PSII-5 Endotoxin-Induced Inflammation on Immune Markers and Steroidogenesis in Granulosa Cells

Abstract

Abstract Reproductive performance is central to profitability in beef cattle operations. Pathogenic stress can result from gram-negative bacteria release of the lipopolysaccharide (LPS) endotoxin which has the potential to accumulate in follicular fluid and impair folliculogenesis and steroidogenesis, leading to reduced fertility. Elevated LPS concentrations observed in acute clinical bacterial infections are recognized to have detrimental effects on female fertility, however, definitive effects of low concentrations of LPS present in subclinical or undetectable infection remains to be fully elucidated. This study investigated the effect of varying concentrations of LPS on mechanisms regulating ovarian function and subsequent steroidogenesis. A granulosa cell line (KGN) was cultured with or without increasing doses of LPS (0.0001 to 10 µg/mL) for 30 min or 48 h. Cells collected 30 min after treatment were analyzed for abundance of phosphorylated proteins and cells collected 48 h after treatment were analyzed for total protein abundance and gene expression. Media were analyzed by radioimmunoassay for steroid hormone concentrations. Quantitative and semi-quantitative PCR was used to evaluate aromatase, beta-catenin, and cytokines IL-6 and IL-8, respectively. No differences were detected among LPS treatment groups compared to vehicle-treated controls for protein abundance of total beta-catenin (P = 0.99) or active beta-catenin lacking phosphorylation at serine 33/37 (P = 0.95) evaluated at 48 h. Similar results were observed in protein abundance of phosphorylated forms of beta-catenin at serine 552 (P = 0.98) and 675 (P = 0.85) evaluated at 30 min. Additionally, estradiol concentrations were not different at 0.0001 to 1.0 µg/mL LPS; however, estradiol was significantly decreased at 10 µg/mL LPS treatment compared to the vehicle-treated control (P = 0.03). Progesterone concentrations were also similar within 0.0001 to 1.0 µg/mL LPS treatment groups with a tendency to decrease at 10 µg/mL LPS treatment (P = 0.08) compared to the vehicle-treated control. Varying concentrations of LPS did not change mRNA expression of beta-catenin (P = 0.28), aromatase (P = 0.32), and IL-6 (P = 0.35). However, a significant increase in cytokine IL-8 mRNA expression was observed at 10 µg/mL LPS compared to the vehicle-treated control (P = 0.04). These findings suggest that inflammatory response against LPS at higher concentrations may influence distinct cytokine expression and steroidogenesis. Conversely, basal expression of key steroidogenic enzymes and ovarian steroidogenic modulators are less affected by low concentrations of LPS. Therefore, immune response of granulosa cells is dependent on concentration of LPS exposure that has the ability to impact fertility by modulating the ovarian hormonal and follicular environment.