A cell culture model of HDL metabolism in human ovarian Granulosa cell line KGN
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Background . High density lipoprotein (HDL) is the sole lipoprotein found in mammalian ovarian follicular fluid (FF) and there is evidence that HDL cholesterol levels and HDL particle remodeling within the ovarian follicle play an important role in the health of the oocyte and resultant embryo. Ovarian Granulosa cells (GCs) are the most likely candidate to affect follicular HDL metabolism in that they line the ovarian follicle, surround the oocyte and, during lutinization and folliculogenesis, take-up large amounts of cholesterol as substrate for steroidogenesis. The purpose of this thesis research is to develop a more physiologic cell culture model of lutinizing GCs and examine the effects of lutinization on cholesterol levels in vitro . Methods . Immature, unlutinized GC cell line KGN was obtained from a biological repository. Morphological characteristics of the KGN cell line were described during culture under previously described conditions. The growth rates of KGN in lipoprotein deficient human serum (HuS) and fetal bovine sera (FBS) were measured. The ability of dibutryl cyclic adenosine monophosphate (dibut-cAMP) and follicle stimulating hormone (FSH) to stimulate lutinization in media containing different lipoprotein composition was measured by the production progesterone as measured by enzyme immunoassay. Cholesterol and cholesteryl ester levels in conditioned media were measured simultaneously by high performance liquid chromatography. HDL was isolated from follicular fluid and characterized. The ability of KGN to alter media supplemented HDL particles was measured by non-denaturing PAGE and western blotting for Apolipoprotein A1. Results . KGN grew best in FBS compared to human serum and preferred media with lipoproteins over lipoprotein deficient media. Dibut-cAMP and FSH caused a dose dependant increase in progesterone production. FSH simultaneously induced a dose dependant decrease in media cholesterol levels whereas Dibut-cAMP treatment had no effect on cholesterol levels. HDL remodeling by the KGN cells was not evident. Conclusion . KGN produces progesterone in response to dibut-cAMP and FSH and is therefore useful for studies of the pre-ovulatory lutinization phase. No evidence of HDL particle remodeling was evident during these studies however there is indication of differences in cholesterol metabolism depending on the stimulus. Further studies are required to refine HDL preparations and examine HDL remodeling and cholesterol metabolism associated gene expression in this model.