Transcriptomic Changes in Response to Form of Selenium on the Interferon-Tau Signaling Mechanism in the Caruncular Tissue of Beef Heifers at Maternal Recognition of Pregnancy.

We have reported that selenium (Se) provided to grazing beef cattle in an inorganic (ISe) form versus a 1:1 mixture (MIX) of inorganic and organic (OSe) forms affects cholesterol biosynthesis in the corpus luteum (CL), the abundance of interferon tau (IFNτ) and progesterone (P4)-induced mRNAs in the caruncular (CAR) tissue of the endometrium, and conceptus length at maternal recognition of pregnancy (MRP). In this study, beef heifers were supplemented with a vitamin-mineral mix containing 35 ppm Se as ISe or MIX to achieve a Se-adequate status. Inseminated heifers were killed at MRP (d 17, n = 6 per treatment) for tissue collection. In CAR samples from MIX versus ISe heifers, qPCR revealed that mRNA encoding the thyroid regulating DIO2 and DIO3 was decreased (p < 0.05) and a complete transcriptomic analysis revealed effects on the interferon JAK-STAT1/2 pathway, including decreased expression of mRNAs encoding the classical interferon stimulated genes IFIT1, IFIT2, IFIT3, IRF1, IRF9, ISG15, OAS2, and RSAD2 (p < 0.05). Treatment also affected the abundance of mRNAs contributing to the immunotolerant environment (p < 0.05). In combination, these findings suggest more advanced preparation of the CAR and developing conceptus for implantation and to evade immune rejection by the maternal system in MIX- vs. ISe-treated heifers.

Influence of form of selenium supplementation and tall fescue endophyte toxicity on growth performance, serum parameters, and tissue masses of grazing beef steers.

To test the hypothesis that average daily gain (ADG) and clinical parameters of steers grazing novel non-toxic (NTE) or toxic KY-31 (TE) endophyte-infected tall fescue would be improved by ad libitum intake of vitamin-mineral mixes (V-M) that contain 27 ppm Se as a 1:1 blend of SELPLEX:sodium selenite (MIX) vs. sodium selenite (ISe), 32 fescue-naïve beef steers partially depleted of Se were randomly assigned to ad libitum consumption ISe vs. MIX for 35 days and fed enough of a NTE/alfalfa/grain diet to achieve 0.57 kg BW gain/day. Then, within Se-form treatments, two steers were randomly assigned to each of four NTE (ISe = 316 ± 31 kg BW, MIX = 315 ± 22 kg BW) or TE (ISe = 316 ± 37 kg BW, MIX = 314 ± 39 kg BW) paddocks for 84 days and had ad libitum access to their respective V-M. The MIXED procedure of SAS was used to assess effects of day, Se-form (ISe, MIX) and endophyte (NTE, TE) treatments, and their interactions. Whole blood Se decreased (P < 0.01) 31% from days 0 to 84 and was 6.2% greater (P < 0.01) for MIX steers. Serum prolactin decreased (P < 0.01) 18% for NTE and 48% for TE steers from days 0 to 84 and was 17% greater (P = 0.01) for MIX vs. ISe for TE steers. Serum alkaline phosphatase activity decreased (P < 0.02) 27% from days 0 to 84 and was 15% greater (P < 0.02) for MIX steers. Serum urea nitrogen increased (P < 0.02) 8.2% from days 0 to 84 for TE but not NTE steers. Average daily gain was less (P < 0.01) for steers grazing TE (-0.18 kg/day) compared with NTE (0.09 kg/d). Although there was increased serum alkaline phosphatase activity and increased serum prolactin for TE + MIX steers compared with TE + ISe steers, MIX supplementation was unable to increase serum prolactin concentrations or ADG to the same levels as steers grazing NTE. Longer adaptation to MIX supplementation ad libitum may be necessary for maximal Se assimilation to restore serum prolactin levels in steers grazing TE.

Form of dietary selenium affects mRNA encoding cholesterol biosynthesis and immune response elements in the early luteal phase bovine corpus luteum.

Widespread regions of the southeast United States have soils, and hence forages, deficient in selenium (Se), necessitating Se supplementation to grazing cattle for optimal immune function, growth, and fertility. We have reported that supplementation with an isomolar 1:1 mix (MIX) of inorganic (ISe) and organic (OSe) forms of Se increases early luteal phase (LP) progesterone (P4) above that in cows on ISe alone. Increased early LP P4 advances embryonic development. Our objective was to determine the effect of form of Se on the transcriptome of the early LP corpus luteum (CL) with the goal of elucidating form of Se-regulated processes affecting luteal steroidogenesis and function. Non-lactating, 3-yr-old Angus-cross cows underwent 45-d Se-depletion, then repletion periods, and then at least 90 d of supplementation (TRT) with 35 ppm Se/d as either ISe (n = 5) or MIX (n = 5). CL were then recovered on day 7 of the estrous cycle, total RNA isolated, and the effect of TRT on the luteal transcriptome evaluated using bovine gene 1.0 ST arrays (Affymetrix, Inc., Santa Clara, CA). The abundance of transcripts in each CL was subjected to one-way ANOVA using Partek Genomic Suite software to determine TRT effects. Microarray analysis indicated a total of 887 transcripts that were differentially expressed and functionally annotated, with 423 and 464 up- and down-regulated (P < 0.05) in MIX vs. ISe CL, respectively. Bioinformatic analysis (Ingenuity Pathway Analysis) revealed the top TRT-affected canonical pathways to include seven specific to cholesterol biosynthesis and two to inflammatory responses. Results from the microarray analysis were corroborated by targeted real-time PCR. MIX CL had increased (P < 0.05) abundance of transcripts regulating cholesterol biosynthesis including DHCR7, DHCR24, and CYP51A1 (fold changes of 1.65, 1.48, and 1.40, respectively), suggesting MIX-induced increases in P4 to be due, in part, to increased availability of substrate to luteal cells. In addition, MIX CL had increased (P < 0.05) abundance of immune-response transcripts including C1QC, FAS, ILR8B, and IL1R1 (fold changes of 2.30, 1.74, 1.66, and 1.63, respectively). SREBF1 mRNA was also increased (1.32-fold, P < 0.05) in the MIX CL, which increases cholesterol synthesis and stimulates IL1B, linking effects of form of supplemental Se (TRT) on cholesterol biosynthesis and immune function in the CL.

In regions with soils deficient in selenium (Se), producers should supplement this trace mineral to the diet of forage-grazing cattle. We previously reported that circulating concentrations of progesterone (P4) are affected by the form of Se supplemented to cows. In this report, we aimed to determine how the form of Se affects the transcriptome of the bovine CL, with the goal of elucidating form of Se effects on luteal steroidogenesis. Cows were supplemented with the industry standard, an inorganic form of Se, or a 1:1 mix of organic and inorganic forms (MIX), with corpora lutea recovered on day 7 of the estrous cycle. The effect of TRT (form of Se) on the luteal transcriptome was then evaluated using bovine gene 1.0 ST arrays (Affymetrix, Inc., Santa Clara, CA), with the results of the microarray analysis corroborated by targeted qPCR. Treatment altered >800 transcripts in the CL, including those regulating cholesterol biosynthesis and immune function. The effect of form of Se on luteal steroidogenesis appears to be the result of changes in cholesterol biosynthesis and uptake by luteal cells, with cross talk between immune and cholesterol regulatory pathways also apparent.

Form of dietary selenium affects mRNA encoding interferon-stimulated and progesterone-induced genes in the bovine endometrium and conceptus length at maternal recognition of pregnancy.

Widespread regions of the southeast United States have soils, and hence forages, deficient in selenium (Se), necessitating Se supplementation to grazing cattle for optimal immune function, growth, and fertility. We have reported that supplementation with an isomolar 1:1 mix (MIX) of inorganic (ISe) and organic (OSe) forms of Se increases early luteal phase (LP) concentrations of progesterone (P4) above that in cows on ISe or OSe alone. Increased early LP P4 advances embryonic development. Our objective was to determine the effects of the form of Se on the development of the bovine conceptus and the endometrium using targeted real-time PCR (qPCR) on day 17 of gestation, the time of maternal recognition of pregnancy (MRP). Angus-cross yearling heifers underwent 45-d Se-depletion then repletion periods, then at least 90 d of supplementation (TRT) with 35 ppm Se per day as either ISe (n = 10) or MIX (n = 10). Heifers were inseminated to a single sire after detected estrus (day 0). On day 17 of gestation, caruncular (CAR) and intercaruncular (ICAR) endometrial samples and the developing conceptus were recovered from pregnant heifers (ISe, n = 6 and MIX, n = 6). qPCR was performed to determine the relative abundance of targeted transcripts in CAR and ICAR samples, with the expression data subjected to one-way ANOVA to determine TRT effects. The effect of TRT on conceptus development was analyzed using a one-tailed Student's t-test. When compared with ISe-treated heifers, MIX heifers had decreased (P < 0.05) abundance of several P4-induced and interferon-stimulated mRNA transcripts, including IFIT3, ISG15, MX1, OAS2, RSAD2, DGAT2, FGF2 in CAR and DKK1 in ICAR samples and tended (P ≤ 0.10) to have decreased mRNA abundance of IRF1, IRF2, FOXL2, and PGR in CAR samples, and HOXA10 and PAQR7 in ICAR samples. In contrast, MIX-supplemented heifers had increased (P < 0.05) mRNA abundance of MSTN in ICAR samples and an increase in conceptus length (ISe: 17.45 ± 3.08 cm vs. MIX: 25.96 ± 3.95 cm; P = 0.05). Notably, myostatin increases glucose secretion into histotroph and contributes to advanced conceptus development. This advancement in conceptus development occurred in the presence of similar concentrations of serum P4 (P = 0.88) and whole blood Se (P = 0.07) at MRP.

In regions with soils deficient in selenium (Se), it is recommended that this trace mineral is supplemented to the diet of forage-grazing cattle. We have previously reported that the form of Se supplemented to cattle affects the function of multiple tissues, including the testis, liver, ovary, and pituitary. The objective of this study was to determine how the form of Se supplemented to heifers to achieve a Se-adequate status affects endometrial function and development of the conceptus at maternal recognition of pregnancy (MRP). Heifers were supplemented with the industry standard, an inorganic form of Se (ISe), or a 1:1 mix of organic and inorganic forms (MIX), with the reproductive tract recovered on day 17 of pregnancy. Real-time PCR was performed to determine the relative abundance of targeted mRNA transcripts in caruncular (CAR) and intercaruncular (ICAR) endometrial samples. The form of supplemental Se affected the abundance of multiple progesterone-induced and interferon-stimulated mRNA transcripts in CAR and ICAR samples, as well as the length of the conceptus that was recovered at MRP (day 17). Overall, our results indicate differences in endometrial function and increased development of the conceptus in cattle provided with MIX vs. ISe, suggesting that the MIX form of supplemental Se may increase fertility in cattle grazing soils deficient in this trace mineral.

Form of Supplemental Selenium Affects the Expression of mRNA Transcripts Encoding Selenoproteins, and Proteins Regulating Cholesterol Uptake, in the Corpus Luteum of Grazing Beef Cows.

Selenium (Se)-deficient soils necessitate supplementation of this mineral to the diet of forage-grazing cattle. Functionally, Se is incorporated into selenoproteins, some of which function as important antioxidants. We have previously shown that the source of supplemental Se; inorganic (sodium selenite or sodium selenate; ISe), organic (selenomethionine or selenocysteine; OSe) or 1:1 mix of ISe and OSe (MIX), provided to Angus-cross cows affects concentrations of progesterone (P4) during the early luteal phase of the estrous cycle. In this study, we sought to investigate (1) the effect of form of Se on the expression of mRNA encoding selenoproteins in the corpus luteum (CL), and (2) whether this previously reported MIX-induced increase in P4 is the result of increased luteal expression of key steroidogenic transcripts. Following a Se depletion and repletion regimen, 3-year-old, non-lactating, Angus- cross cows were supplemented with either ISe as the industry standard, or MIX for at least 90 days, with the CL then retrieved on Day 7 post-estrus. Half of each CL was used for analysis of targeted mRNA transcripts and the remainder was dissociated for culture with select agonists. The expression of three selenoprotein transcripts and one selenoprotein P receptor was increased (p < 0.05), with an additional five transcripts tending to be increased (p < 0.10), in cows supplemented with MIX versus ISe. In cultures of luteal cells, hCG-induced increases in P4 (p < 0.05) were observed in CL obtained from ISe-supplemented cows. The abundance of steroidogenic transcripts in the CL was not affected by the form of Se, however, the abundance of mRNA encoding 2 key transcripts regulating cholesterol availability (Ldlr and Hsl) was increased (p < 0.05) in MIX-supplemented cows. Overall, the form of Se provided to cows is reported to affect the expression of mRNA encoding several selenoproteins in the CL, and that the form of Se-induced effects on luteal production of P4 appears to be the result of changes in cholesterol availability rather than a direct effect on the expression of steroidogenic enzymes within the CL.

Form of Supplemental Selenium in Vitamin-Mineral Premixes Differentially Affects Early Luteal and Gestational Concentrations of Progesterone, and Postpartum Concentrations of Prolactin in Beef Cows.

Soils with marginal to deficient levels of selenium (Se) are widespread in the northwest, northeast, and southeast US. Supplementation to the diet of forage-grazing beef cattle with a vitamin-mineral mix containing additional Se is recommended in these geographic regions. We have reported that the form of supplemental Se provided to Angus-cross beef cows can affect circulating levels of progesterone (P4) on day 6 of the estrous cycle, a time when increased P4 is known to promote fertility. The objectives of this study were to (1) confirm and expand upon our initial report that the form of Se provided to cows affects early luteal-phase concentrations of systemic P4, (2) determine the effects of the form of Se on concentrations of P4 during gestation, and (3) determine the effects of the form of Se on concentrations of prolactin (PRL) during lactation. Throughout this study, Angus-cross beef cows had ad libitum access to a vitamin-mineral mix containing 35 ppm of Se in either an inorganic form (ISe) or a 1:1 mix of inorganic and organic forms (MIX). We observed a MIX-induced increase (p = 0.006) in systemic concentrations of P4 on day 7 but not on days 4 or 10 of the estrous cycle, consistent with our earlier report. We observed a MIX-induced increase (p = 0.02) in the systemic concentration of P4 at months 1, 3, 5, and 7 of gestation, and a MIX-induced decrease (p < 0.05) in systemic concentrations of PRL at months 5 and 6 of lactation. In summary, the form of Se provided to cows can be manipulated to affect the early luteal phase and gestational concentrations of P4, and postpartum concentrations of PRL.

Forms of selenium in vitamin-mineral mixes differentially affect serum alkaline phosphatase activity, and serum albumin and blood urea nitrogen concentrations, of steers grazing endophyte-infected tall fescue.

The goal of this study was to test the hypothesis that sodium selenite (ISe), SEL-PLEX (OSe), vs. a 1:1 blend (MIX) of ISe and OSe in a basal vitamin-mineral mix would differentially affect serological and hepatic parameters of growing steers grazing toxic endophyte-infected tall fescue-mixed forage pasture. Predominately Angus steers (BW = 183 ± 34 kg) were randomly selected from herds of fall-calving cows grazing endophyte-infected tall fescue-mixed pasture and consuming vitamin-mineral mixes that contained 35 ppm Se as ISe, OSe, and MIX forms. Steers were weaned, depleted of Se for 98 d, and subjected to summer-long common grazing of an endophyte-infected tall fescue-mixed pasture (0.51 ppm total ergovaline + ergovalinine; 10.1 ha). Steers were assigned (n = 8 per treatment) to the same Se form treatments upon which they were raised. Se treatments were administered by daily top-dressing 85 g of vitamin-mineral mix onto 0.23 kg soyhulls, using in-pasture Calan gates. The PROC MIXED procedure of SAS was used to assess the effect of Se form treatments on serum parameters at day 0, 22, 43, 64, and 86. After slaughter, the effect of Se treatment on hepatic alkaline phosphatase (tissue nonspecific isoform, TNALP) mRNA, protein, and albumin protein content was assessed using the PROC GLM procedure of SAS. Fisher's protected LSD procedure was used to separate treatment means. Partial correlation analysis was used to evaluate the relationship among whole blood Se concentration and serum parameters, accounting for the effect of time. Across periods, MIX steers had more (P ≤ 0.04) serum albumin than OSe and ISe steers, respectively. However, the relative hepatic bovine serum albumin protein content was not affected (P = 0.28) by Se treatments. Serum alkaline phosphatase activity was greater (P ≤ 0.01) in MIX and OSe steers. Similarly, hepatic TNALP protein content in MIX steers was greater (P = 0.01) than ISe steers. Partial correlation analysis revealed that serum albumin, blood urea nitrogen, and alkaline phosphatase activity were correlated (r ≥ 0.23, P ≤ 0.02) with whole blood Se concentration. In summary, consumption of 3 mg Se/d as OSe or MIX forms of Se in vitamin-mineral mixes increased serum albumin concentration and alkaline phosphatase activity, the reduction of which is associated with fescue toxicosis. We conclude that the organic forms of Se ameliorated the depression of 2 of known serological biomarkers of fescue toxicosis.

Forms of selenium in vitamin-mineral mixes differentially affect the expression of genes responsible for prolactin, ACTH, and α-MSH synthesis and mitochondrial dysfunction in pituitaries of steers grazing endophyte-infected tall fescue.

The goal of this study was to test the hypothesis that sodium selenite (inorganic Se, ISe), SEL-PLEX (organic forms of Se, OSe), vs. a 1:1 blend (MIX) of ISe and OSe in a basal vitamin-mineral (VM) mix would differentially alter pituitary transcriptome profiles in growing beef steers grazing an endophyte-infected tall fescue (E+) pasture. Predominately Angus steers (BW = 183 ± 34 kg) were randomly selected from fall-calving cows grazing E+ pasture and consuming VM mixes that contained 35 ppm Se as ISe, OSe, or MIX forms. Steers were weaned, depleted of Se for 98 d, and subjected to summer-long common grazing of a 10.1 ha E+ pasture containing 0.51 ppm ergot alkaloids. Steers were assigned (n = 8 per treatment) to the same Se-form treatments on which they were raised. Selenium treatments were administered by daily top-dressing 85 g of VM mix onto 0.23 kg soyhulls, using in-pasture Calan gates. As previously reported, serum prolactin was greater for MIX (52%) and OSe (59%) steers vs. ISe. Pituitaries were collected at slaughter and changes in global and selected mRNA expression patterns determined by microarray and real-time reverse transcription PCR analyses, respectively. The effects of Se treatment on relative gene expression were subjected to one-way ANOVA. The form of Se affected the expression of 542 annotated genes (P < 0.005). Integrated pathway analysis found a canonical pathway network between prolactin and pro-opiomelanocortin (POMC)/ACTH/α-melanocyte-stimulating hormone (α-MSH) synthesis-related proteins and that mitochondrial dysfunction was a top-affected canonical pathway. Targeted reverse transcription-PCR analysis found that the relative abundance of mRNA encoding prolactin and POMC/ACTH/α-MSH synthesis-related proteins was affected (P < 0.05) by the form of Se, as were (P ≤ 0.05) mitochondrial dysfunction-related proteins (CYB5A, FURIN, GPX4, and PSENEN). OSe steers appeared to have a greater prolactin synthesis capacity (more PRL mRNA) vs. ISe steers through decreased dopamine type two receptor signaling (more DRD2 mRNA), whereas MIX steers had a greater prolactin synthesis capacity (more PRL mRNA) and release potential by increasing thyrotropin-releasing hormone concentrations (less TRH receptor mRNA) than ISe steers. OSe steers also had a greater ACTH and α-MSH synthesis potential (more POMC, PCSK2, CPE, and PAM mRNA) than ISe steers. We conclude that form of Se in VM mixes altered expression of genes responsible for prolactin and POMC/ACTH/α-MSH synthesis, and mitochondrial function, in pituitaries of growing beef steers subjected to summer-long grazing an E+ pasture.

Glutathione content and expression of proteins involved with glutathione metabolism differs in longissimus dorsi, subcutaneous adipose, and liver tissues of finished vs. growing beef steers.

Increased tissue redox state may result in sub-optimal growth. Our goal was to determine if glutathione (GSH) content and expression of proteins involved with GSH metabolism change in longissimus dorsi (LD), subcutaneous adipose (SA), and liver tissues of growing vs. finishing steer phenotypes. Tissues were taken from 16 Angus steers (BW = 209 ± 29.4 kg) randomly assigned (n = 8) to develop through Growing (final BW = 301 ± 7.06 kg) vs. Finished (final BW = 576 ± 36.9 kg) growth phases, and at slaughter had achieved different rib-eye area (REA) (53.2, 76.8 cm2), marbling scores (296, 668), and 12th rib adipose thickness (0.54, 1.73 cm), respectively (Amino Acids, doi:10.1007/s00726-018-2540-8). GSH content (mg/g wet tissue) was determined by a commercial assay and the relative content of target protein and mRNA in tissue homogenates was determined by Western blot and reverse-transcribed PCR analyses, respectively. The effect of growth phase (Finished vs. Growing) was assessed by ANOVA using the GLM procedure of SAS. The LD of Finished steers had more (P < 0.04) GSH (42%) and GSH synthesizing (GCLC, 61%; GCLM, 21%) and metabolizing (GPX1, 42%; GPX3, 73%; GGT1, 56%) enzymes, and less (P < 0.02) GPX2 (46%), EAAC1 (30%) and glutamine synthetase (GS) (28%), whereas GTRAP3-18 and ARL6IP1 did not differ (P > 0.57). Principal component analysis found that GSH content of LD was associated with REA and marbling score. The SA of Finished steers had less (P < 0.04) GSH (38%), GSH metabolizing (GPX4, 52%; GGT1, 71%) enzyme mRNA, and GTRAP3-18 (123%) and ARL6IP1 (43%), whereas the mRNA content of GSH-synthesizing enzymes and content of EAAC1 and GS did not differ (P > 0.32). The liver of Finished steers had less (P < 0.02) mRNA content of GSH synthesizing (GCLC, 39%; GSS 29%) and metabolizing (GPX1, 30%) enzymes, and more (P < 0.01) GSTM1 metabolizing enzyme (114%). The change in GSH content as steers fattened indicate an increased antioxidant capacity in the LD of Finished steers, and a decreased antioxidant capacity in SA, consistent with changes in enzyme and transporter expression. Changes in liver enzyme and transporter expression were consistent with no change in GSH content. The relationship of EAAC1 regulatory proteins (GTRAP3-18, ARL6IP1) to GSH, EAAC1, and GS content differs and changes as Growing steers develop into Finished phenotypes. These findings provide mechanistic insight into how antioxidant capacity occurs in tissues of economic and metabolic importance as cattle fatten.

Forms of selenium in vitamin-mineral mixes differentially affect serum prolactin concentration and hepatic glutamine synthetase activity of steers grazing endophyte-infected tall fescue.

The goal of this study was to test the hypothesis that sodium selenite (ISe), SEL-PLEX (OSe), vs. an 1:1 blend (MIX) of ISe and OSe in a basal vitamin-mineral (VM) mix would differentially affect metabolic parameters and performance of growing steers grazing toxic endophyte-infected tall fescue mixed forage (E+) pasture. Predominately-Angus steers (BW = 183 ± 34 kg) were randomly selected from herds of fall-calving cows grazing E+ pasture and consuming VM mixes that contained 35 ppm Se as ISe, OSe, and MIX forms. Steers were weaned, depleted of Se for 98 d, and subjected to summer-long common grazing of an E+ pasture (0.51 ppm total ergovaline per ergovalinine; 10.1 ha). Steers were assigned (n = 8 per treatment) to the same Se-form treatments upon which they were raised. Selenium treatments were administered by daily top-dressing 85 g of VM mix onto 0.23 kg soyhulls, using in-pasture Calan gates. The PROC MIXED procedure of SAS was used to assess effect of Se-form treatments on whole blood Se (ng/mL) and serum prolactin (ng/mL) at day 0, 22, 43, 64, and 86, and caudal arterial area (mm2) at day -7, 43, and 86. The effect of Se treatment on ADG (day 86), and liver glutamine synthetase (GS) mRNA, protein, and activity (nmol/mg wet tissue/min) were assessed using the PROC GLM procedure of SAS. Fisher's protected LSD procedure was used to separate treatment means. Whole blood Se increased (P < 0.01) for all treatments from day 0 to 22 and then did not change (P ≥ 0.17), and was greater (P ≤ 0.04) for MIX and OSe steers. Serum prolactin decreased (P < 0.01) over time and was greater (P < 0.05) for MIX and OSe steers. Liver GS mRNA content was 66% and 59% greater (P < 0.05) in MIX and OSe steers, respectively, than ISe steers. Liver GS protein content in MIX steers was 94% more (P < 0.01) than ISe steers. Moreover, MIX and OSe steers had 99% and 55% more (P ≤ 0.01) liver GS activity, respectively, than ISe steers. ADG was not affected (P = 0.36) by Se treatments. We conclude that consumption of 3 mg Se/d as OSe or MIX forms of Se in VM mixes increased 1) whole blood Se content, an indicator of greater whole-body Se assimilation; 2) serum prolactin, the reduction of which is a hallmark of fescue toxicosis; and 3) hepatic GS activity, indicating greater hepatic assimilation of acinar ammonia. However, 4) these positive effects on metabolic parameters were not accompanied by increased growth performance.

Effect of lipopolysaccharide on the expression of inflammatory mRNAs and microRNAs in the mouse oviduct.

Infection with Gram-negative bacteria is a major cause of aberrant inflammation in the oviduct; consequences can include tubal-based infertility and/or ectopic pregnancy. Understanding the inflammatory response is necessary for the development of novel treatment options that counter inflammation-induced infertility. The aim of the present study was to determine the effect of intraperitoneal (i.p.) administration of Escherichia coli-derived lipopolysaccharide (LPS) on the acute expression of inflammatory mRNAs and microRNAs (miRNAs) in the oviduct. On the day of oestrus, 6- to 8-week-old CD1 mice were injected i.p. with 0, 2 or 10µg LPS in 100µL phosphate-buffered saline. Mice were killed 24h later and the oviducts collected for gene expression analyses. The effect of treatment on the expression of mRNAs and miRNAs was evaluated by one-way analysis of variance (ANOVA), with treatment means of differentially expressed (P<0.05) transcripts separated using Scheffé's test. LPS treatment affected 49 of 179 targeted inflammatory mRNAs and 51 of 578 miRNAs (P<0.05). The identity of differentially expressed miRNAs predicted as regulators of chemokine and interleukin ligand mRNAs was then extracted using the microRNA.org database. The results of the present study indicate that systemic treatment with LPS induces a robust inflammatory response in the oviducts of mice, and identify key mRNAs and putative miRNAs modulating this effect.

Pituitary genomic expression profiles of steers are altered by grazing of high vs. low endophyte-infected tall fescue forages.

Consumption of ergot alkaloid-containing tall fescue grass impairs several metabolic, vascular, growth, and reproductive processes in cattle, collectively producing a clinical condition known as "fescue toxicosis." Despite the apparent association between pituitary function and these physiological parameters, including depressed serum prolactin; no reports describe the effect of fescue toxicosis on pituitary genomic expression profiles. To identify candidate regulatory mechanisms, we compared the global and selected targeted mRNA expression patterns of pituitaries collected from beef steers that had been randomly assigned to undergo summer-long grazing (89 to 105 d) of a high-toxic endophyte-infected tall fescue pasture (HE; 0.746 µg/g ergot alkaloids; 5.7 ha; n = 10; BW = 267 ± 14.5 kg) or a low-toxic endophyte tall fescue-mixed pasture (LE; 0.023 µg/g ergot alkaloids; 5.7 ha; n = 9; BW = 266 ± 10.9 kg). As previously reported, in the HE steers, serum prolactin and body weights decreased and a potential for hepatic gluconeogenesis from amino acid-derived carbons increased. In this manuscript, we report that the pituitaries of HE steers had 542 differentially expressed genes (P < 0.001, false discovery rate ≤ 4.8%), and the pattern of altered gene expression was dependent (P < 0.001) on treatment. Integrated Pathway Analysis revealed that canonical pathways central to prolactin production, secretion, or signaling were affected, in addition to those related to corticotropin-releasing hormone signaling, melanocyte development, and pigmentation signaling. Targeted RT-PCR analysis corroborated these findings, including decreased (P < 0.05) expression of DRD2, PRL, POU1F1, GAL, and VIP and that of POMC and PCSK1, respectively. Canonical pathway analysis identified HE-dependent alteration in signaling of additional pituitary-derived hormones, including growth hormone and GnRH. We conclude that consumption of endophyte-infected tall fescue alters the pituitary transcriptome profiles of steers in a manner consistent with their negatively affected physiological parameters.

Progesterone receptor and prostaglandins mediate luteinizing hormone-induced changes in messenger RNAs for ADAMTS proteases in theca cells of bovine periovulatory follicles.

Little is known about the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of extracellular proteases in ovarian follicles of non-rodent species, particularly in theca cells. In the present study, temporal changes in the abundance of mRNA encoding four ADAMTS subtypes and hormonal regulation of mRNA encoding two subtypes were investigated in theca interna cells during the periovulatory period in cattle. Gonadotropin-releasing hormone (GnRH) was injected into animals to induce a luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge, and follicles were obtained at 0 hr post-GnRH (preovulatory) or at 6, 12, 18, or 24 hr (periovulatory). ADAMTS1, -2, -7, and -9 transcript abundance was then determined in the isolated theca interna. ADAMTS1 and -9 mRNA levels were up-regulated at 24 hr post-GnRH, whereas ADAMTS2 mRNA was higher at 12-24 hr post-GnRH and ADAMTS7 mRNA increased transiently at 12 hr post-GnRH compared to other time points. Subsequent in vitro experiments using preovulatory theca interna (0 hr post-GnRH) showed that application of LH in vitro can mimic the effects of the gonadotropin surge on mRNAs encoding ADAMTS1 and -9 and that progesterone/progesterone receptor and/or prostaglandins may regulate the levels of mRNA encoding ADAMTS1 and -9 in theca interna, downstream of the LH surge. Time- and subtype-specific changes in ADAMTS mRNA abundance in vivo, and their regulation in vitro by hormones, indicate that ADAMTS family members produced by theca cells may play important roles in follicle rupture and the accompanying tissue remodeling in cattle. Mol. Reprod. Dev. 84: 55-66, 2017. © 2016 Wiley Periodicals, Inc.

Estrogen Receptor Alpha (ESR1)-Dependent Regulation of the Mouse Oviductal Transcriptome.

Estrogen receptor-α (ESR1) is an important transcriptional regulator in the mammalian oviduct, however ESR1-dependent regulation of the transcriptome of this organ is not well defined, especially at the genomic level. The objective of this study was therefore to investigate estradiol- and ESR1-dependent regulation of the transcriptome of the oviduct using transgenic mice, both with (ESR1KO) and without (wild-type, WT) a global deletion of ESR1. Oviducts were collected from ESR1KO and WT littermates at 23 days of age, or ESR1KO and WT mice were treated with 5 IU PMSG to stimulate follicular development and the production of ovarian estradiol, and the oviducts collected 48 h later. RNA extracted from whole oviducts was hybridized to Affymetrix Genechip Mouse Genome 430-2.0 arrays (n = 3 arrays per genotype and treatment) or reverse transcribed to cDNA for analysis of the expression of selected mRNAs by real-time PCR. Following microarray analysis, a statistical two-way ANOVA and pairwise comparison (LSD test) revealed 2428 differentially expressed transcripts (DEG's, P < 0.01). Genotype affected the expression of 2215 genes, treatment (PMSG) affected the expression of 465 genes, and genotype x treatment affected the expression of 438 genes. With the goal of determining estradiol/ESR1-regulated function, gene ontology (GO) and bioinformatic pathway analyses were performed on DEG's in the oviducts of PMSG-treated ESR1KO versus PMSG-treated WT mice. Significantly enriched GO molecular function categories included binding and catalytic activity. Significantly enriched GO cellular component categories indicated the extracellular region. Significantly enriched GO biological process categories involved a single organism, modulation of a measurable attribute and developmental processes. Bioinformatic analysis revealed ESR1-regulation of the immune response within the oviduct as the primary canonical pathway. In summary, a transcriptomal profile of estradiol- and ESR1-regulated gene expression and related bioinformatic analysis is presented to increase our understanding of how estradiol/ESR1 affects function of the oviduct, and to identify genes that may be proven as important regulators of fertility in the future.

Form of supplemental selenium fed to cycling cows affects systemic concentrations of progesterone but not those of estradiol.

In areas where soils are deficient in selenium (Se), dietary supplementation of this trace mineral directly to cattle is recommended. Selenium status affects fertility, and the form of Se supplemented to cows affects tissue-specific gene expression profiles. The objective of this study was to determine whether the form of Se consumed by cows would affect follicular growth and the production of steroids. Thirty-three Angus-cross cows that had ad libitum access of a mineral mix containing 35 ppm of Se in free-choice vitamin-mineral mixes as either inorganic (ISe), organic (OSe), or a 50/50 mix of ISe and OSe (MIX) for 180 days were used. After 170 days of supplementation, all cows were injected with 25-mg PGF2α to induce regression of the CL and then monitored for behavioral estrus (Day 0). From Day 4 to Day 8 after estrus, follicular growth was determined by transrectal ultrasonography. On Day 6, cows were injected with PGF2α (20 then 15 mg, 8-12 hours apart) to induce regression of the developing CL and differentiation of the dominant follicle of the first follicular wave into a preovulatory follicle. On Day 8, 36 hours after PGF2α (20 mg), the contents of the preovulatory follicle were aspirated by ultrasound-guided follicular puncture. Blood collected on Days 6 and 8 and follicular fluid collected on Day 8 was analyzed for concentrations of progesterone and estradiol. Form of Se supplemented to cows affected (P = 0.04) the systemic concentration of progesterone on Day 6, but not on Day 8. Form of Se did not affect the systemic concentration of estradiol on Day 6 or Day 8. Form of Se tended to affect (P = 0.07) the concentration of progesterone, but not that of estradiol, in the follicular fluid. Form of Se did not affect diameter of the dominant ovarian follicle on Days 4 to 6, but tended to affect (P = 0.08) the diameter of the preovulatory follicle on Day 8. Our results suggest that form of Se fed to cows affects the production of progesterone but not that of estradiol. Further investigation of organic Se-induced increases in progesterone and potentially the effects of increased progesterone on the establishment of pregnancy, especially in cows of lower fertility, is warranted.

Differential Expression of mRNA Encoding Cytokines and Chemokines in the Reproductive Tract after Infection of Mice with Chlamydia trachomatis.

Infection with Chlamydia trachomatis targets epithelial cells within the genital tract which respond by secreting chemokines and cytokines. Persistent inflammation can lead to fibrosis, tubal infertility and/or ectopic pregnancy; many infections are asymptomatic. Most studies have investigated the inflammatory response in the initial stages of infection, less is known about the later stages of infection, especially with a low, potentially asymptomatic, bacterial load. Our objective was to determine the inflammatory mediators involved in clearance of low-grade infection and the potential involvement in chronic inflammation. Six to eight week old C3H/HeJ mice were pretreated with 2.5 mg medroxyprogesterone acetate on day -10 and -3 before infection. Mice (n=3 for 28 d, n=3 for 35 d) were infected with 5 × 102 inclusion-forming units of C. trachomatis, serovar D; vaginal cultures were obtained weekly to monitor infection. Control mice (n=3 for 28 d, n=3 for 35 d) were sham infected. Mice were killed on day 28 (experiment 1) and day 35 (experiment 2) post-infection and vaginal tissue, uterine horns and oviducts collected for analysis of mRNAs encoding inflammatory cytokines and chemokines. Total RNA was isolated and a superarray analysis performed using mouse Cytokines and Chemokines PCR arrays (Qiagen, Valencia, CA). Statistical differences in gene expression were determined using a paired Students t-test. At 28 days after infection, the expression of mRNA encoding 6, 35 and 3 inflammatory genes differed from controls in vaginal, uterine and oviductal tissues, respectively (P<0.05). At 35 days after infection, the expression of mRNA encoding 16, 38 and 14 inflammatory genes differed from controls in vaginal, uterine and oviductal tissues, respectively (P<0.05). Understanding the mechanisms involved in the inflammatory response at later stages of infection should aid in the development of treatment options that minimize the development of asymptomatic, chronic inflammation-induced infertility.

Hepatic transcriptome profiles differ among maturing beef heifers supplemented with inorganic, organic, or mixed (50% inorganic:50% organic) forms of dietary selenium.

Selenium (Se) is an important trace mineral that, due to deficiencies in the soil in many parts of the USA, must be supplemented directly to the diet of foraging cattle. Both organic and inorganic forms of dietary Se supplements are available and commonly used, and it is known that Se form affects tissue assimilation, bioavailability, and physiological responses. However, little is known about the effects of form of dietary Se supplements on gene expression profiles, which ostensibly account for Se form-dependent physiological processes. To determine if hepatic transcriptomes of growing beef (Angus-cross) heifers (0.5 kg gain/day) was altered by form of dietary supplemental Se, none (Control), or 3 mg Se/day as inorganic Se (ISe, sodium selenite), organic (OSe, Sel-Plex®), or a blend of ISe and OSe (1.5 mg:1.5 mg, Mix) Se was fed for 168 days, and the RNA expression profiles from biopsied liver tissues was compared by microarray analysis. The relative abundance of 139 RNA transcripts was affected by Se treatment, with 86 of these with complete gene annotations. Statistical and bioinformatic analysis of the annotated RNA transcripts revealed clear differences among the four Se treatment groups in their hepatic expression profiles, including (1) solely and commonly affected transcripts; (2) Control and OSe profiles being more similar than Mix and ISe treatments; (3) distinct OSe-, Mix-, and ISe-Se treatment-induced "phenotypes" that possessed both common and unique predicted physiological capacities; and (4) expression of three microRNAs were uniquely sensitive to OSe, ISe, or Mix treatments, including increased capacity for redox potential induced by OSe and Mix Se treatments resulting from decreased expression of MiR2300b messenger RNA. These findings indicate that the form of supplemental dietary Se consumed by cattle will affect the composition of liver transcriptomes resulting, presumably, in different physiological capacities.

Why two endothelins and two receptors for ovulation and luteal regulation?

The ovary is a dynamic organ that undergoes cyclic structural and functional changes. Structurally, the internal architecture of the ovary constantly changes as follicles grow, rupture and transform into corpora lutea in a cyclical manner. Functionally, a variety of regulatory ovarian hormones are sequentially produced, and eggs are periodically released. As a highly vascularized organ, the ovarian structures and functions change in response to external stimuli that include but are not limited to pituitary gonadotropins. Following stimulation, the ovary synthesizes and releases autocrine and paracrine signals that play unique roles in regulating its function. Recent studies have identified endothelins as local regulators in the ovary that modulate multiple cyclic events, such as follicle growth, steroidogenesis, oocyte maturation, ovulation, corpus luteum formation and luteolysis. Interestingly, in all mammalian species examined to date, a common observation has been made: the ovary produces two pharmacologically similar endothelins (ET-1 and ET-2) but expresses two functionally different endothelin receptors (ET(A) and ET(B)) that often give rise to opposite physiological outcomes following activation by an endothelin. In this review, the physiological significance of the presence of the two ligand-two receptor endothelin system in the ovary will be discussed.

Hematopoetic prostaglandin D synthase: an ESR1-dependent oviductal epithelial cell synthase.

Oviductal disease is a primary cause of infertility, a problem that largely stems from excessive inflammation of this key reproductive organ. Our poor understanding of the mechanisms regulating oviductal inflammation restricts our ability to diagnose, treat, and/or prevent oviductal disease. Using mice, our objective was to determine the spatial localization, regulatory mechanism, and functional attributes of a hypothesized regulator of oviductal inflammation, the hematopoietic form of prostaglandin D synthase (HPGDS). Immunohistochemistry revealed specific localization of HPGDS to the oviduct's epithelium. In the isthmus, expression of HPGDS was consistent. In the ampulla, expression of HPGDS appeared dependent upon stage of the estrous cycle. HPGDS was expressed in the epithelium of immature and cycling mice but not in the oviducts of estrogen receptor α knockouts. Two receptor subtypes bind PGD2: PGD2 receptor and G protein-coupled receptor 44. Expression of mRNA for Ptgdr was higher in the epithelial cells (EPI) than in the stroma (P < 0.05), whereas mRNA for Gpr44 was higher in the stroma than epithelium (P < 0.05). Treatment of human oviductal EPI with HQL-79, an inhibitor of HPGDS, decreased cell viability (P < 0.05). Treatment of mice with HQL-79 increased mRNA for chemokine (C-C motif) ligands 3, 4, and 19; chemokine (C-X-C motif) ligands 11 and 12; IL-13 and IL-17B; and TNF receptor superfamily, member 1b (P < 0.02 for each mRNA). Overall, these results suggest that HPGDS may play a role in the regulation of inflammation and EPI health within the oviduct.

In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1, PGE2) prevent luteolysis in cows. II: mRNA for PGF2α, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4 prostanoid receptors in luteal tissue.

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every 4h inhibited luteolysis in ewes by altering luteal mRNA for luteinizing hormone (LH) receptors and unoccupied and occupied luteal LH receptors. However, estradiol-17ß or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in cows, but infusion of estradiol+PGE(2) inhibited luteolysis. In contrast, intra-luteal implants containing PGE(1) or PGE(2) in Angus or Brahman cows also inhibited the decline in circulating progesterone, mRNA for LH receptors, and loss of unoccupied and occupied receptors for LH to prevent luteolysis. The objective of this experiment was to determine how intra-luteal implants of PGE(1) or PGE(2) alter mRNA for prostanoid receptors and how this could influence luteolysis in Brahman or Angus cows. On day-13 Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Corpora lutea slices were analyzed for mRNA for prostanoid receptors (FP, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4) by RT-PCR. Day-13 Angus cow luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE(1) and PGE(2) up-regulated FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed. EP1 mRNA was not altered by any treatment. PGE(1) and PGE(2) down-regulated EP2 and EP4 mRNA compared to day-19 Vehicle controls regardless of breed. PGE(1) or PGE(2) up-regulated mRNA EP3B receptor subtype compared to day-19 Vehicle control cows regardless of breed. The similarities in relative gene expression profiles induced by PGE(1) and PGE(2) support their agonistic effects. We conclude that both PGE(1) and PGE(2) may prevent luteolysis by altering expression of mRNA for prostanoid receptors, which is correlated with changes in luteal mRNA for LH receptors reported previously in these same cows to prevent luteolysis.