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 transcriptasepolymerase 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.