[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Apoptosis is a morphologically distinct form of cell death that is involved in the regulation of normal and aberrant cell systems. The complexities of the apoptotic cell death pathway arise from variation in both the cellular specialisation and initial stimulus. The corpus luteum (CL) is an endocrine gland that whilst critical to the maintenance of pregnancy in the rat, regresses at the completion of each oestrous cycle and pregnancy. This regression is facilitated through apoptosis; though, the stimulus and factors involved in the apoptotic pathway are poorly understood. Previous studies suggest that CL regression is not initiated through failure of luteotrophic support, but rather the active production of a luteolytic factor, of which tumour necrosis factor -alpha (TNFα) is one possible candidate. Several publications have reported the participation of the immune system in ovarian events. There is evidence that TNFα expression within the ovary is coordinated between cells of the immune system and the hormonal regulation of the CL. This study has focussed on the role of TNFα in CL apoptosis and the factors involved in this apoptotic pathway. TNFα-induced cell death is governed by the presence of the two TNFα receptors (TNFR) and several second messenger systems that include; the sphingolipids, mitogen-activated protein (MAP) kinases, nitric oxide (NO), nuclear factor-kappaB (NF-κB) and the caspases. These factors and their interactions were assessed in the rat CL during pregnancy and post-partum, and in vitro. Apoptosis was measured through the analysis of DNA fragmentation using DNA 3’ end labelling and single cell electrophoresis (COMET assay). Assessment of mRNA and protein expression was through Real-time RT-PCR and Western blot analysis; proteins were localised within the CL by immunocytochemistry. In addition, specific measurement of sphingolipid expression and nitric oxide (NO) production was by high performance liquid chromatography (HPLC) and NO assay respectively. Following parturition, TNFα mRNA and protein expression increased corresponding to the onset of CL apoptosis and increased expression of the chemotactic factor monocyte chemoattractant protein -1 (MCP-1). Furthermore, CL apoptosis was induced by treatment with recombinant TNFα in a time- and dose-dependent manner. A similar effect was observed in isolated luteal cells. Simultaneously, the functional regression of the CL was assessed by measurement of both progesterone synthesis and steroidogenic acute regulatory (StAR) protein expression. StAR mRNA and protein expression declined toward parturition in vivo. Immunocytochemical studies revealed the presence of TNFα receptors 1 (TNFR1) and 2 (TNFR2) in luteal cells. Furthermore, TNFR mRNA was isolated from CL throughout pregnancy and post-partum. Subsequently, the role of the sphingolipids ceramide and sphingosine was examined during CL apoptosis in vitro. Ceramide and sphingosine were found to be potent apoptotic agents when administered in vitro (50µM). The downstream signal transduction of TNFα and ceramide was assessed through MAP kinase expression. Both TNFα and ceramide increased expression of the pro-apoptotic p38 MAP kinase with no change to the non-apoptotic extracellular signal-related kinase (ERK1&2). Despite previous reports of c-Jun NH2 terminal kinase (JNK) involvement in the cell death pathway, JNK expression was not evident in the rat CL. The caspases are a family of cysteine proteases central to the regulation and execution of apoptosis. General inhibition of the caspase cascade in vitro was effective in preventing apoptosis regardless of the apoptotic stimulus (TNFα, ceramide and sphingosine), suggesting that this pathway is central to CL apoptosis. Specific inhibition of several caspases produced a varying effect; inhibition of caspases 3, 6 and 8 significantly reduced the level of TNFα-induced apoptosis, thus supporting their classification as either regulatory or effector caspases. NO is endowed with the unique ability to initiate and to block apoptosis and this dichotomy extends to the cytotoxic actions of TNFα. Inhibition of NO production by treating CL with L-NAME prevented the onset of apoptosis, whilst NO production increased in response to increasing levels of apoptosis following trophic withdrawal. However, this effect was not seen during TNFα-induced apoptosis, suggesting that the actions of NO are independent of TNFα. The data presented within this study examine multiple elements of the TNFα cell death pathway in a single system. The results suggest that these elements are involved in TNFα signal transduction and furthermore, in rat CL apoptosis. It can be said that TNFα plays an active role in CL regression through the activation of the caspases, the sphingolipids and the MAP kinases.
| Identifer | oai:union.ndltd.org:ADTP/220973 |
| Date | January 2002 |
| Creators | Abdo, Michael A. |
| Publisher | University of Western Australia. School of Anatomy and Human Biology. |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Michael A. Abdo, http://www.itpo.uwa.edu.au/UWA-Computer-And-Software-Use-Regulations.html |
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