Winter survival for many animals living in temperate and polar regions of the Earth relies on the ability to survive the freezing of body fluids. As much as 65-70% of total body water may be converted to ice that accumulates in extracellular spaces. In frogs, this includes ice that fills the abdominal cavity, grows in sheets between skin and muscle layers, and fills small spaces such as the lens of the eye or the ventricles of the brain. As water exits cells to freeze in these extracellular spaces, cells and organs shrink down to a small size and most vital signs disappear – breathing and heart beat stop, the animal can`t move, and brain activity cannot be detected. We study several Canadian animals as models of natural freezing survival: wood frogs, juvenile painted turtles, goldenrod gall fly larvae, and intertidal periwinkle snails. Our studies with freeze tolerant frogs and turtles are targeting a variety of questions that we hope will lead to applied solutions that will generate methods for the cryopreservation of human organs to allow them to be frozen and banked for later transplantation.
Biochemical adaptations that we have identified as supporting freeze tolerance include:
(1) Proteins: Nucleating proteins induce and regulate the process of extracellular freezing. Ice restructuring proteins (also called antifreeze proteins) prevent the recrystallization of small ice crystals into larger and larger ones that can cause physical damage to tissues.
(2) Cryoprotectants: high concentrations of polyhydric alcohols (e.g. glycerol, sorbitol) and sugars (e.g. glucose) are packed into cells to prevent excessive reduction of cell volume, prevent intracellular freezing, and stabilize protein conformation. Other protectants such as trehalose and proline prevent membrane bilayer structure from collapsing when cells shrink to small volumes.
(3) Ischemia tolerance: Because circulation is cut off in frozen animals, cells and organs need to survive without oxygen and nutrient delivery for long periods of time and then recover without injury during thawing. Good antioxidant defenses and elevated chaperone proteins help to protect cell macromolecules and metabolic rate depression greatly reduces cell energy needs while frozen.
Our new work includes a major focus on the role of gene and protein expression in freezing survival including (a) identification of novel genes and their protein products that are found only in freeze tolerant species, (b) exploration of a huge range of other genes/proteins that address many different issues in cell preservation and viability, (c) studies of crucial transcription factors that mediate freeze-responsive gene responses, and (d) analysis of the biochemical mechanisms that regulate gene and protein expression to achieve novel outcomes for freezing survival, including microRNA, protein phosphorylation, and epigenetic controls. For example, wood frogs protect their cells using extremely high levels of glucose as a cryoprotectant – levels that are far in excess of those that are lethal to human diabetics. How do they control and endure extreme hyperglycemia without damage to their macromolecules, cells and organs?