title: Regulation of Fas antigen (Fas, CD95)-mediated apoptosis of bovine granulosa cells by serum and growth factors.
abstract: Our previous studies have shown that bovine granulosa cells cultured in basal media supplemented with 5% fetal bovine serum (BM-FBS) are resistant to apoptosis induced by recombinant Fas ligand (FasL) unless pretreated with interferon-gamma (IFN). Experiments were conducted to test the hypothesis that serum and growth factors alter the susceptibility of granulosa cells to FasL-induced apoptosis. Granulosa cells were cultured in BM-FBS, BM containing insulin, transferrin, selenium, and BSA (BM-ITS), and in BM-ITS supplemented with insulin-like growth factor-I (IGF). Cells were susceptible to FasL-induced killing in BM-ITS (27% killing) but were resistant in BM-FBS and in BM-ITS containing IGF (P < 0.05 vs. killing in BM-ITS). Exposure of phosphatidylserine residues on the outer cell membrane, an early marker of apoptosis, was stimulated by FasL and prevented in the presence of IGF. Neutralization of IGF activity in serum with IGF binding protein 3 reduced the protective effect of FBS on FasL-induced killing (P < 0.05), suggesting that IGF is an inhibitory component in FBS. Cotreatment with IFN overcame the inhibitory effects of serum and IGF on FasL-induced killing (31% and 29% killing, respectively, P > 0.05), but IFN did not potentiate killing of cells cultured in BM-ITS. IFN increased expression of Fas antigen (Fas, the receptor for FasL) mRNA five- to sevenfold (P: < 0. 05) and increased immunostaining for Fas protein similarly in all types of media. Addition of the growth factors epidermal growth factor or basic fibroblast growth factor to BM-ITS also inhibited FasL-induced killing (P < 0.05), whereas keratinocyte growth factor, transforming growth factor, platelet-derived growth factor, FSH, and LH had no effect. In summary, FasL-induced killing is inhibited by FBS and certain growth factors. IFN increased expression of Fas similarly in all types of media but was required for FasL-induced killing only in BM containing FBS or IGF. Therefore, modulation of responsiveness to FasL-induced apoptosis by growth factors and IFN is not directly related to the level of Fas expression.
authors.affiliation: Department of Animal Science, Cornell University, Ithaca, New York 14853, USA. smq1@cornell.edu
authors.forename: S M
authors.initials: SM
authors.lastname: Quirk
authors.forename: R M
authors.initials: RM
authors.lastname: Harman
authors.forename: R G
authors.initials: RG
authors.lastname: Cowan
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002417:Cattle; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D053222:Fas Ligand Protein; D005260:Female; D006062:Gonadotropins / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects*; D006133:Growth Substances / Q000494:pharmacology*; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D019014:fas Receptor / Q000096:biosynthesis / Q000502:physiology*
chemical_list: D016895:Culture Media, Serum-Free; D053222:Fas Ligand Protein; D006062:Gonadotropins; D006133:Growth Substances; D008562:Membrane Glycoproteins; D010718:Phosphatidylserines; D012333:RNA, Messenger; D019014:fas Receptor