261:1346-1350, 1991. Am J Physiol Regulatory Integrative Comp PhysiolJ. P. Costanzo and R. E. Lee, Jr You might find this additional information useful...1 other HighWire hosted article: This article has been cited by [PDF] [Full Text] [Abstract]R713-R719. , September 1, 1998; 275 (3):Am J Physiol Regulatory Integrative Comp PhysiolJ. P. Costanzo, J. A. Mugnano, H. M. Wehrheim and R. E. Lee Jr. frogsOsmotic and freezing tolerance in spermatozoa of freeze-tolerant and -intoleranton the following topics: http://highwire.stanford.edu/lists/artbytopic.dtlcan be found at Medline items on this article's topics Physiology .. Humans Chemistry .. Osmosis Medicine .. Osmotic Fragility Physiology .. Anura Physiology .. Erythrocytes can be found at: Integrative and Comparative PhysiologyAmerican Journal of Physiology - Regulatory,about Additional material and information http://www.the-aps.org/publications/ajpreguThis information is current as of January 9, 2008 . http://www.the-aps.org/.the American Physiological Society. ISSN: 0363-6119, ESSN: 1522-1490. Visit our website at (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by biological organization, ranging from molecules to humans, including clinical investigations. It is published 12 times a yearinvestigations that illuminate normal or abnormal regulation and integration of physiological mechanisms at all levels of publishes originalThe American Journal of Physiology - Regulatory, Integrative and Comparative Physiology on January 9, 2008 ajpregu.physiology.orgDownloaded fromFreeze-thaw injury in erythrocytes of the freeze-tolerant wood frog, Rana sylvatica JON P. COSTANZO AND RICHARD E. LEE, JR. Department of Zoology, Miami University, Oxford, Ohio 45056 COSTANZO,JON P., AND RICHARD E. LEE, JR. Freeze-thaw injury in erythrocytes of the freeze-tolerant wood frog, Rana sylvatica. Am. J. Physiol. 261 (Regulatory Integrative Comp. Physiol. 30): R1346-R1350, 1991.-Erythrocytes from the freeze-tolerant wood frog (Rana sylvatica) were subjected to in vitro tests of freeze tolerance, cryoprotection, and osmotic fragility. The responses of cells from frogs acclimated to 4 or 15°C were similar. Erythrocytes that were frozen in saline hemolyzed at -4°C or lower. The addition of high concentra- tions (150 and 1,500 mM) of glucose or glycerol, cryoprotectants produced naturally by freeze-tolerant frogs, significantly re- duced cell injury at -8°C but concentrations of 1.5 or 15 mM were ineffective. Hemolysis was reduced by 94% with 1,500 mM glycerol and by 84% with 1,500 mM glucose; thus glycerol was the more effective cryoprotectant. Mean fragility values for frog erythrocytes incubated in hypertonic and hypotonic saline were 1,938 and 49 mosM, respectively. Survival in freeze tolerance and cryoprotection experiments was comparable for erythrocytes from frogs and humans, suggesting that these cells may respond similarly to freezing-related stresses. However, the breadth of osmotic tolerance, standardized for differences in isotonicity, was greater for frog erythrocytes than for human erythrocytes. Our data suggest that erythrocytes from R. syl- vatica are adequately protected by glucose under natural con- ditions of freezing and thawing. red blood cell; hemolysis; cryoprotection; osmotic fragility; glu- cose; glycerol SEVERAL SPECIES of temperate-zone vertebrates survive extensive and repeated freezing of their tissues under ecologically relevant conditions. For example, some frogs tolerate up to 65% of their body water as ice and endure prolonged freezing at temperatures as low as -8°C (see Ref. 19 for review). Ice is probably restricted to extracel- lular compartments under natural (i.e., slow) rates of cooling, because it is generally believed that intracellular ice formation is lethal (13). Physiological studies of natural freeze tolerance in frogs have investigated the time course of ice formation (5, 6), organ desiccation rates (8), metabolism in the frozen state (19), dynamics of cardiac function (7), sea- sonal variations in tolerance (6, 18), and behavioral correlates of recovery processes (8). The gross physiolog- ical stresses associated with body freezing include a massive redistribution of water in cellular (13) and organ (8) body compartments, tissue anoxia owing to ischemia (7, 19), and perturbations in the behavior of macromol- ecules and membrane structures (11). These stresses presumably are mitigated by glucose and/or glycerol, cryoprotectants rapidly mobilized from the liver in direct response to ice formation. To date, no studies of freeze-tolerant vertebrates have examined the responses of blood constituents to freezing and thawing. This is surprising because the heritage of cryobiology is founded in research aimed at preserving frozen human erythrocytes. Furthermore, cryoprotec- tants effective in reducing freezing damage to human erythrocytes are produced naturally in freeze-tolerant frogs. Our current objective was to adapt classic experi- ments on human erythrocytes (9, 10, 15) to determine the responses of erythrocytes from the freeze-tolerant wood frog (Rana sylvatica) in tests of freeze tolerance, cryoprotection, and osmotic stress. We replicated these experiments using human cells for comparative purposes. Because the body temperature of R. sylvatica during late winter is variable, and temperature may strongly influ- ence membrane behavior, we tested cells from frogs acclimated to different thermal regimens. MATERIALS AND METHODS Specimens and sample preparation. Male wood frogs were collected from breeding ponds in Adams County, southern Ohio, during March 4-12, 1989. Previous study showed that frogs collected from this population in late winter are fully freeze tolerant (e.g., Ref. 5). Frogs [mean mass 12.6 t 0.3 (SE) g; n = 581 were kept in plastic boxes on damp moss and exposed to 4 or 15°C for 20 t 2 days (range lo-72 days) in total darkness before bleed- ing. Drinking water