Abstract: 410

CASPASE-3 ACTIVATION IN LEYDIG CELL APOPTPSIS INDUCED BY ETHANE DIMETHANESULFONATE.

Jong-Min Kim¹ , Lindi Luo¹ , Barry Zirkin^{1 *}
Division of Reproductive Biology, Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205 ¹

A single injection of ethane dimethanesulfonate(EDS)kills all Leydig cells in the adult rat testis. The molecular mechanism(s) by which EDS elicits its effect remains uncertain. Caspase-3 is involved in the cascade of apoptotic cell death of many cells. The cleavage of pro-caspase-3 (32 kDa) into its active forms (20 kda and 10 kDa) is essential for inducing apoptosis. 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 rats by Percoll gradient centrifugation at 3, 6, 12 or 24 hrs after the rats received an EDS injection (85 mg/kg BW). Apoptosis was analyzed by DNA fragmentation on agarose gels and by the in situ TUNEL assay. Caspase-3 protein was estimated by immuno- histochemistry and Western blot. Low molecular weight DNA fragments were evident by 12 hr post EDS, and the ladder pattern became more evident at 24 hr. At the same times, the intensity of high molecular weight DNA was reduced. Consistent with these results, the number of TUNEL-positive cells increased from 6 to 24 hr following EDS injection. Western blot analysis revealed that the inactive, pro-form of caspase-3 was present at low levels in control Leydig cells, and increased gradually through 6 hr post-EDS. The caspase pro-form began to decrease after 12 hr, while the cleaved, active form appeared at 12 hr and increased through 24 hr post-EDS. Immunocytochemical analysis revealed that caspase-3 in control Leydig cells was localized in the cytoplasm. From 12 through 24 hr post-EDS, the time interval during which the active form of caspase-3 appeared, caspase-3 immunoreactivity became localized in the nuclei. Taken together, these results suggest that Leydig cell apoptosis induced by EDS is mediated by caspase-3 activation (Supported by NIH grant U54 HD36209)

    This abstract is being presented on Monday, August 2 at 3:15 PM at Todd 125.