Identification of synthesis of ApoA-4 Protein in Ovarian Granulosa cells and Follicular Fluid

Abstract

Background . Clinical and experimental studies show that the level of high density lipoprotein is associated with the mammalian female fertility. To further investigate these associations, we have developed an in vitro ovarian granulosa cell luteinization model using granulosa cell (GC) line KGN. Lipoprotein signaling and cholesterol metabolism' focused mRNA expression arrays have been carried out to examine the role of cholesterol homeostasis during the folliculogenesis. In these experiments, we have identified and confirmed a significant upregulation of apolipoprotein A4 (ApoA-4) mRNA in KGN cells following treatment with gonadotropins. However, increased mRNA expression is not always synonymous with protein production and it is thus necessary to confirm whether ApoA-4 protein is actually produced in KGN cells. The goal of this thesis is to identify weather ApoA-4 protein is synthesized in human ovarian granulosa cells using KGN cell culture model of luteinization and to detect the localization of ApoA-4 protein in the KGN cells. Materials and method . KGN cells were grown in T-75 cell culture flasks and on cover slips in cell culture plates in DMEM/F-12 media with 10% fetal bovine serum (FBS). After the cells reached confluence, they were treated DMEMF-12 media containing 10% lipoprotein deficient FBS plus different gonadotropins for 24 hrs. To confirm the luteinization, the media progesterone concentration was measured by EIA. Immunoprecipitation and TCA precipitation techniques were used for enrichment of ApoA-4 protein from KGN media followed by western blotting to identify the presence of ApoA-4 protein in the media. The localization of ApoA-4 protein in the cells after the treatment with different gonadotropins was studied by immunofluorescent staining. Results . Upon stimulation with luteinizing agents, particularly cAMP, KGN cells generate a reproducible single 46 KD protein band by western blotting which corresponds to the positive ApoA-4 protein marker band. The immunofluorescence staining shows the localization of ApoA-4 protein around the nucleus in the luteinized KGN cells. Discussion . All the results support our hypothesis that the ApoA-4 protein may be synthesized de novo by human ovarian granulosa cells during folliculogenesis and that the KGN cell line is a suitable model for examining this expression during luteinization with different luteinizing agents.