Cryoprotection by urea in a terrestrially hibernating frog

The role of urea as a balancing osmolyte in osmotic adaptation is well known, but this 'waste product' also has myriad other functions in diverse taxa. We report that urea plays an important, previously undocumented role in freezing tolerance of the wood frog (Rana sylvatica), a northern woodland species that hibernates terrestrially in sites where dehydration and freezing may occur. Wood frogs inhabiting an outdoor enclosure accumulated urea to 65 mmol l(-1) in autumn and early winter, when soil moisture was scarce, but subsequently urea levels fell to similar to 2 mmol l(-1) as the availability of environmental water increased. Laboratory experiments showed that hibernating R. sylvatica can accumulate at least 90 mmol l(-1) urea under relatively dry, warm conditions. During experimental freezing, frogs synthesized glucose but did not accumulate additional urea. Nevertheless, the concentrations of urea and glucose in some tissues were similar. We tested urea's efficacy as a cryoprotectant by measuring lysis and lactate dehydrogenase (LDH) leakage in samples of R. sylvatica erythrocytes frozen/thawed in the presence of physiological levels of urea or other osmolytes. In conferring protection against freeze/thaw damage, urea was comparable to glycerol and as good as or better than glucose, cryoprotectants found in freeze-tolerant frogs and other animals. Urea treatment also improved the viability of intact tissues frozen in vitro, as demonstrated by post-thaw measures of metabolic activity and LDH leakage. Collectively, our findings suggest that urea functions both as an osmoprotectant and a cryoprotectant in terrestrially hibernating amphibians.