An in vitro study on the myometrial contractility in dairy cattle before calving and after postpartum LPS infusion. Relation to blood progesterone and estradiol-17 [beta] levels

The aims of this study were to investigate the uterine contractility in vitro, and the expression of the oxytocin (OTR), prostaglandin F (FPR), progesterone (PR), and estrogen α (ERα) receptors in late pregnant and postpartum bovine uteri, and whether the systemic levels of progesterone (P4), and 17s-estradiol (E2s) have an influence on them. For these purposes 24 Holstein Friesian cows were used and divided into 2 groups. The first group (na=12) accounted healthy animals at around day 275 of gestation. The second group involved 12 postpartum animals whose afterbirth was spontaneously expelled within 12 hours after calving. After the placental detachment, 5 µg/mL LPS dissolved in 1 L 0.9% NaCl isotonic solution at 37°C were infused transcervically in the uterus. Blood samples were harvested prior to the tissue collection from the jugular vein to examine P4, E2s, metabolite of PGF2α (PGFM) and ionized calcium. The uterine samples were taken through cesarean section (group 1) and laparotomy after euthanasia (group 2) from the large curvature of the pregnant horn. The tissues were placed in Krebs’ solution (KS) at 37°C for the contractility experiments, in formalin for the immuno¬histoche¬mistry, and frozen in liquid nitrogen for the RT-PCR. Four circular and 4 longitudinal myometrial strips were dissected from every uterine sample and mounted in an organ bath filled with KS at 37°C and perfused with 95% O2 and 5% CO2. The spontaneous contractile activity was measured after 1 h of equilibration and during 2.5 h (divided into 5 time periods (T) of 30 min each). Subsequently, every strip was stimulated with increasing concentrations of oxytocin (Oxy), a natural analogue of PGF2α (PGF), calcium chloride (Ca), or nothing (Cont) and the induced response recorded. The parameters area under the curve (AUC), mean amplitude (MA), maximal amplitude (maxA), minimal amplitude (minA), baseline rise (BR), and frequency (FR) were calculated for every T (last 20 min). Retrospectively the animals were regrouped into subgroups attending to their steroid hormones levels. This led to the following subgroups in group 1: E2s high: E2s>400 pg/mL, na=7; and E2s low: E2s<400 pg/mL. Secondly, 4 animals with the highest (P4>5.2 ng/mL) and the lowest (P4<3.5 ng/mL) P4 concentrations were designated P4 high and P4 low, respectively. Similar criteria were used for the animals in group 2. The superior cut-off of E2s concentration was 100 pg/mL and the inferior 60 pg/mL, resulting in 2 subgroups termed E2s high (E2s >100 pg/mL; na=4) and E2s low (E2s<60 pg/mL; na=4). Animals with P4>0.43 ng/mL and P4<0.31 ng/mL were included in P4 high (na=4) and in P4 low (na=4), respectively. The blood P4, E2s, PGFM and ionized calcium values averaged 4.0 ± 1.7 ng/mL, 482.3 ± 63.7 pg/mL, 125.3 ± 63.7 pg/mL, and 0.8 ± 0.3 mmol/L for group 1, and 0.4 ± 0.1 ng/mL, 87.6 ± 39.5 pg/mL, 2154.1 ± 886.0 pg/mL, and 0.9 ± 0.1 mmol/L for group 2, respectively. In group 1, the longitudinal muscle layers achieved higher (P≤0.05) spontaneous contractile values for AUC, MA, maxA, and FR than the circular one; whereas in group 2 the circular layer attained higher median values for BR (P≤0.05), and during T5 for BR (P≤0.05), minA (P≤0.05), maxA (P≤0.05), AUC (P=0.8) and MA (P=0.8). The stimulation with Oxy produced in all cases higher contractile values (P≤0.05) than PGF, Ca and Cont (Oxy>PGF>Ca, Cont). The longitudinal muscle layers of group 1 were numerically more reactive to the stimulation with Oxy and PGF than the circular ones. However, in group 2, the circular muscle layers reached non-significant higher values after the administration of Oxy, but numerically lower values after the stimulation with PGF and Ca compared to the longitudinal ones. Before calving, the longitudinal muscle layers originating from animals with higher concentrations of E2s in vivo exhibited more spontaneous contractile activity. In contrast, longitudinal strips from postpartum animals with lower E2s blood concentrations displayed more spontaneous contractile activity. Prior to calving, both types of strips contracted more if the in vivo P4 concentration was higher. Yet, after calving, the longitudinal muscle layers exhibited higher values for AUC, MA, and maxA, whereas circular strips showed higher minA if they originated from cows with lower blood levels of P4. Concerning the immunohistochemical analysis, PR and ERα were localized in the nuclei of the uterine cells, OTR in cytoplasm and FPR in both locations. PR was only detected in both compact and reticulated stroma (CS and RS, respectively) and in myometrium (MYO). Cells from the surface epithelium (SE), CS, RS, glands (G), vessel wall (VW) and MYO were ERα, OTR and FPR-positive in different proportions (ERα, FPR) or intensities (FPR, OTR). The myometrial expression of ERα, PR, OTR and FPR mRNA transcripts was 16.7 ± 0.4, 9.1 ± 0.8, 16.0 ± 0.7, and 14.2 ± 0.7 ΔCq for group 1, and 22.9 ± 0.7, 31.6 ± 0.6, 24.6 ± 0.9, and 27.3 ± 1.9 ΔCq for group 2, respectively. In both groups, the subgroup E2s high there were differences and tendencies of higher intensity values of cytoplasm stain in VW (group1: P≤0.05; group 2: P=0.09) for OTR, and the PR mRNA expression was numerically enhanced (Group 1: P=0.06; Group 2: P=0.09) compared to E2s low. Additionally, in group 1 there was a tendency for higher ERα mRNA expression (P=0.08) compared to E2s low. In summary, the longitudinal and the circular muscle layer of the bovine uterus around parturition have to be considered as two different entities, because of their distinctive behavior in the organ bath. Moreover, the levels of P4 and E2s might be determinant for the regulation of the contractile activity before and after calving, but seem to influence the uterine layers differently. Further investigations on receptor expression with respect to uterine layer are needed, to elucidate the extent of the influence of the hormonal background on the regulation of the uterine contractility.