The regulation of apoptosis in granulosa cells of bovine ovarian follicles

Ovarian follicle development is dependent on growth, proliferation and apoptosis of follicle cells. The studies presented in this thesis examine the apoptotic pathway mediated by the Fas antigen (Fas) and pro-survival pathways stimulated by insulin-like growth factor (IGF-1) in cultured bovine granulosa cells. The role of Fas in triggering follicular atresia was examined in a serum withdrawal model. After removal of serum from the culture media, increasing numbers of cells became apoptotic over time. Although the level of Fas mRNA remained unchanged after serum removal, Fas protein increased at 3 h and remained elevated through 12 h. Both of the level of FasL mRNA and protein increased 3 h after the withdrawal of serum. Treatment with Fas/FasL antagonists at the time of serum withdrawal reduced the extent of apoptosis suggesting that serum withdrawal-induced apoptosis of granulosa cells is mediated at least partially by Fas/FasL interactions. This supports a potential role of the Fas pathway, activated in the absence of survival factors, to trigger ovarian follicular atresia. IGF-1 inhibited FasL-induced apoptosis in cultured granulosa cells. Signaling pathways which mediate effects by IGF-1 were examined. Inhibition of the phosphoinositide-3′ kinase (PI3K)/Akt pathway by treatment with LY294002 and by overexpression of a dominant negative Akt mutant in granulosa cells prevented the protective effect of IGF-1 against FasL-induced apoptosis. Treatment with the MAPK kinase inhibitor, PD98059, however, did not alter the protective effect of IGF-1. Treatment with IGF-1 and overexpression of a constitutively active Akt mutant (myrAkt) increased progression from G0/G1 to S phase of the cell. I investigated whether cell cycle progression is required for protection from apoptosis. Roscovitine, which blocks cells at the GUS transition, prevented the protective effect of IGF-1 and myrAkt expression against FasL-induced apoptosis. This indicates that the protective effect of IGF-1 against FasL-induced apoptosis only occurs when progression from G1 to S phase of the cell cycle through the PI3K/Akt pathway is not perturbed. In summary, IGF-1 may play a role in preventing the activation of an endogenous Fas pathway and thereby promote growth and survival of granulosa cells during follicle development.