The primary function of the corpus luteum (CL) is to synthesize and secrete progesterone to provide uterine quiescence for the establishment and maintenance of pregnancy. Any premature disruption in the function of the corpus luteum can result in a loss of pregnancy, irregular cyclicity, and a reduction in reproductive efficiency. The inhibition of steroid synthesis (functional regression), and the eventual disruption of cellular homeostasis (structural regression), is collectively described as luteolysis. More recently, cytokines have been implicated in an active role in luteolysis; yet the exact mechanisms by which they elicit their cytotoxic activity remains to be demonstrated. Recent evidence suggests that the signal for cell death, whether it be by TNFalpha, IFNgamma, FAS ligand or other stress-related stimuli, is mediated via the sphingomyelin pathway. Coincedently, these same factors have also been implicated in steroid inhibition and demise of the CL. Activation of the acid-sphingomyelinase by cytokines results in elevated ceramide levels. Subsequently, stress-induced ceramide is believed to preferentially signal through the cytoplasmic stress activated protein kinase (SAPK) a member of the mitogen activated protein kinase cascade (MAPKs). MAPKs are cytoplasmic enzymes responsible for translating the signal generated on the cell surface to the nucleus where they regulate transcription by phosphorylating transcription factors. We hypothesize that cytokines, as well as other cytotoxic stimuli, will initiate cell death via the sphingomyelin pathway and its second messenger ceramide in luteal cells. Furthermore, subsequent increases in ceramide will activate SAPKs which will ultimately phosphorylate specific transcription factors implicated in cellular demise and therefore play a role in the transition from functional inhibition of steroidogenesis to structural regression of the CL. To address this hypothesis we have proposed the following aims: (1) Determine if specific cytokines implicated in the regulation of the functional and structural aspects of luteal regression activate the sphingomyelin pathway. (2) Characterize the pleiotropic responses to cytokines (TNFalpha-INFgamma) as determined by the relative changes in ERKs and/or SAPKs/JNK and (3) the transcription factors c-fos and c-jun in luteal cells. (4) Determine whether or not steroid inhibition alone can activate the sphingomyelin pathway in cultured bovine luteal cells. (5) Determine the role of capase enzymes in ceramine activation. The experiments will utilize well defined primary cultures of bovine luteal cells and employ complimentary cellular and molecular techniques to address the aims. This project is expected to provide novel information on the cellular mechanisms involved in the process of luteolysis.
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