Kenneth B. Storey
Winter survival for numerous cold-blooded animals includes freeze tolerance - the ability to endure the conversion of as much as 65 % of total body water into extracellular ice. Selected molecular adaptations underlying freeze tolerance (e.g. cryoprotectants, ice nucleating proteins) have been widely studied, but the full range of metabolic adjustments needed for freeze endurance remains unknown. Recent studies using gene screening techniques are providing a different approach to the search for biochemical responses that support freezing survival by identifying genes and proteins that are up-regulated by freezing or thawing in freeze tolerant amphibians and reptiles. Screening of a cDNA library from wood frog liver revealed the freeze-induced upregulation of genes coding for the alpha- and gamma-subunits of fibrinogen (a plasma clotting protein), the mitochondrial ADP/ATP translocase, and a novel 10 kD protein containing a nuclear exporting sequence. Furthermore, Northern blotting revealed that these genes were differentially responsive to two of the component stresses of freezing (dehydration and anoxia) indicating that different genes are induced by signals radiating either from cell volume change or oxygen deprivation during freezing. Freeze upregulation of fibrinogen synthesis in liver and other organs appears to be a damage repair response that anticipates a need for enhanced plasma clotting capacity to deal with ice crystal damage to capillary beds. Upregulation of ADP/ATP translocase in frog liver is linked with ischemia resistance and studies with freeze tolerant turtles have shown that other genes encoding proteins involved in mitochondrial energetics (NADH-ubiquinone oxidoreductase subunit 5, cytochrome C oxidase subunit 1) are also upregulated by both anoxia and freezing exposures. These studies are making major advances in our understanding of freeze tolerance as a natural phenomenon and also highlight key new areas that can be targeted by applied interventions for the optimization of medical cryopreservation techniques for cells, tissues and organs.