Spermatogenic cells are susceptible to heat stress and undergo apoptosis. Although a variety of factors appear to be involved in the apoptotic process. the nature of the intracellular signaling pathway is ambiguous. To clarify the process, we chose a simple model in which testes of mice were exposed to mild heating by immersion in hot water at 42degreesC for 15min. In situ DNA fragmentation was detected by a TUNEL method. The release of cytochrome e into the cytoplasm was observed by Western blotting both in heat-treated testis and in isolated spermatogenic cells that had been incubated at 42degreesC for 1 h, but not in Sertoli cells. Minocycline, a semisynthetic tetracycline, is known to reach the brain by permeating the blood-brain barrier and suppresses apoptosis in neuronal cells. Since the testis also has a similar barrier, minocycline was examined as a possible agent to inhibit heat stress-induced apoptosis. The results indicate that minocycline suppressed the release of cytochrome e from mitochondria both in vivo and in vitro and significantly decreased the number of TUNEL-positive cells. These findings suggest that heat stress of testes triggers the release of cytochrome c from mitochondria in spermatogenic cells, leading to the activation of an apoptotic pathway. (C) 2003 Elsevier Inc. All rights reserved.