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 on set of granulosa cell apoptosis. Fas, Fast, and p53 proteins were immunoloc alized, and their contents were determined by Western blotting. Granulosa c ell apoptosis was assessed by DNA fragmentation analyses (DNA ladder) and i n situ terminal deoxynucleotidyl transferase mediated deoxy-UTP-biotin nick end labeling(TUNEL) as well as by flow cytometry. Ovaries not exposed to g onadotropins (control) consisted predominantly of preantral and early (smal l) antral follicles, the latter of which were mostly atretic and demonstrat ed intense TUNEL staining in granulosa cells exhibiting positive immunoreac tivities for Fast and Fas. Granulosa cells isolated from these follicles we re apoptotic, as evident by clear ladder pattern of DNA fragmentation upon electrophoretic analysis and the high percentage (>10%) of the cell populat ion in the A, phase of the cell cycle. After gonadotropin treatment, these features completely disappeared during each of the 3 days of follicular gro wth to the medium to large antral stages. Cell cycle analysis showed signif icantly higher proportion of the cells in S and G(2)/M phases compared with controls, which was accompanied by marked decrease in immunoreactivities f or Fas, Fast, and p53. By days 4 and 5, widespread atresia and extensive gr anulosa cell apoptosis were noted in large antral and preovulatory follicle s and were coincidental to increased expression of p53 and Fas, but not of Fast, as well as an apparent arrest of granulosa cell G(1)/S progression, a s evident by an increased cell population in G(0)/G(1) and a decrease in th e S and G(2)/M. Granulosa cells from equine CG-primed ovaries exhibited mar ked increases in p53 and Fas protein contents and apoptosis after adenovira l p53-sense complementary DNA infection in vitro and were more responsive t o Fas activation by an agonistic Fas monoclonal antibody challenge. Taken t ogether, 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 devel opment. In addition, the control of granulosa cell apoptosis may involve tw o consecutive cellular/molecular events: cell cycle arrest at G(1)/S and ex it from G(0) into A(0) phase, via regulation of the p53 and Fas/FasL death pathways.