http://repub.eur.nl/res/aut/6267/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/10331/ 2004-01-01T00:00:00Z The mechanism by which ethane 1,2-dimethanesulfonate (EDS) selectively kills Leydig cells is poorly understood. To characterize further the cell-specific actions of EDS, we studied biochemical and morphological changes during apoptosis in different Leydig cell and non-steroidogenic cell models.Rat testicular and H540 tumor Leydig cells were killed by 1-2 mM EDS, whereas 20 mM EDS were required for MA-10 cells. This higher concentration of EDS was also necessary for activation of apoptosis in non-steroidogenic Chinese hamster ovary cells, whereas COS-1 monkey kidney cells were resistant. These variable effects of EDS on apoptosis were independent of new protein synthesis and, interestingly, could be delayed by co-incubation with dibutyrl cyclic AMP. Along with cell death, we also observed chromosomal fragmentation and other hallmarks indicative of apoptosis as evidenced by DNA laddering and fluorescent microscopy. Time-lapse photography with a confocal microscope showed that the time of onset, duration and even the sequence of apoptotic events between individual H540 cells was heterogeneous. When the dose of EDS was gradually increased from 2 to 10 mM, the proportion of cells showing normal apoptotic features gradually decreased. Intriguingly, treatment with 10 mM EDS did not result in death for most cells and was marked by an absence of DNA laddering and ultrastructural features of apoptosis and necrosis. However, incubation with 20 mM EDS resulted in necrosis.These results demonstrated that the effects of EDS on cell survival are not specific to Leydig cells, that different cell types have different sensitivities to EDS and that stimulation of the cAMP pathway may mitigate EDS action. The data obtained with H540 cells further revealed that EDS can induce two types of programmed cell death. http://repub.eur.nl/res/pub/9790/ 2001-01-01T00:00:00Z In virtually all studies with MA-10 cells, progesterone RIAs have been used to measure steroid synthesis. To test whether progesterone is a stable end product, we investigated the metabolism of added tritiated progesterone and pregnenolone in MA-10 cells over a period of 3 h. Steroids were then extracted, separated by HPLC, and identified by GC/MS. We found that more than 70% of radiolabeled steroids were converted to at least five different metabolites. A major metabolite (40%) was 5 alpha-pregnan-3 alpha or 3 beta-ol-20one. Similar studies, using radiolabeled T, demonstrated conversion to dihydrotestosterone and two forms of 5 alpha-androstane-diols. These data indicate the presence of active 5 alpha-reductase and 3 alpha- and/or 3 beta-hydroxysteroid dehydrogenase activities in MA-10 cells. Because these results suggest that progesterone is an unstable end product, to gauge the level of active metabolism, we incubated cells in the presence of inhibitors of pregnenolone metabolism and assessed pregnenolone levels by RIA. We discovered that basal levels of steroidogenesis in MA-10 cells were considerably higher than previously estimated. Moreover, dibutyryl cAMP-stimulated steroid production was linear over more than 13 h, in contrast to previous findings that measured progesterone levels. Other consequences of inaccurate assessment of steroidogenic activity in MA-10 cells because of the application of the progesterone assay are discussed.