FREEZE TOLERANCE AND CRYOPROTECTION OF ERYTHROCYTES FROM DRYOPHYTES CHRYSOSCELIS Thesis Submitted to The College of Arts and Sciences of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree of Master of Science in Biology By Loren Geiss Dayton, Ohio August 2018 1 FREEZE TOLERANCE AND CRYOPROTECTION OF ERYTHROCYTES FROM DRYOPHYTES CHRYSOSCELIS Name: Geiss, Loren Vanessa APPROVED BY: ________________________ Carissa M. Krane, Ph.D. Professor, Dept. of Biology, University of Dayton Faculty Advisor ________________________ Amit Singh, Ph.D. Associate Professor, Dept. of Biology, University of Dayton Committee Member ________________________ Yvonne Sun, Ph.D. Assistant Professor, Dept. of Biology, University of Dayton Committee Member ________________________ David L. Goldstein, Ph.D. Professor, Dept. of Biological Sciences, Wright State University Committee Member ii ABSTRACT FREEZE TOLERANCE AND CRYOPROTECTION OF ERYTHROCYTES FROM DRYOPHYTES CHRYSOSCELIS Name: Geiss, Loren Vanessa University of Dayton Advisor: Dr. Carissa M. Krane Dryophytes chrysoscelis (Cope’s gray treefrog) is a freeze-tolerant anuran that survives the freezing of its extracellular fluids. Treefrogs accumulate glycerol and urea in the plasma during cold acclimation and at the onset of freezing. It is hypothesized that glycerol and urea function as cryoprotectants by minimizing osmotically induced cell damage during freezing and thawing; and therefore, the post-freeze viability of red blood cells (RBCs) would improve when frozen in phosphate buffered saline (PBS) containing glycerol or urea. In the present study, erythrocytes were obtained from warm (22°C) and cold-acclimated (4°C) frogs and suspended in PBS. RBCs were frozen in PBS that ranged in osmolarity: PBS at 230 mM is hypoosmotic/hypotonic, 280 mM PBS is isosmotic/isotonic, and PBS was made hyperosmotic/hypertonic by the addition of 150 mM solutes. Post-freeze viability was determined with a hemolysis assay. Viability of erythrocytes from warm frogs was 31.6±4.9% in 230 mM PBS but was enhanced to 59.4±8.7% and 72.9±4.3%, respectively, when cells were frozen with glycerol (p<0.05) or urea (p<0.001). Post-freeze viability of cells from warm-acclimated frogs improved iii from 18.9±1.3% to 47.4±5.2% with the addition of urea to 280 mM PBS (p<0.01). RBCs from cold-acclimated frogs had 45.8±3.4% viability when frozen in 280 mM PBS which improved to 71.6±8.9% or 71.9±1.6%, respectively, when frozen with glycerol (p<0.01) or urea (p<0.001). The viability of RBCs from cold-acclimated frogs was not different when cells were frozen with glycerol, 71.6±8.9%, or left unfrozen (0°C), 88.4±3.5% (p>0.05). There was also no difference in viability between cells from cold-acclimated frogs that were frozen with urea, 71.9±1.6%, or left unfrozen, 86.7±3.8% (p>0.05). These data suggest that glycerol and urea are part of a complex cryoprotectant system in D. chrysoscelis. iv ACKNOWLEDGEMENTS My special thanks are in order to my past and present committee members Dr. Carissa Krane, Dr. Amit Singh, Dr. Yvonne Sun, Dr. David Goldstein, and Dr. Pothitos Pitychoutis for providing me with the time, knowledge, and guidance for this thesis. I would also like to thank Dr. Clara do Amaral who was a mentor and friend throughout my graduate career. I appreciate all the support from James Frisbie and members of the Krane Lab. Lastly, I would like to thank the fellow biology graduate students that have become my friends for their constant support and positivity. v TABLE OF CONTENTS ABSTRACT……………………………………………………………………………...iii ACKNOWLEDGEMENTS……………………………………………………………….v LIST OF FIGURES……………………………………………………………………..viii LIST OF TABLES………………………………………………………………………..ix LIST OF ABBREVIATIONS AND NOTATIONS……………………………………....x CHAPTER 1: LITERATURE REVIEW………………………………………………….1 Discovery of Freeze-Tolerant Anurans……………………….………………....1 Freezing and Thawing……………………………….…………………………..2 Geographic Differences of Freeze-Tolerant Anurans…………………….……..3 Cellular Osmotic Stress During Freezing and Thawing……………………..…..4 Cryoprotectants Mitigate Osmotic Stress………………………………..……....6 Aquaporins and Aquaglyceroporins………………..……………………………9 Aquaporins in Mammals………………………….……………………………12 Transport Mechanisms of AQP / Cell Signaling………………………..……...14 Aquaporins in D. chrysoscelis…………………………..……………………...15 Aquaporin Expression Based on Thermal State of D. chrysoscelis….………...16 Glycerol Induced HC-3 Expression……………………..……………………...17 Post-Freeze Viability of Erythrocytes…………………….……………………18 Hypothesis…………………………….………………………………………..19 vi References……………………………….……………………………………..21 CHAPTER 2: POST-FREEZE VIABILITY OF ERYTHROCYTES FROM DRYOPHYTES CHRYSOSCELIS………………………………………………………..26 Abstract ……………………………………………….……………..………...27 Introduction…………………………………………….………………….…...29 Materials and Methods……………………………….………………………...30 Results………………………………………………….………………………35 Discussion……………………………………………..………………………..38 Acknowledgements..…………………………………..………….……………44 References………………………………………………………….…………..45 CHAPTER 3: FUTURE DIRECTIONS AND BROADER IMPACTS………………....53 References………………………………………………………….…………..60 vii LIST OF FIGURES CHAPTER 1: LITERATURE REVIEW Figure 1. Glycerol regulates water flux during freezing and thawing……………………5 Figure 2. Structure of aquaporins………………………………………………………...11 CHAPTER 2: POST-FREEZE VIABILITY OF ERYTHROCYTES FROM DRYOPHYTES CHRYSOSCELIS Figure 1. Cryoprotection of D. chrysoscelis erythrocytes via 150 mM of solute…….….49 Figure 2. Cryoprotection of D. chrysoscelis erythrocytes with 150 or 1500 mM solutes……………………………………………………………………………………50 Figure 3. Cryoprotection of D. chrysoscelis erythrocytes with acute and long term exposure to solutes……………………………………………………………………….51 Figure 4. Freeze tolerance and cryoprotection of erythrocytes from warm and cold- acclimated D. chrysoscelis………………………………………………………….……52 CHAPTER 3: FUTURE DIRECTIONS AND BROADER IMPACTS Figure 1. Erythrocytes after incubation at 0C or -8C….…………..…………….…….56 viii LIST OF TABLES CHAPTER 1: LITERATURE REVIEW Table 1. Physiological variables of plasma from warm, cold, frozen, and thawed Dryophytes chrysoscelis…………………………………………………………………..8 Table 2. Mammalian aquaporins……………..…………………………………………..13 ix LIST OF ABBREVIATIONS AND NOTATIONS AQP Aquaporin CCCM Complete Cell Culture Media GLP Aquaglyceroporin GPCR G protein-coupled receptor NPA Asparagine-Proline-Alanine PBS Phosphate Buffered Saline RBC Red blood cell x CHAPTER 1 LITERATURE REVIEW Discovery of Freeze-Tolerant Anurans The goal of research is to answer scientific questions based on observations and human curiosity. One scientist, William Schmid, made the observation that some frogs can survive harsh winters by freezing and subsequently thawing when temperatures rise (Schmid 1982). This remarkable observation evoked other scientists to ask how freeze tolerance occurs and begin research that would answer their own questions regarding this phenomenon. Freeze tolerance is a unique strategy utilized by select organisms in environments that experience subfreezing temperatures. The ability of anurans to survive freezing is generally dependent on the proportion of body fluid that freezes and the osmolarity of body fluids (Costanzo et al. 1995). To date there are six known anuran species that are freeze-tolerant: Pseudacris crucifer (spring peeper, formerly Hyla crucifer), Rana sylvatica (wood frog), Pseudacris triseriata (western chorus frog), Dryophytes versicolor (gray treefrog, formerly Hyla versicolor) and its sister species Dryophytes chrysoscelis (Cope’s gray treefrog, formerly Hyla chrysoscelis), and lastly Dryophytes japonicus (Japanese treefrog, formerly Hyla japonica) (Schmid 1982; Storey and Storey 1986; Costanzo et al. 1992; Hirota et al. 2015). These freeze-tolerant anurans survive freezing of up to 65% of their extracellular 1 fluids over a period of days to months, and have a full recovery after thawing (Schmid 1982; Storey and Storey 1988; Storey and Storey 1992; Layne and Jones 2001). It is important to note that freeze tolerance is not limited to the six previously described anuran species. Other animals, such as specific species of turtles (Churchill and Storey 1991b; Costanzo and Claussen 1990; Storey and Storey 1992), snakes (Costanzo et al. 1988; Churchill and Storey 1991a; Storey and Storey 1992), and insects (Zachariassen and Hammel 1976; Storey and Storey 1996; Sinclair 1999, Sinclair et al. 2003), are also freeze-tolerant. A modern definition of freeze tolerance is a strategy of long-term freezing in which organisms have a maximum ice content they can survive when exposed to temperatures that are ecologically-relevant (Storey and Storey 1992). Freezing and Thawing As temperatures drop towards the subzero range, freeze-tolerant anurans burry themselves in damp leaf litter to be protected from wind and low air temperatures. Even in these microhabitats, temperatures can sink to -8°C (Schmid 1982; Storey and Storey 1992). Freeze-tolerant frogs can supercool when exposed to temperatures that are just below the freezing point, but internal ice inoculation occurs when the skin of a supercooled frog makes contact with environmental ice crystals (Layne et al. 1990; Layne 1991; Costanzo et al. 1995). In freeze-tolerant anurans ice crystals only form extracellularly, as intracellular ice formation would disrupt subcellular organization and be lethal (Mazur 1984; Storey and Storey 1992; Costanzo et al. 1995). Upon freezing, anurans become immobile, hypoxic, and ischemic (Storey and Storey 1988). Freeze- tolerant frogs in northern