A granulosa cell culture model of lipoprotein and cholesterol metabolism associated gene expression
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High Density Lipoprotein (HDL) is the sole lipoprotein present within the preovulatory ovarian follicular fluid (FF) and there is increasing evidence that FF-HDL plays an important role in mammalian female fertility. Granulosa cells (GCs) line the follicle and surround the oocyte, acquire and metabolize cholesterol for steroidigenesis and are responsible for most of the cholesterol trafficking to and from the oocyte. The purpose of this study was to simulate the physiologic conditions of the pre-ovulatory ovarian follicle with regard to HDL composition and to investigate GC lipoprotein signaling and cholesterol metabolism related gene expression in an in vitro cell model. Materials and methods . Human granulosa cell line KGN was obtained from Riken Bioresources (Japan). Culture conditions were optimized for growth rate and basal progesterone production. Luteinization was stimulated using dibutyryl cyclic adenosine monophosphate (dbcAMP) and Follicle Stimulating Hormone (FSH). Progesterone production was used as an indicator of luteinization by enzyme immunoassay. Pre beta HDL (the characteristic HDL particle of FF) was isolated through column chromatographic techniques and added to the basal and stimulated KGN cultures. Effect of KGN cells on media supplemented HDL particles was determined using non-denaturing PAGE and western blotting with ApoA1. The changes in gene expression of KGN cells treated under different conditions were determined using a lipoprotein and cholesterol metabolism focused RT-PCR array. Results . F12/DMEM with 10% FBS was determined to be the optimal basal media to culture KGN cells as cells had the best growth rate with FBS and also the lowest level of unstimulated progesterone production. Luteinization with both dbcAMP and FSH exhibited a dose-dependent response in progesterone production. Pre-beta HDL was successfully isolated using DEAE ion exchange chromatography, but the progesterone content from the starting follicular fluid was not successfully eliminated. Changes in HDL particle size upon isolation and purification were not evident by NMR analysis. RT-PCR array results showed significant upregulation in the apolipoprotein ApoA-4, elastase and zinc finger related genes. Conclusions . The KGN cell line produces progesterone in response to luteinization and hence is useful for studies of the pre-ovulatory follicle. The method to isolate HDL requires refinement to eliminate progesterone from follicular fluid as progesterone inhibits the cholesterol biosynthetic pathway and would prevent the KGN cells from utilizing HDL-cholesterol. Further experiments are required to confirm the upregulation of lipoprotein and cholesterol metabolism related genes in this model. If confirmed, these results would indicate the first extrahepatic expression of ApoA-4.