functional pathway. The balance between cholesterol use in steroidogenesis and degradation in the bile acid pathway is not known, but most likely there is a fine balance which may prove crucial to oocyte maturation. In support of this hypothesis are recently published experimental data with rodents where a role for LRH-1 in steroidogenesis, gonadal development, aromatase expression, and progesterone biosynthesis was clearly documented [17,28]. Another recent study has linked LRH-1 with ovulation in a rodent model, demonstrating that this receptor is required for rodent ovarian follicular development and fertility [17]. Given that the ovarian follicle is a highly privileged environ- ment, it is likely that bile acids play a specific role beyond simply acting as detergents and carriers of cholesterol. Full characteriza- tion of the follicular-specific role of bile acids remains to be determined. Methods Ethics statement Follicular fluid, granulosa cells and blood serum were obtained under Beth Israel Deaconess Medical Center (BIDMC) IRB approval (Protocol #2003-P-000289) after appropriate written informed consent from women undergoing in IVF. Discarded, non-fertilized metaphase II human oocytes were obtained under BIDMC IRB approval with waiver of informed consent from IVF cycles in which oocytes failed to fertilize and would have otherwise been discarded. Waiver of informed consent for discarded, non- fertilized oocytes was granted by BIDMC IRB because no patient- identifying information was obtained with the pooled, anonymous samples of discarded tissue, and no protected health information was used. Follicular fluid, granulosa cell, serum, and discarded, non- fertilized oocyte collection and processing All follicular fluid from large (mean diameter .16mm) follicles, granulosa cells and blood plasma retrievals have been performed at the Boston IVF Surgery Center of Waltham, MA as previously described [29]. Discarded, non-fertilized metaphase II human oocytes were obtained under BIDMC IRB approval with waiver of informed consent from IVF cycles in which oocytes failed to fertilize and would have otherwise been discarded. Oocytes were fixed in 4% paraformaldehyde for 20 minutes at RT and stored at 4uC for immunofluorescent analyses. Total bile acid assays Total bile acid concentration in follicular fluid and cell culture media was determined using the BioQuant Total Bile Acids Assay Kit (Enzymatic Cycling, San Diego, CA) as recommended by the supplier. All assays have been repeated in duplicate. Statistical comparisons were made using Microsoft Office Excel 2003 SP2 References 1. Javitt NB (1994) Bile acid synthesis from cholesterol: regulatory and auxiliary pathways. FASEB J 8: 1308–1311. 2. Chiang JYL (1998) Regulation of bile acid synthesis. Front Biosci 3: d176–193. 3. Raufman J-P, Cheng K, Zimniak P (2003) Activation of Muscarinic Receptor Signaling by Bile Acids: Physiological and Medical Implications. Dig Dis Sci 48: 1431–1444. 4. Torchia EC, Stolz A, Agellon LB (2001) Differential modulation of cellular death and survival pathways by conjugated bile acids. BMC Biochem: http:// www.biomedcentral.com/1471-2091/2/11. 5. Ferdinandusse S, Houten SM (2006) Peroxisomes and bile acid synthesis. Biochim et Biophys Acta 1763: 1427–1440. 6. Norlin M, Wikvall K (2007) Enzymes in the Conversion of Cholesterol into Bile Acids. Curr Mol Med 7: 199–218. 7. Nguyen LB, Guorong X, Shefer S, Tint GS, Batta A, et al. (1999) Comparative Regulation of Hepatic Sterol 27-Hydroxylase and Cholesterol 7a-Hydroxylase Ovarian Bile Acid Synthesis version 5.1.2600 (Redmond, WA) with p value ,0.05 considered statistically significant. Western blot and Immunofluorescence Standard technique has been used for western blotting and immunofluorescence [29]. The primary antibodies that have been used are: monoclonal mouse anti-human Farnesoid X receptor (Lifespan Biosciences), polyclonal rabbit anti-human Retinoid X receptor a (Santa Cruz), polyclonal rabbit anti-human Liver X Receptor a (Abcam), monoclonal mouse anti-human Liver Receptor Homolog 1 (R+D Systems), polyclonal goat anti-human CYP7A1 (Santa Cruz), polyclonal goat anti-human CYP7B1 (Santa Cruz), polyclonal goat anti-human CYP8B1 (Santa Cruz), polyclonal goat anti-human CYP27A1 (Santa Cruz), and poly- clonal goat anti-human Inhibin b (Santa Cruz). Granulosa cell culture and bile acids content analysis Primary granulosa cells (passage 4 or less) have been cultured in six wells plates to 70% confluence in DMEM, 10% fetal calf serum (FCS), 2.5 ng/mL bFGF (Sigma Aldrich), and 25 mg/mL Amphotericin B. Cholesterol (Sigma) was added to the cell culture medium as a LDL complex in the following concentrations: 13.95mg/dL, 26.09mg/dL, 46.15mg/dL. After 48 hours incuba- tion with cholesterol, the culture medium from each well has been removed and stored for cholesterol and bile acids content assay. Cells have been also collected to measure the protein content in the individual wells and use it as a base for comparison. Incubation with LDL alone without cholesterol addition has been used as negative control. All measurements have been performed in duplicates and in three independent experiments with different preparations of primary cell cultures. The cholesterol content in the culture mediums was measured by the Cholesterol Liquid Color Assay (StanBio, Boerne TX) as recommended by the supplier. BioQuant Total Bile Acids Assay Kit (San Diego, CA) was used to determine the bile acids content. After removing the culture medium the total cellular protein amount in each well was measured by the Lawry method (Bio-Rad kit) and used as a base for comparison. Granulosa cell gene expression profiling Pooled GC RNA from 10 patients was analyzed for gene expression profile using Agilent gene chip array. Ingenuity Systems Pathway Analysis Software (Redwood City, CA) was used to interpret microchip array data. Author Contributions Conceived and designed the experiments: LPS MN ASP ET AU. Performed the experiments: LPS MN SWY AU. Analyzed the data: LPS MN SWY ASP AU. Wrote the paper: LPS AU. Activities in the Rat, Guinea Pig, and Rabbit: Effects of Cholesterol and Bile Acids. Metab 48: 1542–1548. 8. Hasler JA (1999) Pharmacogenetics of human cytochromes P450. Mol Aspect Med 20: 1–137. 9. Wu Z, Martin KO, Javitt NB, Chiang JYL (1999) Structure and functions of human oxysterol 7a-hydroxylase cDNAs and gene CYP7B1. J Lipid Res 40: 2195–2203. 10. Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, et al. (2000) Molecular Basis for Feedback Regulation of Bile Acid Synthesis by Nuclear Receptors. Mol Cell 6: 507–515. 11. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, et al. (1999) Identification of a Nuclear Receptor for Bile Acids. Science 284: 1362–1365. 12. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, et al. (1999) Bile Acids: Natural Ligands for an Orphan Nuclear Receptor. Science 284: 1365–1368. PLoS ONE | www.plosone.org 5 October 2009 | Volume 4 | Issue 10 | e7333