GRANULOSA-CELL APOPTOSIS INDUCED AT THE PENULTIMATE STAGE OF FOLLICULAR DEVELOPMENT IS ASSOCIATED WITH INCREASED LEVELS OF FAS AND FAS LIGAND IN THE RAT OVARY

Apoptosis of granulosa cells is the cellular mechanism of ovarian foll icular atresia, and cytokines have been implicated as potential atreto genic factors. We therefore investigated the possible role of the cyto kine Fas ligand (FasL) and its receptor Fas in apoptosis during ovaria n follicular atresia induced by gonadotropin withdrawal. Immature rats pretreated with eCG were injected 24 h later with an antiserum genera ted against eCG (antibody group) or preimmune rabbit serum (control), and ovaries were removed 1 and 24 h after treatment. The eCG antiserum caused a dose-dependent inhibition of the significant increase in ova rian weight observed between 24 and 48 h after eCG treatment. In situ detection of fragmented DNA in histological sections identified cell d eath in atretic but not healthy small and medium-sized antral follicle s of the antibody group. Cell death was distributed in a scattered pat tern throughout the granulosa cell layer of small atretic follicles bu t was localized primarily in granulosa cells lining the antral cavity of atretic medium antral follicles. Immunohistochemistry of adjacent h istological sections revealed intense positive immunostaining for Fas and Fast in granulosa cells of atretic small and medium antral follicl es in a pattern coincidental to the localization of cell death. Intens e Fast staining was evident in the theca cells of healthy small antral follicles. An increase in low molecular weight DNA (DNA ''ladders'') indicative of apoptosis was evident in granulosa cells of the antibody group. Western analysis demonstrated increased levels of both Fas and Fast in the granulosa cells of the antibody group. These results demo nstrate that both Fas and Fast are present in ovarian granulosa cells and that Fast may be the signal that induces granulosa cell apoptosis during atresia at the penultimate stage of ovarian follicular developm ent.