Avoidance of Apoptosis in Embryonic Cells of the Annual Killifish Austrofundulus limnaeus Exposed to Anoxia

Embryos of the annual killifish Austrofundulus limnaeus have unequalled ability among vertebrates to survive long-term anoxia. Surprisingly, these embryos can survive for months in anoxia despite a large-scale decrease in ATP levels during the initial hours of anoxic exposure. These conditions are known to trigger apoptotic cell death in mammalian cells as a result of ischemia or anoxia. Anoxia-induced induction of apoptosis was investigated in four developmental stages of A . limnaeus that differ in their tolerance of anoxia, and thus may respond to anoxia uniquely. Exposure to staurosporine was used to determine if A . limnaeus cells were competent to enter apoptosis via cues other than anoxia. Apoptotic cells were identified by TUNEL assays and by measuring caspase 3/7 activity. Exposure to 48 hr of anoxia did not induce an increase in TUNEL-positive cells and generally did not lead to an increase in caspase 3/7 activity. However, treatment of anoxic embryos with 10 μM staurosporine resulted in a significant increase in caspase 3/7 activity in both normoxic and anoxic embryos. These results suggest that apoptosis is avoided in embryos of A . limnaeus following exposure to anoxia at least in part by mechanisms that prevent the activation of caspase 3/7 activity. While this mechanism remains unknown, it may be triggered by a protein kinase that can be experimentally inhibited by staurosporine.