Abstract
The common wood frog, Rana sylvatica, utilizes freeze tolerance as a means of winter survival. Concealed beneath a layer of leaf litter and blanketed by snow, these frogs withstand subzero temperatures by allowing approximately 65–70% of total body water to freeze. Freezing is generally considered to be an ischemic event in which the blood oxygen supply is impeded and may lead to low levels of ATP production and exposure to oxidative stress. Therefore, it is as important to selectively upregulate cytoprotective mechanisms such as the heat shock protein (HSP) response and expression of antioxidants as it is to shut down majority of ATP consuming processes in the cell. The objective of this study was to investigate another probable cytoprotective mechanism, anti-apoptosis during oxygen deprivation and recovery in the anoxia tolerant wood frog. In particular, relative protein expression levels of two important apoptotic regulator proteins, Bax and p-p53 (S46), and five anti-apoptotic/pro-survival proteins, Bcl-2, p-Bcl-2 (S70), Bcl-xL, x-IAP, and c-IAP in response to normoxic, 24 Hr anoxic exposure, and 4 Hr recovery stages were assessed in the liver and skeletal muscle using western immunoblotting. The results suggest a tissue-specific regulation of the anti-apoptotic pathway in the wood frog, where both liver and skeletal muscle shows an overall decrease in apoptosis and an increase in cell survival. This type of cytoprotective mechanism could be aimed at preserving the existing cellular components during long-term anoxia and oxygen recovery phases in the wood frog.
Highlights
Winter survival usually depends on an animal’s ability to utilize physiological and biochemical mechanisms of freeze tolerance, freeze avoidance, or migration
The relative expression levels of the two anti-apoptotic proteins, x-Inhibitor of apoptotic proteins (IAPs) and c-IAP that are responsible for inhibiting the function of Caspase 3, 6, 7 were assessed using western immunoblotting (Fig. 3). x-IAP showed no change in expression, whereas c-IAP showed 0.6-fold increase in expression in response to 24 Hr anoxia (p < 0.05)
The North American Wood frog, R. sylvatica, can survive sub-zero temperatures by utilizing a strategy called freeze-tolerance, in which 65–70% of the total body water is converted into extracellular ice (Storey & Storey, 1984)
Summary
Winter survival usually depends on an animal’s ability to utilize physiological and biochemical mechanisms of freeze tolerance, freeze avoidance, or migration. Complete freezing of bodily fluids can be lethal to most animals and animals that use freeze-avoidance strategies substantially decrease the supercooling point of their bodily fluids and employ anti-freezing strategies to prevent spontaneous nucleation at subzero temperatures (Storey & Storey, 1988). Freeze-tolerant animals allow freezing of the extracellular fluid and utilize extracellular ice nucleating agents (INA) to manage ice growth but stringently prevent freezing of the cytoplasmic fluid (Storey & Storey, 2004a). Natural freeze tolerance is known to occur in terrestrial insects of the Coleoptera, Diptera, Hymenoptera, and Lepidoptera orders (Ring, 1980; Block, 1982; Storey & Storey, 1988), intertidal marine gastropods and barnacles (Aarset, 1982; Murphy, 1983), and several species of terrestrial frogs, one of which is the freeze-tolerant wood frog, Rana sylvatica (Storey & Storey, 1985; Storey & Storey, 1988)
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