Over 80% of the Earth’s surface is exposed to seasonal cold temperatures less than 5.0oC. Ectotherms implement a variety of strategies to survive seasonal, or permanent, cold exposure. Some of the most common overwintering strategies are migration, hibernation, and freeze avoiding behavior. However, freeze tolerance is a minority choice among ectotherms. This strategy permits organisms to survive between 50.0 to 70.0% of their total body water volumes frozen primarily in extracellular spaces for up to several months at a time. Freeze tolerant organisms undergo minimal supercooling of their body fluids to ensure ice formation is slow and produce a wide variety of specific proteins to control the size of ice crystals forming in the body. Freezing gives rise to severe physiological stressors which must be mitigated in order to survive freezing and thawing. While freeze tolerance is not fully understood, a growing body of evidence highlights several core tenants of this complex physiological process. When a non-freeze tolerant organism freezes, osmotic stress caused by the removal of pure water to form ice crystals in the extracellular fluid causes cells to shrink. As ice crystals thaw, water is rapidly reintroduced into the extracellular fluid causing local hypotonicity. Consequently, cells experience a rapid influx of water molecules, inducing acute cell swelling which progresses and ultimately causes cell lysis which leads to irreparable damage to an organism’s tissues and organs. Many freeze-tolerant animals combat dehydration stress by the seasonal accumulation or rapid mobilization at ice-nucleation of colligative cryoprotectants that diffuse across cell membranes through specific integral transmembrane proteins in order to limit cellular volume changes. Cope’s gray treefrog, Dryophytes chrysocelis, is a treefrog capable of freezing 65% of its total body water content for extended periods of time during harsh winter months. This treefrog is unique because it is the only known freeze-tolerant anuran which mobilizes glycerol as a cryoprotectant.
Glycerol diffuses moves across cell membranes through integral transmembrane protein channels called aquaglyceroporins. This thesis presents a comprehensive literature review which focuses primarily on proposed cellular mechanisms that mitigate dehydration stress caused by the formation of pure ice crystals, as well as anoxic and oxidative stresses caused by freezing-induced ischemia and subsequent blood reperfusion during thawing. This thesis also proposes further research to elucidate vital information about the mechanisms permitting D. chrysoscelis’ freeze tolerance. Finally, the biomedical application of human organ and tissue cryopreservation is discussed, and an argument is presented that glycerol may be a superior cryoprotectant to use in future cryopreservation studies.
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Ripley, Samuel A., "An Exploration of the Widely Observed Mechanisms Permitting Freeze Tolerance & the Potential of Cope’s Gray Treefrog, Dryophytes chrysoscelis" (2021). Honors Theses. 333.