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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.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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.

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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.

The objective of this study was to compare conception rates of female beef cattle inseminated at a fixed-time with either conventional (CON) or SexedUltra™ sex-sorted (SU) semen. Treatments included CON or SU with two sires represented within each treatment. Cows (n = 316) and heifers (n = 78) from six locations were randomly assigned treatment. Ovulation was synchronized in all females using the industry-standard 7-d CO-Synch + controlled internal drug release (CIDR) protocol (100 μg GnRH + CIDR [1.38 g progesterone] on d 0, 25 mg PGF2α at CIDR removal on d 7, and 100 μg GnRH on d 10, 54 h (heifers) or 60 h (cows) after CIDR removal). Estrotect™ estrous detection aids were applied at CIDR removal and patch activation was recorded at insemination. Animals were assumed estrual if greater than 50% of the patch coating was removed. The results from this study indicated no main effects of treatment (P = 0.82), sire (P = 0.64), or age (P=0.8) on AI conception rates. Additionally, there were not significant interactions between sire and treatment (P=0.19) or age and treatment (P=0.29). There was however, a significant (P=0.0005) effect of estrous expression on conception rates. Conception rate for estrual females (62.8%) was greater (p=0.0001) than non-estrual females (38.7%) at FTAI regardless of treatment. Furthermore, the conception rates were similar (P = 0.61) between conventional (61.9%) and sex-sorted semen (63.8%) when estrus was expressed prior to FTAI. Larger studies are warranted to determine appropriate timing of insemination with sex-sorted semen in FTAI protocols to maximize pregnancy potential., (Published by Elsevier Inc.)

Hepatic glutamate uptake and conversion to glutamine is critical for whole-body N metabolism, but how this process is regulated during growth is poorly described. The hepatic glutamate uptake activities, protein content of system [Formula: see text] transporters (EAAC1, GLT-1) and regulatory proteins (GTRAP3-18, ARL6IP1), glutamine synthetase (GS) activity and content, and glutathione (GSH) content, were compared in liver tissue of weaned Angus steers randomly assigned (n = 8) to predominantly lean (growing) or predominantly lipid (finished) growth regimens. Steers were fed a cotton seed hull-based diet to achieve final body weights of 301 or 576 kg, respectively, at a constant rate of growth. Liver tissue was collected at slaughter and hepatic membranes fractionated. Total (75%), Na + -dependent (90%), system [Formula: see text]-dependent (abolished) glutamate uptake activity, and EAAC1 content (36%) in canalicular membrane-enriched vesicles decreased as steers developed from growing (n = 6) to finished (n = 4) stages, whereas Na + -independent uptake did not change. In basolateral membrane-enriched vesicles, total (60%), Na + -dependent (60%), and Na + -independent (56%) activities decreased, whereas neither system [Formula: see text]-dependent uptake nor protein content changed. EAAC1 protein content in liver homogenates (n = 8) decreased in finished vs. growing steers, whereas GTRAP3-18 and ARL6IP1 content increased and GLT-1 content did not change. Concomitantly, hepatic GS activity decreased (32%) as steers fattened, whereas GS and GSH contents did not differ. We conclude that hepatic glutamate uptake and GS synthesis capacities are reduced in livers of finished versus growing beef steers, and that hepatic system [Formula: see text] transporter activity/EAAC1 content is inversely proportional to GTRAP3-18 content.

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.

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.

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.

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., (© 2016 Wiley Periodicals, Inc.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

In areas where soils are deficient in Selenium (Se), dietary supplementation of this trace mineral directly to cattle is recommended. Because Se status affects testosterone synthesis and frequency of sperm abnormalities, and the form of Se supplemented to cows affects tissue-specific gene expression, the objective of this study was to determine whether the form of Se consumed by cows during gestation would affect the expression of mRNAs that regulate steroidogenesis and/or spermatogenesis in the neonatal calf testis. Twenty-four predominantly Angus cows were assigned randomly to have individual, 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), starting 4 months prior to breeding and continuing throughout gestation. Thirteen male calves were born over a 3-month period (ISe, n = 5; OSe, n = 4; MIX, n = 4), castrated within 2 days of birth, and extracted testis RNA subjected to transcriptomal analysis by microarray (Affymetrix Bovine 1.0 ST arrays) and targeted gene expression analysis by real-time reverse-transcription PCR (RT-PCR) of mRNAs encoding proteins known to affect steroidogenesis and/or spermatogenesis. The form of dam Se affected (P < 0.05) the expression of 853 annotated genes, including 17 mRNAs putatively regulating steroidogenesis and/or spermatogenesis. Targeted RT-PCR analysis indicated that the expression of mRNA encoding proteins CYP2S1 (cytochrome P450, family 2, subfamily S, polypeptide 1), HSD17B7 (hydroxysteroid (17β) dehydrogenase 7), SULT1E1 (sulfotransferase family 1E, estrogen preferring, member 1), LDHA (lactate dehydrogenase A), CDK5R1 (cyclin-dependent kinase 5, regulatory subunit 1), and LEP (leptin) was affected (P < 0.05) by form of Se consumed by dams of developing bull calves, while AKR1C4 (aldo-keto reductase family 1, member C4) and CCND2 (cyclin D2) tended (P < 0.09) to be affected. Our results indicate that form of Se fed to dams during gestation affected the transcriptome of the neonatal calf testis. If these profiles are maintained throughout maturation, then the form of Se fed to dams may impact bull fertility and the development of Se form-dependent mineral mixes that target gestational development of the testis are warranted.

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 × 10 2 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.

Aged beef cows (≥ 8 yr of age) produce calves with lower birth and weaning weights. In mammals, aging is associated with reduced hepatic expression of glutamine synthetase (GS) and alanine transaminase (ALT), thus impaired hepatic Gln-Glu cycle function. To determine if the relative protein content of GS, ALT, aspartate transaminase (AST), glutamate transporters (EAAC1, GLT-1), and their regulating protein (GTRAP3-18) differed in biopsied liver tissue of (a) aged vs. young (3 to 4 yr old) nonlactating, nongestating Angus cows (Exp. 1 and 2) and (b) aged mixed-breed cows with and without COMPUDOSE (17β-estradiol) ear implants (Exp. 3), Western blot analyses were performed. In Exp. 1, 12 young (3.62 ± 0.01 yr) and 13 aged (10.08 ± 0.42 yr) cows grazed the same mixed forage for 42 d (August-October). In Exp. 2, 12 young (3.36 ± 0.01 yr) and 12 aged (10.38 ± 0.47 yr) cows were individually fed (1.03% of BW) a corn-silage-based diet to maintain BW for 20 d. For both Exp. 1 and 2, the effect of cow age was assessed by ANOVA using the MIXED procedure of SAS. Cow BW did not change ( ≥ 0.17). Hepatic ALT (78% and 61%) and GS (52% and 71%) protein content (Exp. 1 and 2, respectively) was decreased ( ≤ 0.01), whereas GTRAP3-18 (an inhibitor of EAAC1 activity) increased ( ≤ 0.01; 170% and 136%) and AST, GLT-1, and EAAC1 contents did not differ ( ≥ 0.17) in aged vs. young cows. In Exp. 2, free concentrations (nmol/g) of Glu, Ala, Gln, Arg, and Orn in liver homogenates were determined. Aged cows tended to have less ( = 0.10) free Gln (15.0%) than young cows, whereas other AA concentrations did not differ ( 0.26). In Exp. 3, 14 aged (> 10 yr) cows were randomly allotted ( = 7) to sham or COMPUDOSE (25.7 mg of 17β-estradiol) implant treatment (TRT), and had ad libitum access to alfalfa hay for 28 d. Blood and liver biopsies were collected 14 and 28 d after implant treatment. Treatment, time after implant (DAY), and TRT × DAY effects were assessed by ANOVA using the MIXED procedure of SAS. Cow BW was not affected ( ≥ 0.96). Implant increased ( ≤ 0.02) total plasma estradiol by 220% (5.07 vs. 1.58 pg/mL) and GS protein by 300%, whereas the relative content of other proteins was not altered ( ≥ 0.16). We conclude that hepatic expression of ALT and GS are reduced in aged vs. young cows, and administration of 17β-estradiol to aged cows increases plasma estradiol and hepatic GS, but not that of other proteins that support hepatic Glu metabolism.

Background: Reproductive success depends on a functional oviduct for gamete storage, maturation, fertilization, and early embryonic development. The ovarian-derived steroids estrogen and progesterone are key regulators of oviductal function. The objective of this study was to investigate luteal and follicular phase-specific oviductal epithelial cell function by using microarray-based transcriptional profiling, to increase our understanding of mRNAs regulating epithelial cell processes, and to identify novel genes and biochemical pathways that may be found to affect fertility in the future., Methods: Six normally cycling Angus heifers were assigned to either luteal phase (LP, n = 3) or follicular phase (FP, n = 3) treatment groups. Heifers in the LP group were killed between day 11 and 12 after estrus. Heifers in the FP group were treated with 25 mg PGF2α (Lutalyse, Pfizer, NY) at 8 pm on day 6 after estrus and killed 36 h later. Transcriptional profiling by microarray and confirmation of selected mRNAs by real-time RT-PCR analyses was performed using total RNA from epithelial cells isolated from sections of the ampulla and isthmus collected from LP and FP treatment groups. Differentially expressed genes were subjected to gene ontology classification and bioinformatic pathway analyses., Results: Statistical one-way ANOVA using Benjamini-hochberg multiple testing correction for false discovery rate (FDR) and pairwise comparison of epithelial cells in the ampulla of FP versus LP groups revealed 972 and 597 transcripts up- and down-regulated, respectively (P < 0.05). Within epithelial cells of the isthmus in FP versus LP groups, 946 and 817 transcripts were up- and down-regulated, respectively (P < 0.05). Up-regulated genes from both ampulla and isthmus were found to be largely involved in cholesterol biosynthesis and cell cycle pathways, while down-regulated genes were found in numerous inflammatory response pathways., Conclusions: Microarray-based transcriptional profiling revealed phase of the cycle-dependent changes in the expression of mRNA within the epithelium of the oviducts' ampulla and isthmus.

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.

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., (Published by Elsevier Inc.)

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.

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-17β 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., (Published by Elsevier Inc.)

The use of assisted reproduction treatment, especially intracytoplasmic sperm injection (ICSI), is now linked to a range of adverse consequences, the aetiology of which remains largely undefined. Our objective of this study was to determine differences in gene expression of blastocysts generated by ICSI as well as ICSI with artificial oocyte activation (ICSI-A) versus the less manipulative IVF, providing fundamental genetic information that can be used to aid in the diagnosis or treatment of those adversely affected by assisted reproduction treatment, as well as stimulate research to further refine these techniques. Murine blastocysts were generated by ICSI, ICSI-A and IVF, and processed for a microarray-based analysis of gene expression. Ten blastocysts were pooled for each procedure and three independent replicates generated. The data were then processed to determine differential gene expression and to identify biological pathways affected by the procedures. In blastocysts derived by ICSI versus IVF, the expression of 197 genes differed (P < 0.01). In blastocysts derived by ICSI-A versus IVF and ICSI-A versus ICSI, the expression of 132 and 65 genes differed respectively (P < 0.01). Procedural-induced changes in genes regulating specific biological pathways revealed some consistency to known adverse consequences. Detailed investigation of procedure-specific dysfunction is therefore warranted., (Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every four hours inhibited luteolysis in ewes. However, estradiol-17β or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in heifers, but infusion of estradiol+PGE(2) inhibited luteolysis in heifers. The objective of this experiment was to determine whether and how intra-luteal implants containing PGE(1) or PGE(2) prevent luteolysis in Angus or Brahman cows. On day-13 post-estrus, 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. Coccygeal blood was collected daily for analysis for progesterone. Breed did not influence the effect of PGE(1) or PGE(2) on luteal mRNA for LH receptors or unoccupied or occupied luteal LH receptors did not differ (P>0.05) so the data were pooled. Luteal weights of Vehicle-treated Angus or Brahman cows from days-13-19 were lower (P<0.05) than those treated with intra-luteal implants containing PGE(1) or PGE(2). Day-13 Angus luteal weights were heavier (P<0.05) than Vehicle-treated Angus cows on day-19 and luteal weights of day-13 corpora lutea were similar (P>0.05) to Angus cows on day-19 treated with intra-luteal implants containing PGE(1) or PGE(2). Profiles of circulating progesterone in Angus or Brahman cows treated with intra-luteal implants containing PGE(1) or PGE(2) differed (P<0.05) from controls, but profiles of progesterone did not differ (P>0.05) between breeds or between cows treated with intra-luteal implants containing PGE(1) or PGE(2). Intra-luteal implants containing PGE(1) or PGE(2) prevented (P<0.05) loss of luteal mRNA for LH receptors and unoccupied or occupied receptors for LH compared to controls. It is concluded that PGE(1) or PGE(2) alone delays luteolysis regardless of breed. We also conclude that either PGE(1) or PGE(2) prevented luteolysis in cows by up-regulating expression of mRNA for LH receptors and by preventing loss of unoccupied and occupied LH receptors in luteal tissue., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In the last 30 years, remarkable progress has been made in our understanding of the biological role of endothelins in the regulation of reproductive function and fertility. A peptide hormone identified for its ability to regulate blood pressure has now been shown as a potent mediator of several reproductive pathways. Ligand- and receptor-specific roles have been identified and/or postulated during follicular development and ovulation as well as in the function and regression of the corpus luteum. In this review we have attempted to organize endothelin-mediated ovarian processes in a process-specific manner, rather than compile a review of ligand- or isoform-specific actions. Further, we have included a discussion on "post-ovarian" or oviductal function, as well as the future directions that we believe will increase our understanding of endothelin biology as a whole.

The oviduct is a dynamic structure whose function relies upon cyclic changes in the morphology of both ciliated and secretory luminal epithelial cells. Unfortunately, infection of these epithelial cells by sexually transmitted pathogens can lead to pelvic inflammatory disease, ectopic pregnancies and infertility. The disruption of normal, cyclic apoptosis in the oviducal epithelium appears to be a causal factor of oviducal pathology and therefore, these pathways represent a potential target for diagnosis and therapeutic intervention. The objective of this study was to determine the pattern of expression for apoptotic genes in the oviduct of the naturally cycling mouse, generating fundamental information that can be applied to the development of animal models for research and the identification of targets for disease intervention. Whole oviducts were collected from regular cycling mice killed at 1p.m. on each day of the oestrous cycle and the expression of 84 apoptotic genes determined by targeted PCR super-array. Intact and cleaved caspases were then evaluated by western blotting. The expression of mRNA for genes classified as pro-apoptotic (Bad, Bak1 and Bok) and anti-apoptotic (Bag3, Bnip2 and Xiap) was regulated by day (P < 0.05). Differences in the temporal expression of several p53-related genes (Trp53bp2, Trp53inp1 and Trp73), those specific to the TNF superfamily (Tnfrsf10 and Tnfsf10b) and one caspase (Casp14) were also observed (P < 0.05). The cleaved forms of Caspases-3, -6 and -12 were all detected throughout the oestrous cycle. These results represent the first pathway-wide analysis of apoptotic gene expression in the murine oviduct.

Endothelins were first identified as potent vasoactive peptides; however, diversity in the biological function of these hormones is now evident. We have identified a novel role for endothelins: a requirement for these peptides within the oviduct during fertilization and/or early embryo development. In vivo, treatment after ovulation with a dual endothelin receptor antagonist (tezosentan) decreased the number of two-cell embryos that could be collected from within the oviducts. In vitro fertilization experiments showed that gamete viability and their ability to fertilize were not affected by treatment with this antagonist, suggesting that the effect observed in vivo was mediated by the oviduct itself. Expression of mRNA for all three isoforms of the endothelins and both receptor subtypes was detectable within the oviduct. Expression of mRNA for endothelin-3 was regulated by gonadotropins in epithelial cells of the oviduct and increased specifically within the isthmus of this structure. Immunostaining revealed localization of both endothelin receptors A and B to the columnar epithelial cells within the oviduct, suggestive of a local role for endothelins in the regulation of epithelial function and ultimately oviductal secretions. A microarray analysis revealed three likely endothelin-regulated protein networks for future analysis: the TGFbeta, IL-10, and CCAAT/enhancer-binding protein superfamilies. Overall, these results suggest a novel and requisite role for endothelins within the oviduct during fertilization and/or early embryo development.

Endothelin-2 (EDN2)-mediated contraction has been proposed as a final mechanical signal facilitating ovulation. The objectives herein were to determine (1) whether ovarian endothelins were increased before ovulation; (2) whether a specific endothelin-converting enzyme (ECE) was mediating their production; (3) which receptor was facilitating ovarian contraction; and (4) whether receptor-specific antagonism affected ovulation. Follicular development was induced in immature rats with 10 IU pregnant mare serum gonadotrophin (PMSG) and the ovulatory cascade was initiated 48 h later with 10 IU human chorionic gonadotrophin (hCG). In Experiment 1, an immunoassay revealed that the ovarian concentration of endothelin peptide was increased 7-fold 12 h after hCG when compared with 48 h after PMSG (P < 0.05). In Experiment 2, real-time PCR indicated that mRNA for Ece1, but not Ece2, was increased in granulosa cells collected 12 h after hCG when compared with those collected before the ovulatory stimulus (P < 0.05). In Experiment 3, isometric tension analysis revealed that the contractile effect of EDN2 was mediated by endothelin receptor A (EDNRA), not B (EDNRB). In Experiment 4, no effect was observed on the rate of ovulation when rats were treated with an antagonist specific to EDNRA (BQ123) or EDNRB (BQ788), or when mice were treated with BQ123, BQ788 or BQ123 + BQ788. In conclusion, endothelin peptide is produced before ovulation and the contractile action of EDN2 within the ovary is facilitated via EDNRA. In addition, findings of this study indicate synergistic interactions among contractile factors affect ovulatory outcome, while the role of EDNRB alone in the process of ovulation requires further investigation.

Ovulation has long been recognized as one of the most dramatic reproductive processes. Decades of research on how the LH/FSH surge leads to ovulation have made it clear that the surge induces a very complex cascade of changes. Studies of genetically modified mice have pointed to progesterone (P4) and its receptor (PGR) and the prostaglandins (PGs) as critical components of the ovulatory cascade. In cattle, the gonadotropin surge also induces oxytocin (OT), which does not appear to increase in rodent periovulatory follicles. This review is an attempt to summarize studies by our laboratory on the temporal patterns, roles, regulation, and interrelationships among P4/PGR, PGs, and OT in bovine periovulatory follicles. Most of these results are based on an experimental model in which the dominant follicle of the first follicular wave of the estrous cycle is induced to develop into a preovulatory follicle by injection of PGF(2α) on Day 6 of the cycle, followed 36 h later by an injection of GnRH to induce the LH/FSH surge. The results suggest that the effects of the gonadotropin surge on PG production by bovine granulosa cells are mediated by the gonadotropin-induced increase in intrafollicular P4 and that P4 acts by binding to its nuclear receptor and increasing the abundance of mRNA for the enzyme PTGS2 (COX-2). Our data thus far also support the hypothesis that PGs, especially PGE(2), can stimulate progesterone secretion by both follicular cell types and suggest a positive feedback relationship between P4/PGR and the PGs. Additional results suggest a positive feedback loop between P4/PGR and OT. The finding that levels of mRNA for several ADAMTS proteases are regulated by the LH/FSH surge in vivo and by P4/PGR and/or PGs in vitro suggests a role for this family of proteases in remodeling the bovine ovulatory follicle in preparation for ovulation and the formation of the corpus luteum. It is important to remember that a process essential for reproduction, such as ovulation, may involve redundant mechanisms and that these mechanisms may have evolved differently from rodents in larger mammalian species, such as ruminants and humans.

A transgenic mouse line that expresses iCre under regulation of the Cytochrome P(450) 17alpha-hydroxylase/17, 20-lyase (Cyp17) promoter was developed as a novel transgenic mouse model for the conditional deletion of genes specifically in the theca/interstitial cells of the ovary and Leydig cells of the testis. In this report, we describe the development of Cyp17iCre mice and the application of these mice for conditional deletion of the estrogen receptor alpha (Esr1) gene in the theca/interstitial and Leydig cells of the female and male gonad, respectively. These mice will prove a powerful tool to inactivate genes in the gonad in a cell-specific manner.

We have previously proposed endothelin-2 (EDN2) as a granulosa cell-derived contractile signal that facilitates ovulation. Spatially, Edn2 mRNA expression is restricted to granulosa cells of periovulatory follicles. Temporally, mRNA for this contractile peptide is expressed immediately before follicle rupture. The primary objective of this study was to test the hypothesis that hypoxia mediates EDN2 expression in granulosa cells at ovulation, and if it does, to determine the region within the promoter responsible for this effect. To determine the effect of hypoxia on mRNA expression, immature mice were treated with 5 IU of PMSG followed 48 h later by 5 IU of human chorionic gonadotropin (hCG). Granulosa cells were isolated at 9 h after hCG, cultured under normal or hypoxic conditions, and the expression level of mRNA was compared. mRNA expression was increased when granulosa cells were cultured in a hypoxic environment (p<0.05). Subsequent promoter analysis found that the 5' upstream region of the EDN2 promoter (between -1894 bp and -1407 bp) was responsible for hypoxia-mediated changes in EDN2 expression. This promoter region contains multiple sites for potential transcriptional regulation, including that by hypoxia-inducible factor 1 (HIF-1, ACGTG) at -1297 bp. The second objective of this study was to determine whether the progesterone receptor (PR) or cyclooxygenase-2 (COX-2), two key regulators of periovulatory events, controlled EDN2 expression. To accomplish this, gonadotropin-primed mice were treated with RU-486 or indomethacin and expression of mRNA for Edn2 was determined in ovaries collected at 12 h after hCG. Treatment with RU-486 or indomethacin did not affect expression of mRNA for Edn2 (p>0.05). Taken together, we believe that hypoxia, but not the PR or COX-2, regulate gonadotropin-induced EDN2 expression in the periovulatory follicle.

Proper function of the oviduct is critical to reproductive success with regulated contraction and relaxation facilitating transportation of the germ cells to the site of fertilization. Endothelin-2 (EDN2) is a potent vasoconstrictor produced by granulosa cells of the preovulatory follicle at the time of ovulation; however, whether this gonadotropin surge-induced peptide played a role in facilitating germ cell transportation by inducing oviductal contraction was unknown. The objectives of these experiments were (1) to determine whether the endothelin receptor system was present in the oviduct, (2) to test the hypothesis that EDN2 induces oviductal contraction via a specific endothelin receptor subtype, (3) to determine, as a possible alternate source of the ligand, whether mRNA for EDN2 was expressed in cumulus-oocyte complexes (COCs) within the oviduct, and (4) to determine whether EDN2 could overcome prostaglandin E(2) (PGE(2))-induced oviductal relaxation. Microarray and real-time PCR analysis indicated that mRNA for both the endothelin receptor subtypes (ET(A) and ET(B)) was present in the oviduct, whereas immunohistochemical examination revealed that ET(A) protein was the dominant isoform, present in the luminal epithelial cells of the oviduct. Real-time PCR analysis demonstrated that mRNA for EDN2 was expressed in COCs after ovulation. Isometric tension analysis indicated that EDN2 was a potent oviductal constrictor and that the contractile effect of EDN2 was mediated by the ET(A) and not the ET(B) receptor subtype. The oviductal contraction induced by EDN2 also reversed oviductal relaxation induced by PGE(2). In summary, ET(A) receptor-specific EDN2-induced contraction as a facilitator of oviductal function suggests a novel pathway involved in germ cell transport and hence mammalian fertility.

Follicular production of prostaglandins (PGs) is essential for mammalian ovulation, but the factors that mediate production and the cell-specific action(s) of PGE and PGF2alpha during the ovulatory cascade remain largely unknown. The aims of these experiments were: (1) to investigate the potential role of oxytocin (OT) in ovulatory PG production, (2) to determine cellular and temporal patterns of expression of mRNA for specific PG receptors during the periovulatory interval, (3) to determine cell-specific effects of PGE2 on progesterone secretion, and (4) to investigate the potential for an active transport mechanism that may regulate the effect of PGs during the ovulatory cascade, using cattle as the animal model. Heifers were treated sequentially with PGF2alpha and GnRH to induce luteal regression, a follicular phase and the LH/FSH surge (ovulation occurs approximately 30 h after GnRH). In experiment 1, OT increased the secretion of PGE and PGF2alpha by granulosa cells collected from preovulatory follicles (before the LH/FSH surge) and OT production by pieces of follicle wall from periovulatory follicles (after the LH/FSH surge) was regulated by progesterone acting through the progesterone receptor. In experiment 2, levels of mRNA for the PGF2alpha receptor and three PGE receptor subtypes were determined by semi-quantitative RT-PCR in theca interna and granulosa cells from pre- and periovulatory follicles collected at 0, 6, 12, 18 and 24 h post-GnRH. Time- and cell-specific patterns of change in mRNA for PG receptors were observed, suggesting multiple effects of both PGE and PGF2alpha in both theca interna and granulosa cells throughout the ovulatory cascade. Cell-specificity of PG action was confirmed in experiment 3; PGE2 increased the secretion of progesterone by theca interna but not granulosa cells collected from late periovulatory follicles. The results of experiment 4 revealed the expression of mRNA for the bovine PG transporter in theca interna and granulosa cells and its regulation during the periovulatory period, thus revealing the presence of a potential transport mechanism that could regulate cellular distribution of PGs throughout the ovulatory cascade. Taken together, these results provide new insight into mechanisms that regulate the production, distribution and site of action of PGE and PGF2alpha during the ovulatory cascade.

Follicular production of prostaglandins (PGs) is essential for ovulation, but the factors mediating gonadotropin-induced secretion of PGE and PGF(2alpha) remain largely unknown. We tested the hypothesis that gonadotropin-induced changes in progesterone and its receptor (PR) mediate the increase in periovulatory PGs. Heifers were treated with PGF(2alpha) and GnRH to induce luteolysis and the LH/FSH surge (ovulation occurs approximately 30 h after GnRH). Because there are two increases in intrafollicular progesterone/PR mRNA during the bovine periovulatory period, we first examined the temporal pattern of PG production by follicles collected at 0, 3.5, 6, 12, 18, and 24 h after GnRH. Although PGs did not increase in the follicular fluid until 24 h after GnRH, acute secretion of PGs by follicle wall (theca + granulosa cells) was initiated by 18 h and had increased manyfold by 24 h after GnRH. In vitro, FSH and LH induced dramatic transient increases in PG production by follicle wall and granulosa, but not theca, cells isolated from preovulatory follicles (0 h after GnRH). PG accumulation peaked on d 2 of culture, mimicking the secretion pattern after a gonadotropin surge in vivo. In cultures of follicle wall and granulosa cells, the PR antagonist mifepristone (MIFE, 1 microm) inhibited LH-induced PG secretion and the progestin medroxyprogesterone acetate (1 or 10 microm), but not the glucocorticoid dexamethasone (1 or 10 microm), overcame the effect of MIFE on PGs. Semiquantitative RT-PCR revealed that MIFE inhibited LH-induced expression of cyclooxygenase-2 mRNA in granulosa cells in vitro. Again, treatment with medroxyprogesterone acetate overcame the effect of MIFE. Together these results provide strong evidence that periovulatory increases in cyclooxygenase-2 mRNA, PGE, and PGF(2alpha) are mediated by gonadotropin-induced increases in progesterone/PR, indicating that in some species there is an important functional relationship between these pathways in the ovulatory cascade.

Dairy cattle are susceptible to heat stress-induced reductions in fertility; however, direct effects of hyperthermia on specific reproductive functions are difficult to determine in vivo. The objective of this experiment was to examine the effect of elevated temperature in vitro on follicular steroidogenesis, to gain insight into specific follicular responses associated with heat stress. Dominant follicles were obtained from Holstein heifers on day 6 post-estrus (luteal phase; n = 4) or day 8, 36 h after an injection with 25 mg PGF(2alpha) to induce regression of the corpus luteum (follicular phase; n = 4). Pieces of follicle wall were isolated from dominant follicles and cultured for 96 h with 0, 2 or 100 ng/ml LH or FSH at 37, 39 or 41 degrees C. Concentrations of androstenedione, estradiol and progesterone were determined in culture media collected every 24h. During the last 48 h of culture, basal secretion of androstenedione and estradiol by pieces of follicle wall was lower at 41 degrees C than at 37 or 39 degrees C (P < 0.05). In contrast, cumulative secretion of progesterone by pieces of follicle wall in medium alone was higher at 41 degrees C than at 37 or 39 degrees C (P < 0.05). Pieces of follicle wall responded to treatment with both low (2 ng/ml) and high (100 ng/ml) doses of gonadotropins at all temperatures. However, gonadotropin-induced secretion of androstenedione and estradiol was generally lower, whereas gonadotropin-induced secretion of progesterone was higher at 41 degrees C and sometimes at 39 than at 37 degrees C. The changes in basal steroidogenesis and in responses to gonadotropins suggest that follicular cells begin to luteinize at elevated temperatures in vitro. Premature luteinization of follicular cells in vivo has been associated with reduced fertility in cattle with persistent follicles, suggesting that the premature differentiation of follicular cells observed in the current study may be responsible, in part, for the reduced fertility of dairy cattle under heat-stressed conditions.

Extensive remodeling of the extracellular matrix occurs in the ovary during the periovulatory period. Matrix metalloproteinases and their endogenous inhibitors, tissue inhibitors of metalloproteinases, are believed to play integral roles in this highly regulated series of cellular events, but their specific roles remain unclear. Recent cloning studies have identified a novel family of metalloproteinases, the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family. The regulated expression of distinct ADAMTS subtypes has been shown to be required for tissue morphogenesis during embryonic development and for maintaining the integrity of tissues in the adult. In the present studies, we have determined that multiple ADAMTS subtypes are present in the bovine ovary using a reverse transcription-polymerase chain reaction strategy. In particular, ADAMTS-1, -2, -3, -4, -5 (also known as ADAMTS-11), -7, -8, and -9, but not ADAMTS-6, -10, or -12, mRNA transcripts were detected in granulosa cells of nonatretic ovarian follicles and corpora lutea. The levels of mRNA for these ovarian ADAMTS were up- or down-regulated or remained unchanged in the granulosa and/or theca cells of the dominant follicle following the preovulatory surge of gonadotropins, depending on the subtype and/or the cell compartment, and in the corpus luteum during the luteal phase of the estrous cycle. The complex expression patterns observed for the distinct ADAMTS subtypes in the granulosa and theca cells of the periovulatory follicle and in the luteal tissues of the bovine ovary suggest that these novel proteases mediate, at least in part, the remodeling events underlying folliculogenesis and ovulation and the formation, maintenance, and regression of the corpus luteum.

In cattle, sub-luteal circulating progesterone induces an increase in the frequency of LH pulses, prolonged growth of the dominant follicle, increased peripheral estradiol and reduced fertility. The objective of this study was to examine the earliest stages of development of prolonged dominant follicles, to gain insight into the etiology of this aberrant condition. Heifers were treated with an intravaginal progesterone-releasing device (CIDR) from Day 4-8 post-estrus and PGF2alpha was injected on Day 6 and again 12h later (early prolonged dominant group). Follicular phase (CIDR: Day 4-6, with PGF2alpha) and luteal phase (CIDR: Day 4-8, without PGF2alpha) groups served as controls. As expected, peripheral progesterone in heifers of the early prolonged dominant group was intermediate between luteal and follicular phase groups after luteal regression (P<0.05). On Day 7, the frequency of LH pulses was higher in heifers of the follicular phase and early prolonged dominant groups than the luteal phase group (P<0.05). Dominant follicles (n = 4 per group) were collected by ovariectomy on Day 8 and were similar in size among groups (P>0.05). Estradiol and androstenedione concentrations in the follicular fluid at ovariectomy were higher in the follicular phase and early prolonged dominant groups versus the luteal phase group (P<0.01), whereas progesterone did not differ among groups (P>0.05). Granulosa cells and theca interna isolated from dominant follicles were incubated for 3h with or without gonadotropins or frozen for later analysis of mRNA for steroidogenic enzymes. Luteinizing doses (128 ng/ml) of LH and FSH increased secretion of progesterone (P<0.05) but did not affect secretion of estradiol by granulosa cells in all groups. Low (2 or 4 ng/ml) and luteinizing doses of LH increased secretion of androstenedione by theca interna to a similar extent among groups. Expression of mRNA for P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 aromatase (aromatase) and Steroidogenic Acute Regulatory (StAR) protein by granulosa cells did not differ among groups (P>0.05). Levels of mRNA for P450scc, 3beta-HSD, 17alpha-hydroxylase (17alpha-OH) and StAR protein in theca interna were similar in the follicular phase and early prolonged dominant groups (P>0.05), but lower in the luteal phase group (P<0.05-0.1). In summary, the premature follicular luteinization observed in previous studies after prolonged periods of sub-luteal progesterone was absent in early prolonged dominant follicles, exposed to sub-luteal progesterone for 36 h, and their characteristics resembled those of control follicles during the follicular phase.

The objective of this experiment was to determine the effect of mifepristone, a progesterone receptor antagonist, on pregnancy and secretion of steroids, pregnancy-specific protein B (PSPB) and prostaglandins at mid-pregnancy in ewes. Ninety-day pregnant ewes were ovariectomized (OVX) and treatments were initiated 72 h post-OVX. Ewes received (1) vehicle, (2) prostaglandin F2alpha (PGF2alpha, 8 mg/58 kg/bw, i.m.) 84 h post-OVX, (3) mifepristone (50 mg intrajugular at 72, 84, 96, and 108 h post-OVX), (4) mifepristone (50mg) + PGF2alpha, (5) mifepristone (100 mg intrajugular at 72, 84, 96, and 108 h), and (6) mifepristone (100 mg) + PGF2alpha. Ewes treated with vehicle or PGF2alpha alone did not abort (P > or = 0.05). But, 60, 80, 60, and 100% of ewes treated with mifepristone (50 mg), mifepristone (50 mg) + PGF2alpha, mifepristone (100 mg), and mifepristone (100 mg) + PGF2alpha, respectively, aborted (P < or = 0.05). Profiles of progesterone, estradiol-17beta, prostaglandin E (PGE), or PSPB did not differ (P > or = 0.05) among treatment groups. Profiles of PGF2alpha of treatment groups receiving mifepristone with or without PGF2alpha differed (P < 0.05) from vehicle or PGF2alpha alone-treated ewes. It is concluded that progesterone actions are necessary to suppress uterine/placental secretion of PGF2alpha and that maintenance of critical progesterone: estradiol-17beta and PGE:PGF2alpha ratios are necessary for maintenance of pregnancy.

The aromatase inhibitor CGS-16949A was used to determine whether CGS-16949A altered secretion of progesterone, estradiol-17beta, PGE (PGE1 + PGE2), PGF2alpha and PSPB. Ninety day pregnant ewes were ovariectomized and received vehicle, PGF2alpha, CGS-16949A or PGF2alpha+CGS-16949A. None of the ewes treated with PGF2alpha, CGS-16949A or PGF2alpha+CGS-16949A aborted (P > or = 0.05) during the 108-h experimental period. Treatment with CGS-16949A lowered (P < or = 0.05) progesterone in jugular venous plasma but concentrations of progesterone were not affected (P > or = 0.05) by treatment with PGF2alpha. Concentrations of estradiol-17beta and PSPB in jugular venous plasma and PGE in inferior vena cava plasma were decreased (P < or = 0.05) by treatment with CGS-16949A. Concentrations of PGF2alpha in inferior vena cava plasma were not affected (P > or = 0.05) by treatment with CGS-16949A. Decreases in estradiol-17beta occurred before decreases in PSPB, which was then followed by decreases in PGE (P < or = 0.05). It is concluded that these data support the hypothesis that estradiol-17beta regulates placental secretion of PSPB; PSPB regulates placental secretion of PGE; and PGE regulates placental secretion of progesterone during mid-pregnancy in ewes.

The objectives were to evaluate, in anestrous ewes, the effectiveness of a CIDR-G device (0.3 g progesterone) administered for 5 d to induce estrus; and FSH (Folltropin; 55 mg NIH-FSH-P1 equivalent) in saline:propylene glycol (1:4) 24 h before insert removal (Day 0), to increase ovulation rate and prolificacy. Ewes of mixed breeding were assigned at random to 3 treatments: control (C; n = 125), 5 d progesterone (P5; n = 257) and 5 d progesterone plus FSH (P5F; n = 271). Intact rams were joined at insert removal and ewes were observed every 24 h for 3 d. On Day 14, the ovulation rates of all ewes detected in estrus in the treated groups were determined using transrectal ultrasonography. Rams were removed on Day 26 to 31. Ewes were examined for pregnancy then, and again 20 to 25 d later to detect ewes that conceived to the second service period. Percentage of ewes marked by rams was higher in progesterone-treated (77%) than in C (20%; P < 0.01), but did not differ between P5 and P5F. The ovulation rate (1.95+/-0.04) did not differ due to FSH. Conception (68%) and pregnancy (52%) rates were higher in progesterone-treated (P < 0.01) than in C (0%) ewes. Estrous response varied quadratically with time after ram introduction, and the conception rate varied quadratically with the time of observation of onset of estrus. Over two service periods more progesterone-treated than C ewes lambed (65 vs 45%; P < 0.01). Lambs born per ewe exposed (0.7+/-0.1, 1.0+/-0.1, and 1.1+/-0.1 for C, P5 and P5F, respectively) was increased by progesterone (P < 0.05). Litter size to the first service period (1.59+/-0.04) and overall (1.54+/-0.03) did not differ among treatment groups. FSH-treated ewes tended to have more lambs (1.67+/-0.1) than did ewes receiving progesterone alone (1.5+/-0.1; P = 0.06) and than did ewes lambing to the second service period (1.5+/-0.1; P = 0.06). In summary, a 5-d progesterone pre-treatment of anestrous ewes induced estrous cycles and increased the pregnancy rates. A single injection of FSH only tended to increase litter size.

Two experiments were conducted to determine the luteotropin of pregnancy in sheep and to examine autocrine and paracrine roles of progesterone and estradiol-17 beta on progesterone secretion by the ovine corpus luteum (CL). Secretion of progesterone per unit mass by day-8 or day-11 CL of the estrous cycle was similar to day-90 CL of pregnancy (P > or = 0.05). In experiment 1, secretion of progesterone in vitro by slices of CL from ewes on day-8 of the estrous cycle was increased (P < or = 0.05) by LH or PGE2. Secretion of progesterone in vitro by CL slices from day-90 pregnant ewes was not affected by LH (P > or = 0.05) while PGE2 increased (P < or = 0.05) secretion of progesterone. Day 8 ovine CL of the estrous cycle did not secrete (P > or = 0.05) detectable quantities of PGF2alpha or PGE while day-90 ovine CL of pregnancy secreted PGE (P < or = 0.05) but not PGF2alpha. Secretion of progesterone and PGE in vitro by day-90 CL of pregnancy was decreased (P < or = 0.05) by indomethacin. The addition of PGE2, but not LH, in combination with indomethacin overcame the decreases in progesterone by indomethacin (P < or = 0.05). In experiment 2, secretion of progesterone in vitro by day-11 CL of the estrous cycle was increased at 4-h (P < or = 0.05) in the absence of treatments. Both day-11 CL of the estrous cycle and day-90 CL of pregnancy secreted detectable quantities of PGE and PGF2alpha (P < or = 0.05). In experiment 1, PGF2alpha secretion by day-8 CL of the estrous cycle and day-90 ovine CL of pregnancy was undetectable, but was detectable in experiment 2 by day-90 CL. Day 90 ovine CL of pregnancy also secreted more PGE than day-11 CL of the estrous cycle (P < or = 0.05), whereas day-8 CL of the estrous cycle did not secrete detectable quantities of PGE (P > or = 0.05). Trilostane, mifepristone, or MER-25 did not affect secretion of progesterone, PGE, or PGF2alpha by day- 11 CL of the estrous cycle or day-90 CL of pregnancy (P > or = 0.05). It is concluded that PGE2, not LH, is the luteotropin at day-90 of pregnancy in sheep and that progesterone does not modify the response to luteotropins. Thus, we found no evidence for an autocrine or paracrine role for progesterone or estradiol-17 36 on luteal secretion of progesterone, PGE or PGF2alpha.

Experiments were conducted in beef cows without a primary CL, in which pregnancy had been maintained with exogenous progestogen. In preliminary trials, replacement CL induced ipsilateral to the embryo and after, rather than before, d 36 of pregnancy, maintained more pregnancies after withdrawal of exogenous progestogen (13/13 vs 2/6; P < 0.05). In Exp. 1, in cows with replacement CL induced by treatment with hCG on d 28 of pregnancy, treatment with flunixin meglumine on d 31 through 37 did not increase maintenance of pregnancy. Experiment 2 was conducted to evaluate directly the effects of concentrations of PGF2alpha and estradiol-17beta during d 31 through 35 of pregnancy on maintenance of pregnancy by replacement CL induced between d 28 and 31. In cows that maintained pregnancy while progestogen was provided, maintenance of pregnancy after withdrawal of exogenous progestogen tended to be greater with high (5/5) than with low (2/6; P < 0.10) concentrations of PGF2alpha and greater with low (6/7) than with high (2/6; P = 0.10) concentrations of estradiol-17beta. Secretion of progesterone by replacement CL was greater (P < 0.05) in cows with high than in those with low concentrations of PGF2, during d 31 through 35. Prostaglandin F2alpha may facilitate attachment of the bovine embryo (d 30 to 40) in a manner similar to that reported for implantation in other species. Cows that did not form CL in response to hCG on d 28 to 31 responded well when retreated after d 36. Again, maintenance of pregnancy was greater when replacement CL were induced after (9/9) rather than before d 36 (8/16; P < 0.05).

An experiment was performed to evaluate the effect of the side of ovulation with respect to the previously gravid uterine horn on fertility of cows inseminated at one of two periods postpartum. All cows were treated with an intravaginal progesterone insert for 7 d and received estradiol benzoate (2 mg, i.m.) at the time of device insertion, prostaglandin F2alpha (25 mg, i.m.) at the time of device removal, and estradiol benzoate (1 mg, i.m.) 30 h after device removal. All cows were inseminated 28 to 30 h after the second treatment with estradiol benzoate, regardless of observed estrus. Cows treated in Period 1 received inserts at 16 to 20 d postpartum and were inseminated at 25 to 29 d postpartum. Cows treated in Period 2 received inserts at 26 to 30 d postpartum and were inseminated at 35 to 39 d postpartum. Diameter of the largest follicle at insert removal was greater in cows treated in Period 2 (10.1 +/- 0.3; mm +/- SEM) than in cows treated in Period 1 (9.1 +/- 0.3; P < .05). Diameter did not differ with the side of ovulation in respect to the previously gravid uterine horn. Diameter was greater in cows 5 to 9 (10.3 +/- 0.3) than in cows 3 to 4 (9.0 +/- 0.3) or 10 to 13 (9.4 +/- 0.6) yr of age (P < .01). The proportion of cows that ovulated from the ovary contralateral to the previously gravid uterine horn was greater (P < .05) than of those that ovulated from the ipsilateral ovary, and the incidence of ovulation was reduced in cows 3 to 4 yr of age (P < .01). Conception rate tended to be greater for ovulation from the ipsilateral compared with the contralateral ovary, relative to the previously gravid uterine horn (P < .10) and for ovulation from the right than the left ovary (P < .06). Conception rate was less if cows ovulated a follicle that was < 9 mm than a follicle > or = 9 mm in diameter at insert removal (P < .01) and was greater in cows inseminated in June than in April or May (P < .05). In conclusion, in cows in which estrus was synchronized at 25 to 39 d postpartum, ovulation from either the ovary ipsilateral to the previously gravid uterine horn, or the right ovary, tended to increase fertility.

Treatment with PGF2alpha plus estradiol-17beta aborts 90-day pregnant ewes, whereas PGF2alpha or estradiol-17beta alone does not abort ewes. The objective of this experiment was to evaluate whether tamoxifen, an estrogen receptor antagonist, estradiol-17beta, prostaglandin F2alpha (PGF2alpha), indomethacin, or some of their interactions affected ovine uterine/placental secretion of PGF2alpha, estradiol-17beta or prostaglandins E (PGE), because a single treatment with PGF2alpha and estradiol-17beta given every 6 h aborts 90-day pregnant ewes. Concentrations of PGF2alpha in uterine venous blood were increased (P < or = 0.05) by estradiol-17beta, PGF2alpha + estradiol-17beta, and PGF2alpha + tamoxifen, and decreased (P < or = 0.05) by indomethacin or PGF2alpha + indomethacin at 72 h when compared to the 0 h samples. Concentrations of PGE in uterine venous blood were decreased (P < or = 0.05) by indomethacin and PGF2alpha + indomethacin and increased (P < or = 0.05) by PGF2alpha + estradiol-17beta at 72 h when compared to the 0 h samples. Concentrations of PGF2alpha in inferior vena cava blood at 6 h were increased (P < or = 0.05) by PGF2alpha either alone or in combination with indomethacin, tamoxifen, or estradiol-17beta, which is due to the PGF2alpha injected. Concentrations of PGF2alpha in inferior vena cava blood in PGF2alpha + estradiol-17beta-treated 88- to 90-day pregnant ewes increased (P < or = 0.05) linearly over the 72-h sampling period and averaged 4.0 + 0.4 ng/ml. Concentrations of PGF2alpha in inferior vena cava blood of control, PGF2alpha, tamoxifen, PGF2alpha + indomethacin, PGF2alpha + tamoxifen, and estradiol-17beta-treated ewes did not differ (P > or = 0.05) and averaged 0.4 + 0.04 ng/ml. Profiles of PGE in inferior vena cava blood of 88- to 90-day pregnant ewes treated with vehicle, PGF2alpha, estradiol-17beta, tamoxifen, tamoxifen + PGF2alpha, or estradiol-17beta + PGF2alpha did not differ (P > or = 0.05). Concentrations of PGE in inferior vena cava blood of 88- to 90-day pregnant ewes treated with indomethacin or PGF2alpha + indomethacin were lower (P < or = 0.05) than in control ewes. Concentrations of estradiol-17beta in jugular venous plasma of PGF2alpha + estradiol-17beta-treated 88- to 90-day pregnant ewes increased linearly and differed (P < or = 0.05) from controls. Profiles of estradiol-17beta in jugular venous plasma of PGF2alpha, indomethacin, tamoxifen, and PGF2alpha + tamoxifen and PGF2alpha + indomethacin, estradiol-17beta, and controls did not differ (P > or = 0.05). It is concluded that treatment with a single injection of PGF2alpha and estradiol-17beta given every 6 h causes a linear increase in PGF2alpha and estradiol-17beta.

Ninety-day pregnant sheep were ovariectomized and received vehicle or trilostane every 12 h through 132 h, starting at 72 h postovariectomy. All trilostane-treated ewes aborted (P < or = 0.05) between 36 and 50 h after initiation of treatment. Profiles of progesterone in jugular venous blood differed (P < or = 0.05) and was lower (P < or = 0.05) in trilostane-treated ewes. Profiles of estradiol-17beta in jugular venous plasma of trilostane-treated ewes differed (P < or = 0.05) from controls. Estradiol-17beta increased after the first two treatments, followed by a return 2 h later to pretreatment levels (P > or = 0.05), which was followed by a sustained increase (P < or = 0.05) in estradiol-17beta. Profiles of PGF2alpha in inferior vena cava plasma of trilostane-treated ewes differed and were greater (P < or = 0.05) and occurred with the sustained increase in estradiol-17beta and the onset of most of the abortions. Profiles of PGE in inferior vena cava plasma between control and trilostane-treated 90-day pregnant ewes did not differ (P > or = 0.05). It is concluded that abortions occur at midpregnancy in sheep when the estradiol-17beta : progesterone ratio changes sufficiently to cause a sustained increase in estradiol-17beta and PGF2alpha but without changing placental secretion of PGE.

Ninety-day pregnant ewes were either laparotomized, ovaries left in situ or bilaterally ovariectomized, and a jugular venous catheter and an inferior vena cava catheter via the saphenous vein were installed. Seven days later, placenta slices were collected and incubated in vitro for 4 h. Secretions of progesterone, PGE, estradiol-17beta and pregnancy-specific protein B (PSPB) in vitro by placenta from ovariectomized ewes were increased (P < or = 0.05) by 2.7-, 3.6-, 2.2-, and 2.4-fold, respectively, when compared to placenta slices from intact 90-day pregnant ewes. Secretion of PGF2alpha in vitro was unchanged (P > or = 0.05). Ovariectomy decreased (P < or = 0.05) jugular venous progesterone for 78 h followed by a quadratic increase (P < or = 0.05), whereas progesterone remained unchanged (P > or = 0.05) in intact ewes over the 162-h sampling period. Ovariectomy increased (P < or = 0.05) PGE in inferior vena cava plasma over the last half of the 162-h sampling period, whereas concentration of PGF2alpha did not change (P > or = 0.05). Increases in PGE occurred before the increase in progesterone. Concentrations of PSPB in inferior vena cava plasma of ovariectomized pregnant ewes increased (P < or = 0.05) during the last half of the 162-h sampling period, but not in intact ewes (P > or = 0.05). PSPB increased before PGE and progesterone. Concentrations of estradiol-17beta in jugular venous plasma of ovariectomized pregnant ewes increased (P < or = 0.05) during the last half of the sampling period, but not in intact ewes (P > or = 0.05). Increases in estradiol-17beta occurred before increases in PSPB. It is concluded that these data support the hypothesis that estradiol-17beta may control placental secretion of PSPB; PSPB may regulate placental secretion of PGE; and PGE may regulate placental secretion of progesterone.

One objective of this experiment was to evaluate our hypotheses that estradiol-17beta regulates secretion of pregnancy specific protein B (PSPB) and that secretion of progesterone during pregnancy is regulated by a prostanoid by examining the effects of prostaglandin F2alpha (PGF2alpha), a luteolyic agent; indomethacin, a prostanoid synthesis inhibitor; tamoxifen, an estrogen receptor antagonist; estradiol 17-beta; and interaction of these factors on the incidence of abortion and progesterone and PSPB secretion. Another objective was to determine if there is a luteal source of PSPB. Weights of corpora lutea were decreased (P < or = 0.05) by PGF2alpha, indomethacin, PGF2alpha + tamoxifen, PGF2alpha + indomethacin, and PGF2alpha + estradiol-17beta but not (P > or = 0.05) by tamoxifen or estradiol-17beta alone. No ewe treated with PGF2alpha alone aborted (P > or = 0.05). Forty percent of ewes treated with PGF2alpha + estradiol-17beta aborted (P < or = 0.05), but ewes were not aborted by any other treatment within the 72-h sampling period. Profiles of progesterone in jugular venous blood differed (P < or = 0.05) among control, indomethacin-, tamoxifen-, and PGF2alpha + indomethacin-treated ewes. Progesterone in jugular venous blood of control ewes decreased (P < or = 0.05) by 24 h, followed by a quadratic increase (P < or = 0.05) from 24 to 62 h. Progesterone in jugular venous blood of indomethacin-, PGF2alpha-, PGF2alpha- + tamoxifen-, PGF2alpha + indomethacin-, PGF2alpha + estradiol-17beta-, and tamoxifen-treated ewes was reduced (P < or = 0.05) by 18 h and did not vary (P > or = 0.05) for the remainder of the 72-h sampling period. Progesterone in vena cava and in uterine venous blood was reduced (P < or = 0.05) at 72 h in PGF2alpha-, indomethacin-, tamoxifen-, PGF2alpha + indomethacin-, PGF2alpha + tamoxifen-, and PGF2alpha + estradiol-17beta-treated ewes. Weights of placentomes did not differ among treatment groups (P > or = 0.05). Profiles of PSPB in inferior vena cava blood differed (P < or = 0.05) among control, estradiol-17beta-, indomethacin-, tamoxifen-, PGF2alpha + indomethacin-, and PGF2alpha + tamoxifen-treated 88- to 90-day pregnant ewes. Concentrations of PSPB in inferior vena cava blood were increased (P < or = 0.05) in indomethacin-, estradiol-17beta-, tamoxifen-, PGF2alpha + tamoxifen-, and PGF2alpha + indomethacin-treated 88- to 90-day pregnant ewes within 6 h and did not vary (P > or = 0.05) for the remainder of the 72-h sampling period. Concentrations of PSPB in uterine venous blood of indomethacin-, tamoxifen-, PGF2alpha + tamoxifen-, and PGF2alpha + indomethacin-treated ewes were greater (P < or = 0.05) at 72 h than at 0 h. PSPB in ovarian venous blood did not differ (P > or = 0.05) adjacent or opposite to the ovary with the corpus luteum. It is concluded from these data that estrogen regulates placental secretion of PSPB and that a prostanoid, presumably prostaglandin E, regulates placental secretion of progesterone during 88-90 days of gestation in sheep and that there is no luteal source of PSPB.