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dc.contributor.authorBoparai, Ravneet
dc.date.accessioned2016-03-29T17:19:07Z
dc.date.available2016-03-29T17:19:07Z
dc.date.issued2010
dc.identifier.isbn9781124243849
dc.identifier.other759959637
dc.identifier.urihttp://hdl.handle.net/10477/45978
dc.description.abstractBackground. There is increasing evidence that high density lipoprotein (HDL) metabolism is important to mammalian female fertility. HDL is the sole lipoprotein in human ovarian follicular fluid (FF) and FF-HDL levels have been shown to be associated with embryo morphology outcomes during in vitro fertilization (IVF). As a mechanism-based follow-up, we are developing an in vitro ovarian granulosa cell (GC) culture model containing a physiologic HDL composition. Focused RT-PCR arrays of lipoprotein signaling and cholesterol metabolism associated genes indicate that apolipoprotein A-IV ( ApoA-IV ) was greatly upregulated in response to follicle stimulating hormone (FSH) or cell permeable cyclic adenosine monophosphate (cAMP). The aim of this thesis was to refine the HDL composition of this cell culture model, confirm the upregulation of ApoA-IV and to explore changes in conditioned media cholesterol composition in response to gonadotropins. Methods and Materials. HDL was purified from FF by sequential polyanion precipitation with MnCl 2 /Heparin and affinity chromatography on cibachron blue 3GA sepharose. Ovarian GC line KGN and human pre-ovulatory primary GCs were cultured in media supplemented with purified HDL (15 μg/mL ApoA-I) and stimulated for 24 hours with FSH, cAMP and human chorionic gonadotropin (hCG). Conditioned media progesterone was measured by EIA to confirm luteinization. Media cholesterol and cholesterol ester levels were analyzed by HPLC. GC ApoA-IV mRNA levels were measured by quantitative PCR. Results. FF-HDL purification efforts increased ApoA-I specific activity by 38 fold and removed high levels of FF progesterone but also caused an increase in the mean HDL particle size. In KGN cells, FSH, cAMP and hCG significantly increased media levels of progesterone and cholesterol esters and upregulated cellular ApoA-IV mRNA levels by >2000, 30 and 15 fold respectively. huGCs showed ten times higher progesterone production compared to KGN cells, however no upregulation of ApoA-IV, relative to unstimulated control huGCs, was observed. Conclusion. ApoA-IV mRNA is upregulated in response to gonadotropins in KGN granulosa cells and this upregulation coincides with increased media cholesterol esters which we hypothesize is the result of cholesterol efflux. The failure of huGCs to upregulate ApoA-IV may be due to the fact that huGCs are preluteinized in vivo during IVF treatment. Direct comparisons between huGC and KGN ApoA-IV mRNA levels as well as examination of ApoA-IV protein levels would help clarify these findings.
dc.languageEnglish
dc.sourceDissertations & Theses @ SUNY Buffalo,ProQuest Dissertations & Theses Global
dc.subjectPure sciences
dc.subjectBiological sciences
dc.subjectApoa4
dc.subjectFollicular fluid
dc.subjectGranulosa cells
dc.titleGonadotropin stimulated apolipoprotein A-IV gene expression in human ovarian granulosa cells in vitro
dc.typeDissertation/Thesis


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