Activation of the Akt/protein kinase B signaling pathway is associated with granulosa cell survival

Follicles from the hen ovary that have been selected into the preovulatory hierarchy are committed to ovulation and rarely become atretic under normal physiological conditions. In part, this is attributed to the resistance of the granulosa layer to apoptosis. The present studies were conducted to ev aluate the role of the phosphatidylinositol (PI) 3-kinase/Akt signaling pat hway in hen granulosa cell survival and, by implication, follicle viability . Cloning of the chicken akt2 homologue revealed a high degree of amino aci d homology to its mammalian counterparts within the catalytic domain, plus complete conservation of the putative Thr(308) and Ser(474) phosphorylation sites. Treatment of granulosa cells from the three largest preovulatory fo llicles with insulin-like growth factor (ICF)-I and, to a lesser extent, tr ansforming growth factor (TGF)-alpha induces rapid phosphorylation of Akt, and such phosphorylation is effectively blocked by the PI 3-kinase-inhibito r LY294006. Serum withdrawal from cultured cells for 33-44 h initiates olig onucleosome formation, an indicator of apoptotic cell death, whereas cotrea tment with ICF-I prevents this effect. Moreover, treatment of cultured cell s for 20 h with LY294006 induces apoptosis. The potential for nonspecific c ell toxicity following LY294006 treatment is considered unlikely because of the ability of either LH or 8-bromo cAMP cotreatment to block LY294006-ind uced cell death. Finally, both ICF-I and TGF-alpha also activate mitogen-ac tivated protein (MAP) kinase signaling, at least in part, through the phosp horylation of Erk. However, treatment with neither U0126 nor PD98059 (inhib itors of MAP kinase kinase) induced cell death in cultured granulosa cells, despite the ability of each inhibitor to effectively block Erk phosphoryla tion. Taken together, these results provide evidence for a role of the Akt signaling pathway in promoting cell survival within the preovulatory follic le granulosa layer. In addition, the data indicate the importance of an alt ernative survival pathway mediated via gonadotropins and protein kinase A i ndependent of Akt signaling.