Caspase-3 activation is required for Leydig cell apoptosis induced by ethane dimethanesulfonate
Abstract
Previous studies have shown that ethane dimethanesulfonate (EDS) causes the apoptotic death of Leydig cells. The molecular mechanism by which EDS elicits its effect remains uncertain. The present study tested the hypothesis that caspase-3 is involved in the EDS-induced death of rat Leydig cells. Leydig cells were isolated from adult Sprague Dawley at 3, 6, 12, or 24 h after the rats received an EDS injection. Low mol wt DNA fragments that are characteristic of apoptosis were evident by 12 h post-EDS, and the ladder pattern was more pronounced at 24 h. During this same time period, the number of terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling (TUNEL)-positive cells increased. Western blot analysis revealed that procaspase-3 was present only at low levels in control Leydig cells, and increased through 6 h post-EDS. By 12 h, procaspase-3 was reduced, whereas the cleaved, active caspase-3 forms appeared at 12 h and increased through 24 h post-EDS. Caspase-3 activity was blocked by caspase-3 inhibitor. In vitro, EDS treatment induced Leydig cell apoptosis. In the presence of cell-permeable caspase-3 inhibitor, however, apoptosis was significantly suppressed, providing further evidence for the involvement of caspase-3 in EDS-induced Leydig cell apoptotic death. Immunohistochemical analysis revealed weak staining for caspase-3 in the cytoplasm of control Leydig cells. From 12-24 h post-EDS, the time interval during which the active forms of…
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