Ovarian Bile Acid Synthesis Figure 2. Presence of enzymes and receptors regulating the classical and alternative pathways of bile acid synthesis. In the cytosol of granulosa cells, specific positive staining was registered for CYP7A1 (Panel A, image 1), CYP27A1 (Panel A, image 2), and CYP7B1 (Panel A, image 3). Specific positive staining for Inhibin b was used to confirm granulosa cell type (Panel A, image 4). In oocytes, specific positive staining was identified for CYP7A1 (Panel C, image 1), CYP7B1 (Panel C, image 2), and CYP8B1 (Panel C, image 3). The receptors known to regulate the bile acid synthesis pathway were also present in granulosa cells and oocytes. In granulosa cells, specific positive staining was registered for FXR (Panel B, image 1), LRH-1 (Panel B, image 2), and RXRa (Panel B, image 3). Using western blot of granulosa cells, a specific reacting band for the receptor LXRa was observed (Panel B, image 4). In human oocytes, specific positive staining was also demonstrated for the receptors FXR (Panel D, image 1), RXRa (Panel D, image 2), and LRH-1 (Panel D, image 3). Secondary antibody controls are presented in images 4 of Panels C and D. doi:10.1371/journal.pone.0007333.g002 Enzymes in the Bile Acid Synthesis Pathway are Present in the Ovarian Follicle Initially, we applied gene chip array to search for the presence of mRNA transcripts of these enzymes in granulosa cells from 10 patients. The assay identified the presence of mRNA for the key enzymes in the classical and the alternative pathways of bile acid synthesis: CYP7A1, CYP27A1, CYP7B1, and CYP8B1 (Figure 2a). Next, the presence of these genes was verified at the protein level by immunofluorescence (IF) with specific antibodies. Granulosa cells from four different patients were analyzed for the presence of CYP7A1, CYP27A1, CYP7B1, and CYP8B1. Specific positive staining was registered for enzymes from both the classical and alternative pathways of bile acid synthesis, including CYP7A1, the rate-limiting enzyme in the classical pathway, CYP27A1, the first and rate-limiting enzyme of the alternative pathway, and CYP7B1 from the alternative pathway of bile acid synthesis (Figure 2b, panel A). The enzyme CYP8B1 was not detected in granulosa cells. Although the result was negative, it served as IF specificity control (not shown). It is possible that the oocytes which reside within the ovarian follicle also participate in the synthesis of bile acids. We analyzed discarded, non-fertilized human oocytes by IF with specific antibodies for the key enzymes in both bile acid synthesis pathways. We identified specific positive staining of CYP7A1, CYP7B1, and CYP8B1 (Figure 2b, panel C). The immunostaining for CYP27A1 from the alternative pathway was negative (not shown). Bile acid enzyme data from IF in granulosa cells and oocytes are summarized in Table 1. The Bile Acid Synthesis Pathway is Functional in the Ovarian Follicle To test for bile acids synthesis in granulosa cells we cultured primary human cumulus granulosa cells (CGC) and exposed them to media containing different cholesterol concentrations (13.95mg/dL, 26.09mg/dL, 46.15mg/dL) loaded in human low density lipoprotein (LDL) particles. After 48 hours culture medium and cells have been analyzed for presence of bile acids at time 0 Table 1. Presence of bile acids synthesis enzymes in human ovarian follicle. Classical Pathway Enzyme Granulosa Cells CYP7A1 CYP8B1 CYP27A1 CYP27A1 CYP7B1 Alternative Pathway Enzyme present absent present present present Oocytes present present absent absent present Protein presence was analyzed with enzyme specific antibodies and immunofluorescence. Present - the enzyme is present at a protein level; absent - the enzyme is not present at a protein level. doi:10.1371/journal.pone.0007333.t001 PLoS ONE | www.plosone.org 3 October 2009 | Volume 4 | Issue 10 | e7333