X-linked inhibitor of apoptosis protein (XIAP) in granulosa cells is regulated by gonadotropins during follicular development, although the current understanding of the mechanisms by which XIAP suppressed granulosa cell apoptosis is incomplete. In the present study, we investigated the possible involvement of the phosphatidylinositol 3-kinase (PI 3-K) survival pathway in the regulation of granulosa cell fate. Using a fully characterized in vivo model to study the induction of follicular development and atresia in immature rats, we have demonstrated that gonadotropin treatment increased granulosa cell XIAP and phospho-Akt protein contents and suppressed apoptosis. In addition, gonadotropin withdrawal [equine CG (eCG)-primed rats treated with an anti-eCG antibody] induced granulosa cell apoptosis and significantly decreased ovarian weight. The increased apoptosis was accompanied by marked decreases in XIAP expression and phosphorylation of Akt protein. Infection of granulosa cells from eCG-primed rats with adenoviral sense XIAP [lacZ as a control; multiplicity of infection, 1-5] resulted in XIAP overexpression and increased phospho-Akt content, whereas XIAP antisense expression (multiplicity of infection, 10-40) decreased granulosa cell phospho-Akt level and induced apoptosis. Addition of the specific PI 3-K inhibitor LY294002 to the granulosa cell cultures decreased Akt phosphorylation and induced apoptosis in a dose-dependent manner. Taken together, these results demonstrate for the first time the importance and regulation of the PI 3-K survival pathway by XIAP in the control granulosa cell apoptosis.