Carissa M. Krane, Ph.D.
Dryophytes chrysoscelis, commonly known as Cope’s gray treefrog, is a freeze tolerant anuran that freezes up to 65% of extracellular fluid during winter to survive. Glycerol is presumably used as a cryoprotectant during a period of cold-acclimation to protect cells from permanent damage due to hypoosmotic stress upon freezing and thawing. The passage of glycerol and water during cold-acclimation is mediated through aquaglyceroporin HC-3 in the nucleated erythrocytes (RBCs) of D. chrysoscelis. This thesis analyzes the mechanisms in which D. chrysoscelis prepares for cold-acclimation and glycerol synthesis. Cortisol is a stress hormone known to respond to osmolarity and metabolic challenges and regulate aquaporins; however, the role of cortisol in regulating anuran HC-3 protein expression and subcellular localization, and implications for mediating anticipatory glycerol synthesis and freeze tolerance remain to be determined. We hypothesize that cortisol exposure regulates HC-3 protein expression and subcellular localization. Freshly isolated RBCs were cultured in complete cell culture media (CCCM) and cortisol for 2, 4, and 6 hours at two separate concentrations, 1.0 and 0.1 μg/ml. Another group of RBCs was incubated with CCCM for 24 hours in culture before the 4 and 8-hour incubation with cortisol concentrations of 0.01, 0.1, and 1.0 μg/ml. Densitometric analyses of immunoblots specific for HC-3 for RBCs that had undergone the 24-hour incubation before cortisol exposure showed a 3.7-fold increase in native HC3 from RBCs cultured in 0.01 μg/ml cortisol for 4 hours, 40-fold increase in native HC-3 from RBCs cultured in 0.1 μg/ml cortisol for 4 hours, and 21-fold increase in native HC3 from RBCs cultured in 1.0 μg/ml cortisol for 4 hours compared to RBCs cultured in 0 μg/ml for 4 hours. HC-3 protein abundance increased in RBCs exposed to 0.01 μg/ml cortisol for 8 hours by 2.4-fold from the 4-hour time point. The abundance of Page | ii glycosylated HC-3 (60-150kDa) increased by 3.1-fold, 5.8-fold, and 5.3-fold for RBCs exposed to 0.01, 0.1, and 1.0 μg/ml cortisol for 4 hours. The abundance of glycosylated HC-3 increased by 0.8-fold for RBCs treated with 0.01 μg/ml cortisol for 8 hours. A variation in HC-3 protein abundance was observed for freshly isolated RBCs. The subcellular localization and fluorescent intensity (arbitrary units) of the HC-3 protein were analyzed via scanning laser confocal microscopy and immunocytochemistry using ImageJ software. Perinuclear localization of the HC-3 protein was oberved in RBCs exposed to 0.1 and 1.0 μg/ml cortisol for 4 hours and membrane localization for RBCs exposed to 0.1 μg/ml cortisol for 8 hours. Fluorescent analysis of RBCs exposed to 0.01 and 1.0 μg/ml cortisol for 8 hours exhibit enhanced HC-3 intensity in the cytosol compared to control RBCs. Therefore, there is a potential correlation between cryoprotective glycerol, freeze tolerance, and the role of cortisol in regulating HC-3 protein expression and subcellular localization.
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LaBello, Maria P., "Cortisol Regulation of Aquaglyceroporin HC-3 Protein Expression in the Erythrocytes of the Freeze Tolerant Tree Frog Dryophytes chrysoscelis" (2019). Honors Theses. 218.