Caspase-3 activation is required for Leydig cell apoptosis induced by ethane dimethanesulfonate

Previous studies have shown that ethane dimethanesulfonate (EDS) causes the apoptotic death of Leydig cells. The molecular mechanism by which EDS elic its its effect remains uncertain. The present study tested the hypothesis t hat caspase-3 is involved in the EDS-induced death of rat Leydig cells. Ley dig cells were isolated from adult Sprague Dawley at 3, 6, 12, or 24 h afte r the rats received an EDS injection. Low mol wt DNA fragments that are cha racteristic of apoptosis were evident by 12 h post-EDS, and the ladder patt ern was more pronounced at 24 h. During this same time period, the number o f terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end l abeling (TUNEL)-positive cells increased. Western blot analysis revealed th at 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 v itro, EDS treatment induced Leydig cell apoptosis. In the presence of cell- permeable caspase-3 inhibitor, however, apoptosis was significantly suppres sed, providing further evidence for the involvement of caspase-3 in EDS-ind uced Leydig cell apoptotic death. Immunohistochemical analysis revealed wea k 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 caspase-3 appeared, caspase-3 immunoreactivity increased and became localized to the nuclei. Apoptosis and caspase-3 were colocalized in Leydig cells by a hist ological method that combined TUNEL and caspase-3 immunohistochemistry, in these studies, TUNEL-positive cells all exhibited intense nuclear caspase-3 immunoreactivity, whereas TUNEL-negative cells exhibited weak caspase-3 im munoreactivity in the cytoplasm. Taken together, these results indicate tha t Leydig cell apoptosis induced by EDS is mediated by caspase-3 activation, and suggest that the translocation of the active caspase-3 forms to the nu cleus may be involved.