| In many species, the process of ovarian follicle selection is the rate-limiting step by which a species-specific number of follicles are designated to form mature oocytes in the reproductively active female. Selected follicles then transition from an undifferentiated to a differentiated state. This thesis addresses the hypothesis that coordinately timed cell-signaling events are requisite for the onset of differentiation, resulting from a release from inhibitory signaling in combination with stimulatory cell signaling events. The hen provides an excellent model to study granulosa cell differentiation based on large follicle size and easy access to pure populations of granulosa cells from all stages of follicle development.;Treatment of undifferentiated granulosa cells with transforming growth factor (TGF)beta1 increased FSHR mRNA and enhanced FSH-induced progesterone production. Dissimilar results were obtained with the related protein, activin, as high levels of the endogenous activin binding protein, follistatin, precluded activin-mediated transcription. The differentiation-inducing effects of TGFbeta1 were attenuated by activation of Erk signaling. While Erk signaling inhibited TGFbeta1 induced differentiation, it up-regulated TGFbeta1 mRNA, indicating that alleviation of Erk signaling is requisite for TGFbeta1 induced differentiation. Results also indicate that Erk up-regulates MAPK phosphatases (MKPs) MKP-1 and -3, as well as dual specificity phosphatase (DUSP)-5. In turn, Erk signaling is terminated, allowing TGFbeta1 to initiate differentiation.;While Erk signaling inhibits differentiation in granulosa cells from undifferentiated follicles, it becomes required for differentiation in preovulatory follicles. This transition occurs post-follicle selection, as results from the 9-12mm (most recently selected) follicle indicate that Erk signaling inhibits steroidogenesis at this stage. Significantly, while previous work has demonstrated that protein kinase C (PKC) inhibits differentiation in granulosa cells from prehierarchal follicles, results herein demonstrate that PKC activity is required for differentiation of granulosa cells in preovulatory follicles. Unlike Erk signaling, the requirement for PKC signaling occurs concurrent with follicle selection, as cells treated with a PKC inhibitor in combination with gonadotropin failed to produce progesterone in the most recently selected follicle.;In summary, these data support the hypothesis that alleviation from inhibitory signaling is required for granulosa cell differentiation. However, these results also indicate that the removal of inhibitory signaling is transient, as indicated by the requirement for Erk and PKC signaling in the latter, preovulatory stage. |
