Cholesterol and bile acid metabolism in an ovarian granulosa cell model of folliculogenesis
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Background: High density lipoprotein (HDL) cholesterol levels predict embryo quality during in vitro fertilization (IVF) in the maturing human ovarian follicle. The cholesterol essential for oocyte development is solely provided via bidirectional communication with the surrounding granulosa cells. This study investigates lipoprotein signaling and cholesterol metabolism associated gene expression in an in vitro cell culture model of ovarian granulosa cells. Methods: The granulosa cell line KGN was stimulated with follicle stimulating hormone and cell permeable dibutyryl cAMP in physiologic media, to simulate folliculogenesis. Luteinization was confirmed by measuring media progesterone levels by enzyme immunoassay (EIA). Gene expression in response to FSH and cAMP treatments was measured using a focused real time-polymerase chain reaction (RT-PCR) array for eighty-eight genes involved in lipoprotein signaling and cholesterol metabolism pathways. Quantitative PCR (qPCR) was used in an attempt to confirm significant changes in expression of a novel gene, CYP7A1. Cellular localization of the enzyme CYP7A1 was studied by immunofluorescence and the level of the enzymatic product of CYP7A1:7&agr;-hydroxy cholesterol along with the major bile acids was analyzed by LC-MS. Results: RT-PCR arrays showed significant increase in expression of 71 genes involved in cholesterol synthesis, steroidogenesis, HDL transport and bile acid synthesis. The upregulation of CYP7A1 – the gene coding for the key regulatory enzyme of the bile acid synthetic pathway, is a novel finding. Hence the upregulation of CYP7A1 was sought to be confirmed by gene-specific qPCR. However CYP7A1-specific qPCR could not confirm the upregulation observed by the PCR array. No difference in the levels of 7&agr;-hydroxy cholesterol or any of the major bile acids was observed by LC-MS analysis. Immunofluorescence studies suggested the enzyme CYP7A1 to be present mainly in the perinuclear region. Conclusion: The results demonstrate that, beyond steroidogenesis, granulosa cells are actively engaged in cholesterol biosynthesis and uptake. However qPCR analysis could not confirm upregulation of CYP7A1, the key regulatory gene for bile acid synthesis, LC-MS analysis did not suggest any significant changes in the levels of bile acids or their metabolites. Our results therefore suggest that the expression of CYP7A1 is unchanged in KGN cells in response to the luteinizing treatments.