The global effect of follicle-stimulating hormone and tumour necrosis factor a on gene expression in cultured bovine ovarian granulosa cells

Background: Oocytes mature in ovarian follicles surrounded by granulosa cells. During follicle growth, granulosa cells replicate and secrete hormones, particularly steroids close to ovulation. However, most follicles cease growing and undergo atresia or regression instead of ovulating. To investigate the effects of stimulatory (follicle-stimulating hormone; FSH) and inhibitory (tumour necrosis factor alpha; TNF alpha) factors on the granulosa cell transcriptome, bovine ovaries were obtained from a local abattoir and pools of granulosa cells were cultured in vitro for six days under defined serum-free conditions with treatments present on days 3-6. Initially dose-response experiments (n = 4) were performed to determine the optimal concentrations of FSH (0.33 ng/ml) and TNF alpha (10 ng/ml) to be used for the microarray experiments. For array experiments cells were cultured under control conditions, with FSH, with TNF alpha, or with FSH plus TNF alpha (n = 4 per group) and RNA was harvested for microarray analyses. Results: Statistical analysis showed primary clustering of the arrays into two groups, control/FSH and TNF alpha/TNF alpha plus FSH. The effect of TNF alpha on gene expression dominated that of FSH, with substantially more genes differentially regulated, and the pathways and genes regulated by TNF alpha being similar to those of FSH plus TNF alpha treatment. TNF alpha treatment reduced the endocrine activity of granulosa cells with reductions in expression of FST, INHA, INBA and AMH. The top-ranked canonical pathways and GO biological terms for the TNF alpha treatments included antigen presentation, inflammatory response and other pathways indicative of innate immune function and fibrosis. The two most significant networks also reflect this, containing molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor beta signalling, and these were up regulated. Upstream regulator analyses also predicted TNF, interferons. and beta 1 and interleukin 1 beta. Conclusions: In vitro, the transcriptome of granulosa cells responded minimally to FSH compared with the response to TNF alpha. The response to TNF alpha indicated an active process akin to tissue remodelling as would occur upon atresia. Additionally there was reduction in endocrine function and induction of an inflammatory response to TNF alpha that displays features similar to immune cells.