Conceptus signaling markers in immune cells enhance pregnancy prediction in beef cattle

Abstract

In beef cattle, estrous synchronization aiming a second artificial insemination (AI) requires a reliable estimation of the pregnancy status 20 days (D20) after the first AI. The hypothesis is that the expression of interferon-stimulated genes (ISGs; ISG15, OAS1, RSAD2, and IFI44) and cytokines (IL1β and IL10) in mononuclear (PBMC) and polymorphonuclear (PMN) cells is regulated by interferon-τ (IFN-τ) and predicts the pregnancy status. PBMC and PMN were isolated from non-pregnant beef cows (N = 9), 10-12 days post-ovulation (D0), and stimulated with 100 ng/mL recombinant ovine (ro) IFN-τ or with pooled uterine flush (UF) from D18 pregnant cows. Both roIFNT and UF stimulated the expression of ISG15, RSAD2, and IFI44 in PBMC and PMN. Expression of IL1β was reduced by UF in both PBMC and PMN. On another experiment, PMN were isolated, and luteal blood perfusion was measured on D20 post-timed-AI in beef females. The accuracy of ISG expression and luteal blood perfusion to predict the pregnancy outcome was determined by ROC curve analysis. All gene combinations were tested, and the best association for increased accuracy (92.7%) and reduction of false-negative results (0.9%, 2/233) was obtained through the combination of the four ISGs (ISG15, OAS1, RSAD2, and IFI44). The criterion was that if the expression levels of at least one of the four genes were greater than the predefined cutoffs, the animal would be considered pregnant. In conclusion, the expression of ISGs and IL1β was upregulated by roIFNT and UF from pregnancy cows. The combined PMN expression of classical (ISG15 and OAS1) and unusual (RSAD2 and IFI44) ISGs provided the greatest predictive accuracy of the pregnancy status on D20 in females with active CL by Doppler and is a potential tool to be used in reproductive programs for beef cattle.

Keywords: ISG; Immune cells; Interferon-tau; Pregnancy prediction; Uterine flush.

Fig. 1

Schematic representation of the experimental model. (A) In the Exp. 1 and 2, non-pregnant Nelore heifers (N = 12) were submitted to blood sampling collection between D10-D12 post-ovulation (D0 = day of ovulation), for the isolation of mononuclear (PBMC) and polymorphonuclear (PMN) cells. Isolated PBMC and PMN were stimulated with roIFNT (100 ng/mL, IFNT group) or uterine flush from day 18 pregnant cows (UF-Conceptus) for 24 h (PBMC) or 3 h (PMN) at 37 °C in 5% CO₂. The groups without treatment [Control or UF from cows on day 18 of the diestrus phase (UF-Control)] served as controls. After the incubation, the cells were directed to RNA extraction, and gene expression of ISGs (ISG15, RSAD2, and IFI44) was determined by qPCR. (B) For Exp. 3, Nelore females (nulliparous, N = 103; primiparous, N = 53; pluriparous, N = 91) were submitted to timed-AI (TAI) on day 0. On D20 post-TAI, PMN was isolated from the peripheral blood of inseminated and non-inseminated cows. After isolation, PMN was directed to RNA extraction, and gene expression of ISGs (ISG15, OAS1, RSAD2, and IFI44) was determined by qPCR for the accuracy of pregnancy predictors. Illustration created using the Biorender software (https://www.biorender.com/).

Fig. 2

Experiment 1. Mean ± SEM for relative expression (reference genes, PBMC: GAPDH/PPIA; PMN: GAPDH/ACTB) and fold change of ISG15, RSAD2, and IFI44 genes by qPCR in PBMC (Panel A and C; N = 9) cultured for 24 h and PMN (Panel B and D; N = 10) cultured for 3 h, and treated (100 ng/mL roIFNT) or untreated (Control) with recombinant ovine interferon-τ (roIFNT). ^*AB An asterisk or different letters above the bar indicates a significant difference (P ≤ 0.05) between the groups or the transcripts.

Fig. 3

Experiment 1. Mean ± SEM for relative expression (reference genes, PBMC: GAPDH/PPIA; PMN: GAPDH/ACTB) and fold change of pro-inflammatory (IL1β) and anti-inflammatory (IL10) cytokine genes by qPCR in PBMC (Panel A and C; N = 9) cultured for 24 h and PMN (Panel B and D; N = 10) cultured 3 h, and treated (100 ng/mL roIFNT) or untreated (Control) with recombinant ovine interferon-tau (roIFNT). ^#A hatch tag above the bar indicates a tendency to significance (0.05 < P ≤ 0.1) between the transcripts. ^AB Different letters above the bar indicates a significant difference (P ≤ 0.05) between the groups or the transcripts.

Fig. 4

Experiment 2. Mean ± SEM for relative expression (reference genes, PBMC: GAPDH/PPIA; PMN: GAPDH/ACTB) and fold change of ISG15, RSAD2, and IFI44 genes by qPCR in PBMC (Panel A and C; N = 10) cultured for 12 h and PMN (Panel B and D; N = 8) cultured for 3 h in UF from day 18 of pregnant cows (UF-Conceptus) or UF from non-pregnant cows (UF-Control). ^*AB An asterisk or different letters above the bar indicates a significant difference (P ≤ 0.05) between the groups or the transcripts.

Fig. 5

Experiment 2. Mean ± SEM for relative expression (reference genes, PBMC: GAPDH/PPIA; PMN: GAPDH/ACTB) and fold change of pro-inflammatory (IL1β) and anti-inflammatory (IL10) cytokine genes by qPCR in PBMC (Panel A and C; N = 10) cultured for 12 h and PMN (Panel B and D; N = 8) cultured for 3 h in UF from day 18 of pregnant cows (UF-Conceptus) or UF from non-pregnant cows (UF-Control). ^*AB An asterisk or different letters above the bar indicates a significant difference (P ≤ 0.05) between the groups or the transcripts.

Fig. 6

Experiment 3. Mean ± SEM for relative expression (reference genes, PMN: GAPDH/ACTB) of RSAD2 and IFI44 genes by qPCR in PMN from pregnant and non-pregnant nulliparous (N = 103), primiparous (N = 53), and pluriparous (N = 91) bovine females 20 days post-timed-AI (TAI). The main effects of group (G), category (Cat), and interaction group*category (G*Cat) that were significant are shown. ^XY Bars with a different letter indicate a significant difference (P ≤ 0.05) among the parity order in non-pregnant animals. ^ABC Bars with a different letter indicate a significant difference (P ≤ 0.05) among the parity order, regardless of the pregnancy status.

Fig. 7

Experiment 3. ROC (Receiver Operating Characteristic) curves of the ISGs on D20 post-timed-AI (TAI) in nulliparous (N = 103), primiparous (N = 53), and pluriparous (N = 91) bovine females. The horizontal and vertical axes represent false positive rate (1 - specificity) and sensitivity, respectively. RSAD2 and IFI44 provided the most adequate prediction of pregnancy when compared to classical usual ISGs (ISG15 and OAS1) in primiparous and pluriparous bovine females.

Fig. 8

Summary of Results. In the Experiments 1 and 2 (in vitro studies), relative expression indicated that the treatments upregulated the ISGs (ISG15, RSAD2, and IFI44) and downregulated the pro-inflammatory cytokine IL1β in PBMC and PMN immune cells. According to the in vivo study Experiment 3 (in vivo study), we propose that an association between the expression of classical usual (ISG15 and OAS1) and unusual (RSAD2 and IFI44) ISGs in females with active CL through Doppler ultrasonography can be used as a high-accurate predictor of pregnancy in cows on day 20 post-timed-AI (TAI). Blue arrows represent downregulation and red arrows represent upregulation of genes. Illustration created using the Biorender software (https://www.biorender.com/).