In earlier studies, we have shown that the mitochondria-dependent (intrinsic) pathway is the key pathway for male germ cell apoptosis across species. To characterize the upstream signaling pathways that activate this death pathway, here we investigated the contributions of ERK (extracellular signal-regulated kinase) and JNK (c-Jun NH2-terminal kinase) to male germ cell apoptosis in rats after hormone deprivation or heat stress. In the hormone deprivation model, rats were given a daily injection of vehicle for 14 days or GnRH-antagonist, acyline (1.6 mg/kg BW) for 2, 5, and 14 days. In the hyperthermia model, scrota of rats were exposed once to a temperature of 22°C (control) or 43°C for 15 minutes and killed at ½, 2, and 6 hours after heat treatment. Testicular hyperthermia but not hormone withdrawal led to sustained activation of ERK. Immunocytochemistry further revealed ERK activation only in the Sertoli cells involving exclusively heat-susceptible early (I-IV) and late (XII-XIV) stages within ½ hour of heating. By 6 hours, immunostaining for active ERK was, however, evident, mostly in germ cells. Co-staining for TUNEL and phospho-ERK confirmed activation of ERK only in those germ cells undergoing apoptosis. Heat-induced germ cell apoptosis was also preceded by JNK activation, as evidenced by an increase in phospho c-Jun in testis lysates within ½ hour of heating. Unlike ERK, phospho-c-Jun immunostaining was detected only in those heat-susceptible germ cells. Because activation of both ERK and JNK is known to induce BCL-2 phosphorylation leading to its inactivation, we next examined whether the increased germ cell apoptosis after heat stress is associated with BCL-2 phosphorylation. Compared with control, where no staining was detected, we found marked increase in serine-phosphorylated form of inactive BCL-2 only in heat-susceptible germ cells. Co-staining for TUNEL and phospho-BCL-2 confirmed phosphorylation of BCL-2 only in those germ cells undergoing apoptosis. The BCL-2 phosphorylation was not apparent during hormone deprivation-induced apoptosis. These results suggest that (1) ERK and JNK promote male germ cell apoptosis via inactivation of BCL-2 through serine phosphorylation, and (2) ERK signaling is decisively different between these two paradigms and may represent an important step in the signal transduction pathway by which heat stress induces male germ cell apoptosis.
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