Luteolysis is multi-faceted involving numerous effectors that utilize multiple cellular signaling mechanisms. These effectors and the mechanisms by which they elicit their responses are not by themselves sufficient to explain the process of luteolysis in its entirety. Thus, other mediators must be involved. Cytokines, once thought to be secondary in the luteolytic process, are proving to be active players. Recent evidence suggests that cytokines (FasL and TNFalpha) signal via intermediates of the sphingomyelin pathway including sphingomyelinases (acid or neutral) and ceramide. The sphingomyelin pathway is a novel pathway involved in modulating cell signaling and cell death. There is also evidence that ceramide can regulate gonadotropin-induced steroidogenesis. Whether this is a direct effect, a by-product of altered membrane fluidity or due to disruption of pro-survival signaling is unknown. It is also possible that these events are pivotal to the structural involution of the CL. We hypothesize that ceramide generated at multiple sites within luteal cells mediates luteolysis. More specifically, an increase in the levels of ceramide via acid sphingomyelinase hydrolysis in the inner and/or outer leaflet of the plasma membrane alters the hierarchy of lipid ordering in the cellular membranes. Consequently, gonadotropin and PI(3)kinase signaling is inhibited, accelerating loss of function. Concomitantly, an increase in ceramide at the level of the mitochondria via ceramide synthase results in perturbation of the mitochondrial membrane contributing to loss of function and activation of the apoptosome. This hypothesis will be tested by the following Specific Aims: 1) determine if acid sphingomyelinase is functionally required in mouse CL for the disruption of steroidogenesis and induction of apoptosis using in vitro and in vivo models, 2) determine the mechanism(s) by which ceramide generated at the plasma membrane contributes to cytokine-induced loss of function or luteal cell death 3) determine at what cellular level TNFa inhibits gonadotropin-stimulated progesterone production in the CL, 4) ascertain whether increased levels of ceramide result in inhibition of the PI(3)-kinase by sequestering PI(3)-kinase in the caveolae fraction of luteal cells, and 5) establish if PGF2u or cytokine-mediated luteal cell apoptosis require involvement of the mitochondria and whether or not ceramide potentiates the dysregulation of mitochondrial function.
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