and time 48 hours. The experiment was repeated in triplicate. We measured a CGC-specific dose-dependent increase in production of bile acids in response to increasing cholesterol in CGC media (Figure 3). The results of this experiment clearly demonstrate that CGC respond to changes in cholesterol concentration by synthesizing bile acids. This suggests that the enzymes which we have identified to be present in the granulosa cells are part of a functional pathway in these cells. The presence of all major constituents of the classical pathway, including elements never found outside the liver is curious and novel. The relative contributions of the classical and alternative pathways to the observed bile acid synthesis remains to be determined. Receptors Known to Regulate the Bile Acid Synthesis Pathway are Present in the Ovarian Follicle The bile acid synthesis pathway is known to be regulated by nuclear receptors. We examined primary human granulosa cells Figure 3. Bile acid production in primary cultured human cumulus granulosa cells (CGC). Cholesterol entering granulosa cells may be used to form sex steroids or bile acids (Panel a). With increasing cholesterol concentration in media, CGC demonstrate dose-dependent bile acid content (Panel b). doi:10.1371/journal.pone.0007333.g003 Ovarian Bile Acid Synthesis and discarded, non-fertilized oocytes for presence of FXR, RXRa, LXRa, and LRH-1 by protein-specific antibodies and IF. FXR, which is known to have as its ligand bile acids, was found to be present in the cytosol of granulosa cells (Figure 2, panel B) as well as in the oocytes (Figure 2, panel D) [11–13]. To our knowledge, this is the first report of this receptor in the human ovary. Furthermore, by staining granulosa cells and oocytes with RXRa and LRH-1 specific antibodies we observed specific positive staining for both receptors in three independent experiments with cells from three different patients (Figure 2, panels B and D). The receptor LXRa has never been previously described in the human ovary [6,23–25]. By applying western blot (WB) with LXRa specific antibodies we consistently observed specifically reacting 64kDa band that correspond to LXRa in granulosa cell lysates from four different healthy patients (Figure 2, panel B). Data for presence of receptors from the bile acids pathways are summarized in Table 2. Discussion Our results strongly argue for the presence of all of the components of the bile acid synthesis pathway, including the key enzymes in bile acid synthesis as well as the receptors that regulate expression of these enzymes, and the end product bile acids. To our knowledge, this is the first report of this pathway in the human ovary. Although it is widely accepted that the classical pathway of bile acid synthesis is present only in the liver, our data clearly suggests that the key enzymes from both the classical and alternative synthesis pathways are present in the ovarian granulosa cells and oocytes. To our knowledge, these findings are the first demon- stration for presence of CYP7A1, CYP27A1, CYP7B1, and CYP8B1 at both mRNA and protein level in granulosa cells. This is also the first demonstration of the presence of these enzymes in the oocytes as proteins. CYP7A1 is considered to be a liver-specific enzyme, although it has been reported to be present in human prostate [6]. CYP27A1 has been identified in a wide range of extrahepatic tissues, but has not been previously reported in the human ovary [26]. CYP8B1 has only been identified in the liver prior to our experimental results [27]. CYP7B1 is known to have wide tissue distribution, but has not been previously identified in the human ovarian follicle [9]. The presence of bile acids and the key enzymes of both pathways in ovarian follicles suggests for the presence of a fully functional ovarian pathway locally producing bile acids. Presently it is not clear whether bile acids have been synthesized mainly by the granulosa cells, oocytes, or both. Our results suggest that granulosa cells have the key enzymes to synthesize bile acids from cholesterol, and that this is an active Table 2. Presence of receptors known to regulate the bile acids synthesis pathways. Receptor FXR RXR alpha LRH1 LXR alpha Granulosa Cells present, IF present, IF present, IF present, WB Oocytes present, IF present, IF present, IF absent Protein presence was analyzed with receptor specific antibodies and either immunofluorescence (IF) or western blot (WB). (Present) – the receptor is present at a protein level; (absent) – the receptor is not present at a protein level. doi:10.1371/journal.pone.0007333.t002 PLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7333