Evaluating lipopolysaccharide-induced oxidative stress in bovine granulosa cells.

PURPOSE: The purpose of this study was to evaluate the capacity of bovine granulosa cells to generate reactive oxygen intermediates in response to lipopolysaccharide. We hypothesized that granulosa cells increase reactive oxygen intermediates in response to Gram-negative lipopolysaccharide in a similar manner to immune cells. METHODS: Bovine peripheral blood mononuclear cells and granulosa cells were cultured in the presence of lipopolysaccharide. Oxidative stress was evaluated using the fluorescent marker dye CellROX, and oxidative stress-related genes were measured using real-time RT-PCR. RESULTS: As expected, peripheral blood mononuclear cells increased oxidative stress in response to lipopolysaccharide as measured by accumulation of the fluorescent marker dye CellROX. While granulosa cells demonstrate the capacity to increase accumulation of CellROX dye in response to a positive control menadione, lipopolysaccharide had no effect on accumulation of CellROX dye. The expression of GSR, SOD1, and SOD2 were variable in peripheral blood mononuclear cells treated with lipopolysaccharide but were consistently upregulated when co-incubated with the antioxidant, N-acetyl cysteine. The expression of oxidative stress-related genes was not altered in granulosa cells, with the exception of elevated SOD1 following lipopolysaccharide exposure in the absence of antioxidant. CONCLUSIONS: Combined, these data suggest that while reactive stress is important in pathogen killing and inflammation in immune cells, granulosa cells do not increase oxidative stress in response to lipopolysaccharide.

Characterisation of peripheral blood mononuclear cell populations in periparturient dairy cows that develop metritis.

Bacterial contamination of the uterus following calving is ubiquitous in the dairy cow, 40% of cows develop postpartum uterine infection, including metritis. While predisposing factors like twinning and dystocia are associated with metritis, it is unclear why some cows remain healthy following calving and others develop uterine infection, negatively impacting animal health, milk production and economic return. Here, we profiled peripheral blood mononuclear cells of cows before calving and during postpartum metritis. We hypothesized that peripheral blood mononuclear cell function and proportions would be altered during the prepartum period in cows that develop postpartum metritis. Using flow cytometry we observed reduced proportions of peripheral CD3+/CD4+, CD4+/CD62L+, FOXP3+ and CD21+ populations from -10 to 40 days relative to calving associated with metritis, while the proportion of peripheral CD3+/CD4+ lymphocytes were specifically reduced in the prepartum period before the onset of metritis. Peripheral blood mononuclear cells from cows with metritis had a perturbed capacity to secrete IL-1ß or IFNγ in response to in vitro stimulus; cells collected during the prepartum period from cows that would go on to develop metritis failed to increase IL-1ß secretion in response to stimulation, while IFNγ secretion was altered at calving and postpartum in cows with metritis compared to healthy herd mates. No effect of metritis was observed in the capacity of cows to mount a humoral immune response to antigen administered on the day of calving. The studies discussed here suggest that while minor changes to the prepartum immune system are observed in cows that develop metritis, changes observed in the postpartum period are more prevalent and likely a consequences of disease and not causative. Future studies to modulate the prepartum immune system may help to limit postpartum metritis.