159 CHANGES IN PROTEIN EXPRESSION PROFILES IN BOVINE ENDOMETRIAL EPITHELIAL CELLS (bEEC) FOLLOWING E. COLI LIPOPOLYSACCHARIDE CHALLENGE
Y. Z. Guo A , C. Piras B , A. Soggiu B , M. Chanrot A C , R. Båge A , G. Andersson D , P. Reinaud E , G. Charpigny E , O. Sandra E , J. F. Valarcher F , P. Roncada G and P. Humblot AA Department of Clinical Sciences, SLU, Uppsala, Sweden;
B Department of Veterinary Sciences & Public Health, University of Milan, Milan, Italy;
C Faculty of Veterinary Science, Rajamangala University of Technology, Thailand;
D Department of Animal Breeding and Genetics, SLU, Uppsala, Sweden;
E INRA, UMR1198, Biology of Development & Reproduction, France;
F Department of Virology, Immunobiolgy and Parasitology, SVA, Uppsala, Sweden;
G Istituto Sperimentale Italiano L. Spallanzani, Milan, Italy
Reproduction, Fertility and Development 27(1) 170-170 https://doi.org/10.1071/RDv27n1Ab159
Published: 4 December 2014
Abstract
E. coli is one of the most frequent bacteria involved in uterine diseases. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria involved in the pathogenic processes leading to postpartum metritis and endometritis in cattle. It also causes inflammation of the endometrium. Increase of cell proliferation by LPS is part of the inflammatory process and has been reported in human epithelial and immune cells (Martin et al. 2000 J. Immunol. 165, 139–147) and from bovine endometrial epithelial cells (bEEC) (Guo et al. 2014 Reprod. Fertil. Dev. 26, 165–166). The aim of this study was to investigate possible changes in protein expression in relation with the proliferative response of bEEC after challenge with E. coli-LPS. In vitro culture of bEEC was performed from 3 cows. On passage 5, bEEC from each individual were exposed to 0, 8, and 16 µg mL–1 LPS for 72 h. At time 0 and 72 h later, attached cells were counted and for each time and LPS dosage, cells were frozen for proteomic analyses. The variation of cells number over time was analysed by ANOVA (SAS 9.1, proc GLM; SAS Institute, Inc., Cary, NC, USA). All samples were analysed (every sample run in triplicate) by 2-D gel electrophoresis coupled to matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/time-of-flight (TOF) mass spectrometry (MS) and shotgun nLC-MS/MS analysis. As reported before, a significant increase in cell number was observed for cells treated with 8 µg mL–1 LPS (P ≤ 0.001), whereas changes in cell number were highly variable and nonsignificant for 16 µg mL–1 LPS. From each sample, ~800 proteins were visualised. Results from 2-D gel coupled to MALDI-TOF/TOF were very reproducible (same responses between individual cows) and revealed changes in protein profiles very much related (from P < 0.05 to P < 0.01) to proliferative phenotypes for seven proteins. From shotgun analysis, 27 proteins were found significantly differentially expressed (P < 0.05 to P < 0.01) following exposure to LPS (21 up-regulated and 6 down-regulated). Among the 21 found as up-regulated, 20 were differentially expressed both for the 8 and 16 µg mL–1 LPS, whereas 5 out of 6 were down-regulated for both dosages. Differentially expressed proteins were associated to cell proliferation, apoptosis, oxidative stress, regulation of histones, allergy, and general cell metabolism pathways. Candidate proteins need to be confirmed from larger series of individuals and relevant pathways further studied.
Research was partially funded by RMUSTV.
