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

Abstract

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.

Extreme tolerance and developmental buffering of UV‐C induced DNA damage in embryos of the annual killifish Austrofundulus limnaeus

“…Recent work with A. limnaeus embryos has suggested that levels of the phosphorylated active form of AKT (pAKT), a known apoptotic programmed cell death repressor (Hers et al, ), are highest at 4 and 32 dpf (Meller et al, ). The low amount of TUNEL‐positive nuclei we observed in 4 and 32 dpf embryos after UV‐C irradiation, even at the highest dose for the 32 dpf embryos, agrees with pervious work that suggests apoptosis may be suppressed at these stages (Meller and Podrabsky, ). Although our breeding stock do not routinely arrest at DI, the tendency to suppress apoptosis at this developmental stage may remain and could be a feature that is shared with DII embryos.…”

Physiological strategies during animal diapause: lessons from brine shrimp and annual killifish

“…A similar lack of calcium-induced opening has been observed for mitochondria of the ghost shrimp, Lepidophthalmus louisianensis (Holman and Hand, 2009). While no direct evidence exists concerning opening of the transition pore in embryos of A. limnaeus, they are highly resistant to anoxia-induced apoptosis (Meller and Podrabsky, 2013), and thus may share similar resistance to mitochondrial-induced apoptosis. Additionally, it will be important to explore the cell-signaling pathways involved in the induction and breakage of diapause.…”

Mechanisms of animal diapause: recent developments from nematodes, crustaceans, insects, and fish

“…stress is also of central importance to survival of dormant embryos (81,124,127,128,188). However, relatively little attention has been focused on maintenance of genome integrity during embryonic diapause, and how DNA damage may affect survival of dormant embryos.…”

“…The extensive types of chaperonelike proteins expressed during diapause are major contributors to tolerance to environmental stress during diapause, but more work is needed to clarify the range of their functions and why so many families and isoforms are apparently required (28,84,101). Similarly, additional insights about molecular safeguards against unwanted cell death during diapause would be a productive avenue to explore further (81,124,126). The dispersed cell stage in embryos of the killifish A. limnaeus may provide new insights here (188).…”