Involvement of the Fas/Fas ligand system in p53-mediated granulosa cell apoptosis during follicular development and atresia.

In the present study we have examined the presence of Fas, Fas ligand (FasL), and p53 in rat granulosa cells during follicular development and atresia, especially in relation to the granulosa cell cycle progression and the onset of granulosa cell apoptosis. Fas, FasL, and p53 proteins were immunolocalized, and their contents were determined by Western blotting. Granulosa cell apoptosis was assessed by DNA fragmentation analyses (DNA ladder) and in situ terminal deoxynucleotidyl transferase mediated deoxy-UTP-biotin nick end labeling (TUNEL) as well as by flow cytometry. Ovaries not exposed to gonadotropins (control) consisted predominantly of preantral and early (small) antral follicles, the latter of which were mostly atretic and demonstrated intense TUNEL staining in granulosa cells exhibiting positive immunoreactivities for FasL and Fas. Granulosa cells isolated from these follicles were apoptotic, as evident by clear ladder pattern of DNA fragmentation upon electrophoretic analysis and the high percentage (>10%) of the cell population in the A0 phase of the cell cycle. After gonadotropin treatment, these features completely disappeared during each of the 3 days of follicular growth to the medium to large antral stages. Cell cycle analysis showed significantly higher proportion of the cells in S and G2/M phases compared with controls, which was accompanied by marked decrease in immunoreactivities for Fas, FasL, and p53. By days 4 and 5, widespread atresia and extensive granulosa cell apoptosis were noted in large antral and preovulatory follicles and were coincidental to increased expression of p53 and Fas, but not of FasL, as well as an apparent arrest of granulosa cell G1/S progression, as evident by an increased cell population in G0/G1 and a decrease in the S and G2/M. Granulosa cells from equine CG-primed ovaries exhibited marked increases in p53 and Fas protein contents and apoptosis after adenoviral p53-sense complementary DNA infection in vitro and were more responsive to Fas activation by an agonistic Fas monoclonal antibody challenge. Taken together, these findings are consistent with the well accepted concept that gonadotropin plays a central role as a survival factor in the regulation of granulosa cell Fas/FasL and p53 expression during ovarian follicular development. In addition, the control of granulosa cell apoptosis may involve two consecutive cellular/molecular events: cell cycle arrest at G1/S and exit from G0 into A0 phase, via regulation of the p53 and Fas/FasL death pathways.