Post-Freeze Viability of Erythrocytes from D. chrysoscelis


Post-Freeze Viability of Erythrocytes from D. chrysoscelis



Loren Vanessa Geiss



Cope’s gray treefrog, Dryophytes chrysoscelis, is a freeze tolerant frog that survives the freezing of extracellular fluids during winter. Throughout the fall months the frogs are exposed to a gradual decrease in temperature, becoming cold-acclimated. Cold-acclimated treefrogs have elevated plasma levels of glycerol and urea, solutes that function as cryoprotectants by reducing the intra- and extracellular flux of water during freezing and thawing. To minimize cell damage, glycerol and urea need to be taken up by erythrocytes through a transmembrane protein, aquaglyceroporin HC-3. 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. Cells were suspended in phosphate buffered saline (PBS) ± solute and incubated for 30 minutes at -8°C. Percent hemolysis was calculated as the amount of hemoglobin leaked from erythrocytes over the total amount hemoglobin in the erythrocyte sample. PF viability was 2.6-fold greater in cells from cold-acclimated frogs compared to cells from warm-acclimated frogs. Cells from warm- and cold-acclimated frogs enhanced PF viability when frozen in PBS containing urea (2.8-fold, p<0.005 and 0.6-fold, p<0.001, respectively). PF viability of cells frozen in PBS containing glycerol improved when cells were from cold-acclimated frogs (0.6-fold, p<0.005). Although results support the involvement of glycerol and urea in the cryoprotection of D. chrysoscelis, enhanced viability of cells from cold-acclimated frogs, compared to cells from warm-acclimated frogs, suggest that changes other than glycerol and urea accumulation are involved in the freeze tolerance of D. chrysoscelis.

Publication Date


Project Designation

Graduate Research

Primary Advisor

Carissa M. Krane

Primary Advisor's Department



Stander Symposium project

Post-Freeze Viability of Erythrocytes from D. chrysoscelis