Freeze Tolerance and Cryoprotection of Erythrocytes from Warm and Cold Acclimated D. chrysoscelis
Authors: Geiss Loren , do Amaral Clara , Frisbie James , Goldstein David , Krane Carissa 
Published: June 2021

Dryophytes chrysoscelis (Cope's gray treefrog) is a freeze tolerant frog that survives the freezing of extracellular fluids throughout winter. Prior to freezing, D. chrysoscelis goes through a cold acclimation period during which the temperature, hours of daylight, and food intake decrease. Cold‐acclimated frogs have elevated plasma levels of glycerol and urea, solutes that function as cryoprotectants by minimizing osmotically induced cell damage during freezing and thawing. To minimize damage, urea and glycerol must be taken up by erythrocytes. Erythrocytes from cold‐acclimated frogs have enhanced abundance and membrane localization of an aquaglyceroporin, HC‐3, through which the intra‐ and extracellular flux of glycerol and urea occur. It is hypothesized that erythrocytes frozen in solution containing glycerol/urea would have greater post‐freeze (PF) viability than cells frozen without glycerol or urea. It is further hypothesized that cells frozen with naturally accumulating, and HC‐3 permeating, solutes (glycerol and urea) would have enhanced PF viability compared to cells frozen with solutes that do not accumulate during cold acclimation (glucose, NaCl, sorbitol). In this study, erythrocytes were obtained from warm‐acclimated (22°C) or cold‐acclimated (4°C) frogs, suspended in phosphate buffered saline (PBS) ± solutes, then incubated at 0 or −8°C for 30 minutes. A separate aliquot of cells was frozen in liquid nitrogen for a 100% hemolysis control. Hemolysis was determined by a colorimetric assay. The percentage of free hemoglobin from the total hemoglobin in the erythrocyte sample was calculated and was normalized to the 100% hemolysis control. Viability of the liquid nitrogen sample was taken as 0%. Viability of cells from warm‐acclimated frogs was reduced by 85% when cells were incubated at −8°C in PBS as oppose to 0°C (p<0.001). Cell viability was reduced approximately 50%, when cells from cold‐acclimated frogs were incubated at −8°C compared to 0°C (p<0.001). Erythrocytes from cold‐acclimated frogs have a 2.6‐fold increase in PF viability compared to cells from warm‐acclimated frogs. Cells from warm‐acclimated frogs had a 2.8‐fold increase in PF viability with the addition of urea in the PBS (p<0.005). Cells from cold‐acclimated frogs had a 0.6‐fold increase in PF viability with the addition of urea or glycerol to the PBS (p<0.001 or p<0.005, respectively). The addition of glucose, NaCl, or sorbitol to PBS did not significantly alter PF viability of cells from warm‐ or cold‐acclimated frogs (p>0.05). Enhanced PF viability of cells from cold‐acclimated frogs, compared to cells from warm‐acclimated frogs suggests cold acclimation aids in the freeze tolerance of D. chrysoscelis by mechanisms other than glycerol/urea accumulation. The results of the cryoprotection assay support the involvement of glycerol and urea in the complex cryoprotectant system of D. chrysoscelis.Support or Funding InformationThis research was supported by NSF IOS‐1121457 (CMK and DLG), Schuellein Chair in the Biological Sciences to CMK, and Schuellein Graduate Research Fellowship to LG.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.