The objective was to characterize effects of Escherichia coli LPS (given i.v.) on corpus luteum (CL) and embryonic viability in early pregnant cattle. Eight non-lactating German Holstein cows were given 0.5 µg/kg LPS on 35 ± 3 day (mean ± s.e.m.) of pregnancy, whereas seven heifers, 41 ± 6 day pregnant, were given 10 mL saline (control group). Transrectal B-mode examinations of the CL were done at −1, 3, 6, 12, 24, 48, 72 and 96 h relative to treatment. Blood samples were collected at −1, 0.5, 1, 2, 3, 4, 6, 9, 12, 24, 48, 72 and 96 h. At 12 and 48 h, the CL was biopsied. None of the cows still in the experiment 10 day after LPS (n = 7) had embryonic loss. In LPS-treated cows, luteal area decreased (from 4.1 to 3.1 cm2; P ≤ 0.05) within 6 h and until 48 h. Luteal blood flow decreased by 39% (P ≤ 0.05) within the first 6 h after LPS, but returned to pre-treatment values by 48 h. Plasma P4 decreased by 62% (P ≤ 0.05), reached a nadir (2.7 ± 0.6 ng/mL) at 12 h after LPS and was not restored to pre-treatment (P ≤ 0.05). In luteal tissue, mRNAs for STAR and for FGF1 were lower (P ≤ 0.05) in LPS than in saline-treated cattle at 12 h, with no difference between groups at 48 h. Levels of mRNAs for CASP3 and FGF2 were not different between groups (P > 0.05) at 12 or 48 h after treatment. In conclusion, LPS transiently suppressed CL function, but did not induce embryonic mortality.
K Herzog, L Debertolis, J P Kastelic, M Schmicke, S E Ulbrich and H Bollwein
J Lüttgenau, B Möller, D Kradolfer, O Wellnitz, R M Bruckmaier, A Miyamoto, S E Ulbrich and H Bollwein
Lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria, has detrimental effects on the structure and function of bovine corpus luteum (CL) in vivo. The objective was to investigate whether these effects were mediated directly by LPS or via LPS-induced release of PGF2α. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and isolated perfused for 240 min. After 60 min of equilibration, LPS (0.5 μg/ml) was added to the medium of five ovaries, whereas an additional six ovaries were not treated with LPS (control). After 210 min of perfusion, all ovaries were treated with 500 iu of hCG. In the effluent perfusate, concentrations of progesterone (P4) and PGF2α were measured every 10 and 30 min, respectively. Punch biopsies of the CL were collected every 60 min and used for RT-qPCR to evaluate mRNA expression of receptors for LPS (TLR2, -4) and LH (LHCGR); the cytokine TNFA; steroidogenic (STAR, HSD3B), angiogenic (VEGFA 121, FGF2), and vasoactive (EDN1) factors; and factors of prostaglandin synthesis (PGES, PGFS, PTGFR) and apoptosis (CASP3, -8, -9). Treatment with LPS abolished the hCG-induced increase in P4 (P≤0.05); however, there was a tendency (P=0.10) for increased release of PGF2α at 70 min after LPS challenge. Furthermore, mRNA abundance of TLR2, TNFA, CASP3, CASP8, PGES, PGFS, and VEGFA 121 increased (P≤0.05) after LPS treatment, whereas all other factors remained unchanged (P>0.05). In conclusion, reduced P4 responsiveness to hCG in LPS-treated ovaries in vitro was not due to reduced steroidogenesis, but was attributed to enhanced apoptosis. However, an impact of luteal PGF2α could not be excluded.
J Lüttgenau, K Herzog, K Strüve, S Latter, A Boos, R M Bruckmaier, H Bollwein and M P Kowalewski
When given intravenously (iv), lipopolysaccharide (LPS) transiently suppresses the structure and function of the bovine corpus luteum (CL). This is associated with increased release of prostaglandin (PG) F2α metabolite. The underlying regulatory mechanisms of this process remain, however, obscure. Therefore, the aims of this study were: i) to investigate the expression of the LPS receptor toll-like receptor 4 (TLR4) and 2 (TLR2) in the bovine CL during early, mid- and late luteal phases; and ii) to further dissect the mechanisms of LPS-mediated suppression of luteal function. As revealed by semi-quantitative qPCR and immunohistochemistry, both receptors were detectable throughout the luteal lifespan. Their mRNA levels increased from the early toward the mid-luteal phase; no further changes were observed thereafter. The TLR4 protein seemed more highly represented than TLR2. The cellular localization of TLRs was in blood vessels; weaker signals were observed in luteal cells. Additionally, cows were treated either with LPS (iv, 0.5 μg/kg BW) or with saline on Day 10 after ovulation. Samples were collected 1200 h after treatment and on Day 10 of the respective subsequent (untreated) cycle. The mRNA expression of several possible regulatory factors was investigated, revealing the suppression of PGF2α receptor (PTGFR), STAR protein and 3β-hydroxysteroid dehydrogenase, compared with controls and subsequent cycles. The expression of TLR2 and TLR4, interleukin 1α (IL1A) and 1β (IL1B) and of PGF2α and PGE2 synthases (HSD20A and mPTGES respectively) was increased. The results demonstrate the presence of TLR2 and TLR4 in the bovine CL, and implicate their possible involvement in the deleterious effects of LPS on its function.
K Herzog, K Strüve, J P Kastelic, M Piechotta, S E Ulbrich, C Pfarrer, K Shirasuna, T Shimizu, A Miyamoto and H Bollwein
The objective was to characterize the effects of Escherichia coli lipopolysaccharide (LPS) endotoxin (given i.v.) on luteal structure and function. Seven nonlactating German Holstein cows, 5.1±0.8 years old (mean±s.e.m.), were given 10 ml saline on day 10 (ovulation=day 1) of a control estrous cycle. On day 10 of a subsequent cycle, they were given 0.5 μg/kg LPS. Luteal size decreased (from 5.2 to 3.8 cm2, P≤0.05) within 24 h after LPS treatment and remained smaller throughout the remainder of the cycle. Luteal blood flow decreased by 34% (P≤0.05) within 3 h after LPS and remained lower for 72 h. Plasma progesterone (P4) concentrations increased (P≤0.05) within the first 3 h after LPS but subsequently declined. Following LPS treatment, plasma prostaglandin (PG) F metabolites concentrations were approximately tenfold higher in LPS-treated compared with control cows (9.2 vs 0.8 ng/ml, P≤0.05) within 30 min, whereas plasma PGE concentrations were nearly double (P≤0.05) at 1 h after LPS. At 12 h after treatment, levels of mRNA encoding Caspase-3 in biopsies of the corpus luteum (CL) were increased (P≤0.05), whereas those encoding StAR were decreased (P≤0.05) in cattle given LPS vs saline. The CASP3 protein was localized in the cytoplasm and/or nuclei of luteal cells, whereas StAR was detected in the cytosol of luteal cells. In the estrous cycle following treatment with either saline or LPS, there were no significant differences between groups on luteal size, plasma P4 concentrations, or gene expression. In conclusion, LPS treatment of diestrus cows transiently suppressed both the structure and function of the CL.