Although people dream of being able to freeze whole bodies and later return them to life, currently successful biomedical cryopreservation is restricted to relatively small structures like blood or embryos. However, there are animals, including several amphibians and reptiles, that survive northern winters in part by an ability to tolerate being frozen. This research will examine the physiological mechanisms responsible for freeze tolerance in one of those species, Cope's gray treefrog. This frog is unusual among freeze-tolerant vertebrates in that it accumulates glycerol in its body fluids as a cryoprotective agent; glycerol is also commonly used in biomedical preservative solutions. Specific questions to be addressed are how glycerol moves in and out of tissues of the treefrogs as they acclimate to cold and actually freeze, and how the glycerol movement is related to the distribution of water among tissues, which is critical to preventing ice formation inside cells where it would lead to lethal damage. In particular, experiments will investigate the role of aquaporins in accomplishing that glycerol and water movement. Aquaporins are proteins known to form passageways that allow water and, sometimes, other small molecules like glycerol to cross cell membranes, and it is hypothesize that they are critical to freeze tolerance. The objectives are to enhance the understanding of this fascinating natural phenomenon, and to use a natural model of freeze tolerance to elucidate principles that can be applied to biomedical cryopreservation. In the process, this work will provide training to undergraduate students at multiple levels of analysis, from principles of comparative biology to whole animals and molecular biology. This training will encourage integrative thinking and application of diverse methods to the study of animal physiology.
