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
S E Ulbrich, K Schulke, A E Groebner, H D Reichenbach, C Angioni, G Geisslinger and H H D Meyer
Prostaglandins (PGs) are important regulators of reproductive processes including early embryonic development. We analyzed the most relevant PG in bovine uteri at different preimplantation pregnancy stages when compared with non-pregnant controls. Additionally, endometrium and trophoblast tissues were examined regarding specific enzymes and receptors involved in PG generation and function. Simmental heifers were artificially inseminated or received seminal plasma only. At days 12, 15, or 18, post-estrus uteri were flushed for PG determination by liquid chromatography–tandem mass spectrometry. Endometrium and trophoblast tissues were sampled for RNA extraction and quantitative real-time PCR analysis. At all days and points of time examined, the concentration of 6-keto PGF1α (stable metabolite of PGI2) was predominant followed by PGF2α>PGE2>PGD2≈TXB2 (stable metabolite of TXA2). At days 15 and 18, PG increased from overall low levels at day 12, with a much more pronounced increase during pregnancy. The PGF2α/PGE2 ratio was not influenced by status. The highest PG concentration was measured at day 15 with 6-keto PGF1α (6.4 ng/ml) followed by PGF2α (1.1 ng/ml) and PGE2 (0.3 ng/ml). Minor changes in endometrial PG biosynthesis enzymes occurred due to pregnancy. Trophoblasts revealed high transcript abundance of general and specific PG synthases contributing to uterine PG. As PGI2 and PGF2α receptors were abundantly expressed by the trophoblast, abundant amounts of PGI2 and PGF2α in the uterine lumen point towards an essential role of PG for the developing embryo. High amounts of PG other than PGE2 in the preimplantation uterus may be essential rather than detrimental for successful reproduction.
V A van der Weijden, J T Bick, S Bauersachs, G J Arnold, T Fröhlich, B Drews and S E Ulbrich
The uterine microenvironment during pre-implantation presents a pro-survival milieu and is essential for embryo elongation in ruminants. The European roe deer (Careolus capreolus) pre-implantation embryo development is characterised by a 4-month period of reduced development, embryonic diapause, after which the embryo rapidly elongates and implants. We investigated the uterine fluid proteome by label-free liquid chromatography tandem mass spectrometry at four defined stages covering the phase of reduced developmental pace (early diapause, mid-diapause and late diapause) and embryo elongation. We hypothesised that embryo development during diapause is halted by the lack of signals that support progression past the blastocyst stage. Three clusters of differentially abundant proteins were identified by a self-organising tree algorithm: (1) gradual reduction over development; (2) stable abundance during diapause, followed by a sharp rise at elongation; and (3) gradual increase over development. Proteins in the different clusters were subjected to gene ontology analysis. ‘Cellular detoxification’ in cluster 1 was represented by alcohol dehydrogenase, glutathione S-transferase and peroxiredoxin-2. ATP-citrate synthase, nucleolin, lamin A/C, and purine phosphorylase as cell proliferation regulators were found in cluster 2 and ‘cortical cytoskeleton’, ‘regulation of substrate adhesion-dependent cell spreading’ and ‘melanosome’ were present in cluster 3. Cell cycle promoters were higher abundant at elongation than during diapause, and polyamines presence indicates their role in diapause regulation. This study provides a comprehensive overview of proteins in the roe deer uterine fluid during diapause and forms a basis for studies aiming at understanding the impact of the lack of cell cycle promoters during diapause.
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.
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.