Apo A-IV and LXR mRNA expression in gonadotropin stimulated human ovarian granulosa cell line KGN.

摘要

Background. Follicular fluid high density lipoprotein (FF-HDL) has been shown to be associated with the quality of embryos derived from in vitro fertilization (IVF). In vitro studies examining the ovarian granulosa cell line KGN indicate these cells express ApoA4 mRNA (an HDL Apolipoprotein) in response to gonadotropins. ApoA4 is a downstream target of the master nuclear transcription factor LXR. The goal of this thesis is to investigate if LXR genes are regulated by gonadotropins and if LXR activation is sufficient to elicit ApoA4 production in this model.;Materials and Methods. The human ovarian granulosa cell line KGN was cultured in defined DMEM/F12 media containing 10% lipoprotein deficient fetal bovine serum and 15 mug/mL of FF-HDL isolate. Cells were treated for 24 hours with 1mM dibutryl cyclic AMP (dbcAMP) or 100 ng/mL follicle stimulating hormone (FSH) with or without the LXR agonist TO901317. Media progesterone was measured by EIA to confirm luteinization and the cholesterol/cholesteryl ester content was measured by HPLC. The mRNA expression of ApoA4, LXRalpha (NR1H3) and LXRbeta (NR1H2) were measured by quantitative reverse transcriptase polymerase chain reaction (qPCR) relative to the expression of the housekeeping gene GAPDH.;Results. In KGN, gonadotropin treatment increases ApoA4 expression by 1.5 to 2 fold while LXR alpha or LXR beta expression is not significantly altered. Treatment with the LXR agonist alone causes 3 fold increased ApoA4 expression and augments gonadotropin induced expression to >10-fold.;Discussion. LXR gene expression does not appear to be altered during folliculogenesis however LXR activation is sufficient to induce ApoA4 expression in ovarian granulosa cells.