PSIV-4 - Impact of lipopolysaccharide endotoxin on growth and viability of ovarian granulosa cells

Abstract: Lipopolysaccharide (LPS) endotoxin from Gram-negative bacteria has been demonstrated to negatively impact reproductive parameters leading to ovarian dysfunction. Ovarian granulosa cells express the toll-like receptor 4 complex necessary to elicit inflammatory immune responses in the presence of LPS. Quantifiable amounts of LPS have been detected in follicular fluid from slaughterhouse-derived ovaries with further deleterious effects on granulosa cell steroidogenesis. Therefore, the objective of this study was to investigate the effects of LPS on granulosa cell proliferation in vitro. We hypothesized that increasing concentrations of LPS would negatively impact granulosa cell proliferation dynamics. A human granulosa cell line (KGN) was plated at 60% confluency in 96-well tissue culture plates and treated with phosphate buffered saline-vehicle or LPS (0.01, 0.1, 1, or 10 µg/mL) in the presence or absence of follicle-stimulating hormone (FSH). Cells were incubated for 48 h at 37 ºC and 5% CO₂ in an IncuCyte incubator that utilizes real-time automated microscopy to continuously capture images of cells and measure cell confluency. Individual well images were collected every 6 h. The study was performed in 3 independent experiments with 8 replicates per treatment group in each experiment. Cells were allowed to equilibrate in the IncuCyte for 6 h prior to image collection. Cell confluency for each treatment group was normalized to vehicle-treated control confluency at 6 h. A significant difference was observed at the initial 6 h timepoint, with 1 and 10 µg/mL LPS treatment groups demonstrating lower cell confluency compared with vehicle-treated controls (P < 0.05). However, no significant difference was observed in 0.01 (P = 0.65) and 0.1 (P = 0.53) µg/mL LPS treatment groups compared to vehicle-treated controls at 6 h timepoint. Interestingly, the deleterious effects of LPS on confluency and cell growth parameters were mitigated with the addition of FSH. No observable differences were detected in the two highest LPS doses concurrently incubated with FSH, 1 and 10 µg/mL LPS (P = 0.55 and P = 0.62, respectively), compared with vehicle-treated controls. From 12 h until the end of the incubation period, cell confluency in 1 and 10 µg/mL LPS treatment groups remained significantly different compared to FSH-treated cells (P < 0.05). Comparably, no differences were detected among FSH-stimulated groups, regardless of LPS dose or time (P = 0.99). Results indicate that higher LPS concentrations (1 and 10 µg/mL) negatively impacts cell confluency and proliferation in vitro, however, FSH has the ability to protect granulosa cells against the detrimental effects that LPS has on cell confluency. These data suggest that FSH is critical in regulating cell growth and proliferation despite the action of inflammatory pathways induced by bacterial endotoxin.