Molecular Changes Associated with Anoxia Tolerance in \u3ci\u3eAustrofundulus limnaeus\u3c/i\u3e embryos

Embryos from the annual killifish Austrofundulus limnaeus have a unique and unequalled ability among vertebrates to withstand extended periods of anoxia (maximum lethal time to 50% mortality of 65 days at 25°C). In addition, tolerance of anoxia is gained and subsequently lost during the normal development of this species. Thus, anoxia tolerant and anoxia sensitive individuals can be compared within the same species, making A. limnaeus an excellent model for studying the molecular changes associated with survival of oxygen deprivation in vertebrates. The aim of this project is to analyze the molecular changes associated with anoxia tolerance in the embryos of A. limnaeus. Understanding how the cells of these embryos become tolerant to anoxia will aid in identifying novel therapeutic targets to reduce cell death following periods of ischemia in heart, brain or other tissues. Three major analyses were used to investigate the molecular changes associated with anoxia tolerance in this species. The first was a cell cycle and cell cycle arrest analysis using flow cytometry along with an immunoblot analysis of both positive and negative regulators of cell cycle progression. The second was a cell death analysis utilizing caspase-3/7 activity as well as TUNEL staining. The third was an immunoblot analysis of three different post-translational modifers (ubiquitin, SUMO-1 and SUMO-2/3). The overall findings from this study indicate that the embryos of A. limnaeus do indeed experience some degree of cellular stress (i.e. increase in ubiquitinated proteins, increase in p53 expression, evidence of DNA damage from TUNEL staining and increases in caspase activity) in response to anoxic treatment, even in their most protective state of diapause II. However, despite these observations, the whole organisms are still able to recover from anoxia and do not succumb to death. The overall low levels of TUNEL-positive cells and caspase activity relative to the positive controls indicates that the damage accrued in response to anoxic treatment is minimal. It appears that embryos are able to either sacrifice a certain portion of cells or they are able to repair the damage required for resumed development following anoxia