was inhibited 74% by the addition of rbIFN-τ (P < 0.0001) and was ablated completely by PD98059. Parallel results were obtained for cyclooxygenase (COX)-2 protein expression. PDBu induced (P < 0.05) activation of the Raf-1/MEK-1/ERK-1/2 pathway, which was obligatory for the expression of COX-2 and secretion of PGF but was not altered by cotreatment with rbIFN-τ. PDBu induced (P < 0.05) transcription of c-jun and c-fos mRNAs within 30 min; induction was inhibited (P < 0.05) by cotreatment with PD98059 but not by cotreatment with rbIFN-τ. Treatment of BEND cells with rbIFN-τ also did not attenuate PDBu-induced degradation of IκBα, suggesting that the IκBα/NFκB pathway is not a site of IFN-τ inhibition of PGF. However, rbIFN-τ did block transcription of the COX-2 gene induced by PDBu within 30 min. In conclusion, COX-2 expression and PGF secretion induced by PDBu is mediated through the Raf-1/MEK-1/ERK-1/2 pathway, but this pathway is not disrupted by rbIFN-τ. Because rbIFN-τ inhibits COX-2 mRNA within 30 min, we hypothesized that transcription factors activated by rbIFN-τ rapidly and directly attenuate COX-2 gene expression, thereby suppressing secretion of PGF.

Effects of high-sulfur water and clinoptilolite on health and growth performance of steers fed forage-based diets

Sulfur-induced polioencephalomalacia (sPEM), a neurological disorder affecting ruminants, is associated with consumption of diets with increased S (high-S). High-S water is commonly found in many western states and is a major source of dietary S for grazing cattle. Consumption of high-S water has been associated with sPEM and decreased performance. Identification of a feed supplement that would counteract the negative effects of high-S water would decrease the incidence of sPEM and prevent performance reductions in regions with problematic water sources. The objectives of this study were to 1) determine the effects of administering high-S drinking water to forage-fed feedlot steers on health and performance, and 2) determine the effectiveness of clinoptilolite, a clay mineral with increased cation-exchange capacity, in negating the effects of high-S drinking water. Yearling steers (n = 96; 318.2 +/- 2.1 kg of BW) were randomly assigned to 1 of 4 treatments for a 77-d trial period: control with low-S water (566 mg of SO(4)/L), high-S water (3,651 mg of SO(4)/L), or high-S water plus clinoptilolite supplemented at 2.5 or 5.0% of the diet DM. Feed and water consumption were measured daily, and all steers were weighed on d -2, -1, 29, 53, 76, and 77. Plasma samples were collected on d 0, 58, and 77, and liver samples on d 0 and 77. There was a greater (P <or= 0.046) frequency of sPEM in high-S steers than control steers, but no differences among high-S treatment groups. In total, 12 cases of sPEM were confirmed by the presence of cortical lesions in steers consuming high-S water. Daily DMI (P = 0.002) and daily water intake (P = 0.001) were less in high-S water steers than control steers. No differences (P >or= 0.546) in ADG or G:F were observed. Plasma Cu decreased (P = 0.029) to a greater magnitude in high-S water steers than the control steers over the 77-d trial period. Mineral analyses of hepatic tissue from randomly selected healthy steers from each treatment group (n = 10 per treatment) showed an interaction (P <or= 0.034) of sample time and treatment for Cu, Se, and Zn concentrations. These results suggest that clinoptilolite does not negate the effects of high-S water, and administration of high-S water decreases herd health through an increased incidence of sPEM and reduced nutritional status.

Effects of dietary aflatoxin on the health and performance of growing barrows.

Aflatoxins, especially aflatoxin B1 (AFB1), can be greater in dried distillers grains with solubles (DDGS) because it can be concentrated during the ethanol production process. Increased use of DDGS in swine diets could potentially lead to an increased incidence of aflatoxicosis, a disease associated with decreased feed intake, reduced BW gain, and impaired liver function. The objective of this study was to determine the effects of AFB1 on the health, performance, and serum profile of growing barrows. Ninety Duroc × Yorkshire crossbred barrows were purchased (age = 35 ± 5 d; BW = 14.2 ± 3.0 kg), allocated to 9 pens with 10 pigs per pen, and randomly assigned to receive diets containing 0 µg/kg of AFB1 (CON), 250 µg/kg of AFB1 (LO), or 500 µg/kg of AFB1 (HI) for 7, 28, or 70 d in a 3 × 3 factorial arrangement of treatments. Feed intake was measured daily, and pigs were weighed and blood samples collected weekly. Serum was analyzed for concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (BILI), and blood urea nitrogen (BUN). Both ADFI and ADG were negatively affected (P ≤ 0.001) by AFB1 treatment. Average daily feed intake was less (P < 0.05) in HI barrows than in CON barrows from wk 5 to 10 and was less (P < 0.05) in LO barrows than in CON barrows in wk 5 and again from wk 8 to 10. Also, ADFI was less (P = 0.022) in HI barrows than LO barrows in wk 10. Decreased ADG (P < 0.05) was observed in HI barrows than in CON barrows in wk 8 and 10; no differences (P ≥ 0.665) in ADG were noted between CON and LO barrows. There was no effect (P ≥ 0.080) of AFB1 treatment on ALT or BILI concentrations. However, both AST and BUN were affected (P < 0.05) by AFB1 treatment. Serum AST was greater (P = 0.010) in LO barrows than CON barrows in wk 5, and serum BUN was greater (P = 0.004) in CON barrows than LO barrows in wk 3. Results from this study demonstrate that the performance and health of young growing barrows were affected by consumption of an AFB1-contaminated diet, especially when fed for a more extended period.

Complementary deoxyribonucleic acid sequence encoding bovine ubiquitin cross-reactive protein : A comparison with ubiquitin and a 15-kDa ubiquitin homolog.

Pregnancy in the cow depends on secretion of interferon-tau (IFN-τ) by the conceptus (trophoblast and embryo) and the actions of this cytokine on the uterine endometrium. A novel 17-kDa uterine protein that is regulated by IFN-τ during early pregnancy and crossreacts with ubiquitin antiserum on Western blots, has been named bovine ubiquitin cross-reactive protein (bUCRP). We suspected that bUCRP might be structurally related to ubiquitin, and to a human UCRP (ISG15 product) that has been described in several cell lines to be regulated by Type I IFNs. In this study, immunoscreening of a bovine endometrial cDNA library with ubiquitin antiserum resulted in the isolation of cDNAs encoding bUCRP. Nucleotide sequence of the bUCRP cDNA shared 70% identity with hUCRP and 30% identity with a tandem ubiquitin repeat. Computer translation revealed that bUCRP shared the Leu-Arg-Gly-Gly (LRGG) C-terminal sequence with ubiquitin and hUCRP that has been implicated in the modulation of intracellular proteins. However, some ubiquitin residues known to function in the ligation (Arg-54) to targeted proteins and in the processing of conjugates through the proteasome (His-68), have been lost through mutation in bUCRP. Lys-48, known to function in formation of ubiquitin polymers, was present in hUCRP, but mutated to Arg in bUCRP. Because bUCRP is secreted and retains the LRGG sequence, it may have both intracellular and secreted endocrine roles in maintaining pregnancy. Bovine UCRP also may have very different intracellular roles when compared with ubiquitin and hUCRP because of mutations in residues known to form polymers and to target proteins to degradation.

Diet Alters Both the Structure and Taxonomy of the Ovine Gut Microbial Ecosystem

We surveyed the ruminal metagenomes of 16 sheep under two different diets using Illumina pair-end DNA sequencing of raw microbial DNA extracted from rumen samples. The resulting sequence data were bioinformatically mapped to known prokaryotic 16S rDNA sequences to identify the taxa present in the samples and then analysed for the presence of potentially new taxa. Strikingly, the majority of the microbial individuals found did not map to known taxa from 16S sequence databases. We used a novel statistical modelling approach to compare the taxonomic distributions between animals fed a forage-based diet and those fed concentrated grains. With this model, we found significant differences between the two groups both in the dominant taxa present in the rumen and in the overall shape of the taxa abundance curves. In general, forage-fed animals have a more diverse microbial ecosystem, whereas the concentrate-fed animals have ruminal systems more heavily dominated by a few taxa. As expected, organisms from methanogenic groups are more prevalent in forage-fed animals. Finally, all of these differences appear to be grounded in an underlying common input of new microbial individuals into the rumen environment, with common organisms from one feed group being present in the other, but at much lower abundance.