title: Apoptosis of bovine granulosa cells after serum withdrawal is mediated by Fas antigen (CD95) and Fas ligand.
abstract: Ovarian follicular atresia occurs by apoptosis of granulosa and theca cells. The Fas antigen (Fas), a cell surface receptor that triggers apoptosis when activated by Fas ligand (FasL), may be involved in this process. A possible role of the Fas pathway in mediating serum withdrawal-induced apoptosis of granulosa cells was examined. Granulosa cells collected from 5- to 10-mm bovine follicles were cultured in DMEM-F12 containing serum for 3 days, deprived of serum, and live cells were counted at various times after serum withdrawal. Cell death increased significantly 6 h after serum withdrawal (21% +/- 7%; P: < 0.05 vs. 0 h) and continued to increase until 24 h (43% +/- 6%). No further increases in cell death were observed through 72 h. Detection of the translocation of phosphatidylserine to the outer surface of the cell membrane by annexin V binding indicated that cells died by apoptosis. Quantitative reverse transcriptase-polymerase chain reaction assays showed no changes in Fas mRNA levels but a 4.7-fold increase in FasL mRNA 3 h after serum withdrawal (P: < 0.05 vs. 0 h). FasL mRNA remained elevated through 24 h and returned to basal levels at 48 h. Immunohistochemical staining showed that both Fas and FasL protein increased on the cell surface within 3 h and remained elevated through 12 h (the last time point tested). Binding of FasL to Fas was blocked with two reagents that bind to the extracellular domain of FasL: an anti-FasL antibody and Fas:Fc, a chimeric protein consisting of the Fc portion of human immunoglobulin G and the extracellular domain of human Fas. Cell death 24 h after serum withdrawal was reduced 55% +/- 10% and 34% +/- 12% by anti-FasL antibody and Fas:Fc, respectively (P: < 0.05 vs. no blocking protein). In conclusion, serum withdrawal-induced apoptosis of bovine granulosa cells is mediated at least partially by Fas/FasL interactions. These results are consistent with a potential role of Fas in an autocrine or paracrine pathway to trigger ovarian follicular atresia.
authors.affiliation: Department of Animal Science, Cornell University, Ithaca, NY 14853, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Hu
authors.forename: R G
authors.initials: RG
authors.lastname: Cowan
authors.forename: R M
authors.initials: RM
authors.lastname: Harman
authors.forename: D A
authors.initials: DA
authors.lastname: Porter
authors.forename: S M
authors.initials: SM
authors.lastname: Quirk
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016895:Culture Media, Serum-Free; D053222:Fas Ligand Protein; D005260:Female; D006107:Granulosa Cells / Q000502:physiology*; D007150:Immunohistochemistry; D008024:Ligands; D008562:Membrane Glycoproteins / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019014:fas Receptor / Q000096:biosynthesis / Q000502:physiology*
chemical_list: D016895:Culture Media, Serum-Free; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008024:Ligands; D008562:Membrane Glycoproteins; D019014:fas Receptor