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Programmed cell death is an important regulatory event in spermatogenesis. However, the molecular events governing apoptosis have not been characterized. Using the Leydig cell-specific toxin ethane dimethanesulfonate (EDS) to withdraw androgen support, we have investigated the relationship between apoptosis and apoptosis-related genes. Adult male Sprague-Dawley rats were injected (i.p.) with 100 mg/kg EDS and killed at times of androgen depletion 2, 5, and 8 days postinjection. A 24-fold increase in the apoptotic index 8 days after EDS administration was demonstrated in tissue sections by in situ end-labeling of fragmented DNA. Leydig cell death and androgen withdrawal were confirmed by the absence of 3β-hydroxysteroid dehydrogenase in testes from animals treated with EDS for 2 days. After androgen withdrawal, there were no significant changes in the levels of clusterin, Bcl-xl, Bak, and Bad …