1 Freeze Tolerance and Cryoprotection Of

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

Loren Geiss

Dayton, Ohio

August 2018

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

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.

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.

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

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 0C or -8C….…………..…………….…….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

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

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 populations may remain frozen for the duration of winter whereas other populations may experience fluctuations in temperature throughout the

2 winter months and thus go through multiple freeze/thaw cycles. In either case, thawing occurs faster at the core of the frog compared with peripheral tissue, as the core has a higher concentration of cryoprotectants. The cardiac system is the first process to be initiated. As thawing extends outwards, the lungs regains function followed by nerve excitability and then limb movement (Rubinsky and Storey 1994; Costanzo et al. 1995).

Geographic Differences of Freeze-Tolerant Anurans

Apart from D. japonicus, which reside in Japan, the remaining five freeze-tolerant frogs inhabit a geographic range extending from Alaska, through Canada, and throughout the northeastern portion of the United States. D. chrysoscelis, a diploid organism, has gone through an allopatric speciation event giving rise to its sister tetraploid species, D. versicolor. These two species have additional populations throughout the southeastern

United States.

The latitudinal range that the gray treefrog complex, D. versicolor and D. chrysoscelis, inhabits promoted studies that examined the freeze tolerance of frogs from different geographic locations because the frogs are exposed to a variation of cold temperatures. Frogs of the D. versicolor species from a northern population in Ontario,

Canada survive freezing at lower temperatures for longer periods of time compared with frogs from a more southern population, Indiana (Storey and Storey 1985; Layne and Lee

1989; Costanzo et al. 1992). The conditions the frogs were exposed to prior to freezing varied between populations (e.g. rate of the decrease in temperature, amount of daylight, and decrease in food availability). These conditions are collectively known as cold acclimation.

It is possible that differences in the parameters involved in cold acclimation set the stage for how frogs respond to varying degrees of subfreezing temperatures. Going through a mild cold acclimation period, as is the case with frogs from southern populations, may limit the physiological responses of the frogs in terms of viability upon freezing and thawing. A harsh or more rapid cold acclimation period may physiologically set the frogs up to survive more intense freezing events as is seen in northern D. versicolor frogs. To understand the differences in the cold acclimation regime and freeze tolerance, frogs from both diploid and tetraploid species were collected from a few populations that varied in latitudinal range. All frogs were acclimated under the same conditions. Regardless of the species or collection site, the gray treefrogs did not differ in physiological responses to freezing (Irwin and Lee 2003). This experiment provides support that cold acclimation is important precursor event that aids in post-freeze viability in the gray treefrog complex.

Cellular Osmotic Stress During Freezing and Thawing

Freezing and thawing create physiological challenges to an anuran at the cellular level. The formation of ice crystals in the extracellular fluid causes mechanical stress to the cells. Metabolic stress occurs as blood circulation slows and limits oxygen delivered to cells. A third stressor induced by freezing and thawing is osmotic stress. The present work focuses on osmotic stress and the mechanisms that freeze-tolerant anurans, specifically D. chrysoscelis, utilize to mitigate this stress. Upon freezing, extracellular ice formation reduces the amount of free water and increases the extracellular solute concentrations creating a hypertonic environment for cells. This change in effective osmolarity could cause cells to lose water resulting in cell shrinkage, loss of membrane

4 integrity, and potentially cell death. The accumulation and distribution of osmolytes within an organism reduces the amount of intracellular water loss and cell death. (Figure

1; Storey and Storey 1997; Costanzo and Lee 2005; Yancey 2005; Storey and Storey

2013). During thawing, the melting of ice crystals in the extracellular fluid potentially creates a hypotonic environment for cells. The additional osmolytes would further aid in the survival of cells at the time of thawing by limiting water movement into the cell that would otherwise result in cell swelling and lysis. To understand freeze tolerance at the organismal and cellular level, the type and source of osmolytes must be examined, as well as cell membrane permeability to the specific osmolyte. In the context of freeze tolerance, these osmolytes may function as cryoprotectants.

Figure 1. Glycerol regulates water flux during freezing and thawing. At subfreezing temperatures, some extracellular fluid freezes which increases the osmolarity of the remaining extracellular fluid. Water is osmotically driven out of cells, causing cell shrinkage and death. Intracellular glycerol retains water within the cell during freezing. Extracellular glycerol prevents osmotic swelling of cells during thawing. Image adapted from Storey and Storey, 2013.

Cryoprotectants Mitigate Osmotic Stress

Cryoprotectants are solutes that aid in the survival of cells during freezing and thawing events. Known cryoprotectants utilized by freeze-tolerant anurans are generally organic solutes with a low molecular mass and high stability, and are metabolic end- products, making them readily available, as with glucose in R. sylvatica (Costanzo and

Lee 2005; Storey and Storey 2004). Upon freezing, R. sylvatica have plasma glucose concentrations up to 35-fold higher than unfrozen frogs (Costanzo et al. 1993; Costanzo and Lee 2005).

There is evidence of glycerol and urea being accumulated in D. chrysoscelis.

Some studies on D. chrysoscelis report anticipatory accumulation of glycerol during the cold acclimation period prior to freezing (Schmid 1982; Storey and Storey 1985;

Costanzo et al. 1992; Layne and Jones 2001; Irwin and Lee 2003; Zimmerman et al.

2007), whereas other studies report plasma glycerol levels are elevated at the onset of freezing, after cold-acclimation (Layne and Stapleton 2009; do Amaral et al. 2018). One study investigated the accumulation of plasma glycerol, glucose, and urea levels as well as changes in plasma osmolarity between warm-acclimated, cold-acclimated, frozen, and thawed treefrogs (Table 1; do Amaral et al. 2018). Plasma urea levels increased in cold- acclimated frogs compared with warm-acclimated frogs, whereas plasma osmolarity, glucose, or glycerol levels did not significantly change. Plasma urea levels in frozen or thawed frogs did not vary from urea levels in cold-acclimated frogs. Plasma osmolarity, glycerol, and glucose levels all increase in frozen frogs compared with cold-acclimated frogs. However, plasma glycerol was the only variable that remained elevated in thawed frogs (do Amaral et al. 2018). From a functional perspective, it would be more beneficial

6 to D. chrysoscelis to have anticipatory accumulation of glycerol because glycerol transport into cells occurs more rapidly under warmer conditions and occurs more slowly as the temperature approaches the freezing point (Goldstein et al. 2010). Regardless if plasma glycerol levels become elevated during cold acclimation or upon freezing, hepatic glycogen stores are the likely source of glycerol (Storey and Storey 1984; Irwin and Lee

2003). The liver is also the source of urea, which comes from protein degradation. When temperatures decrease and environmental resources, like food, become limited triglycerides may be broken down for energy during which glycerol is metabolic by- product.

The blood stream is the likely mechanism for these solutes to be distributed throughout the frog. Circulating blood is filtered by the kidneys and these solutes exhibited at elevated concentrations, compared with blood from warm-acclimated frogs, then some of the solutes may hypothetically be filtered into the urine, thereby losing the cryoprotective values. However, functional analysis of kidneys from cold-acclimated D. chrysoscelis suggests reduced glomerular filtration rates and urine flow in comparison to kidneys from warm-acclimated frogs and therefor solutes are retained in the blood

(Zimmerman et al. 2007). Data are supportive of glycerol and urea being accumulated and distributed throughout the treefrog via blood plasma.

Aquaporins and Aquaglyceroporins

One potential mechanism for these solutes to enter and exit cells is through specialized proteins known as aquaglyceroporins (GLPs). Aquaporins (AQPs) are a family of proteins that facilitate the osmotically driven transmembrane flux of water.

Although simple diffusion allows for the osmotically driven movement of water directly through the lipid bilayer, this mechanism is too slow to support normal physiological processes such as cellular absorption and secretion of water that occurs along the urinary tract (Krane and Kishore 2003). Physiological rates of transmembrane water flux are maintained in part by AQPs. The first AQP was discovered in 1992 by Peter Agre and was denoted CHIP28, channel-like integral membrane protein of the 28 kDa size, but now is known as AQP1 (Preston et al. 1992). There are currently multiple known AQPs expressed in a variety of cell types. These proteins range from 27-37 kDa before post translational modifications (Kitchen et al. 2015). Some AQPs are further classified as aquaglyceroporins (GLPs). GLPs are permeable to water as well as small solutes such as

9 glycerol and urea (Borgnia and Agre 2001; Thomas et al. 2002; Krane and Kishore 2003;

Hara-Chikuma and Verkman 2006; Krane and Goldstein 2007).

The basic structure of all AQPs and GLPs is similar. An individual AQP protein consists of six transmembrane alpha helices with cytoplasmic tails (Figure 2a). This pattern results in three extracellular loops (A, C, and E) and two intracellular loops (B and D). Loops B and E fold into the cell membrane and each contains an Asparagine-

Proline-Alanine (NPA) motif; this NPA motif is highly conserved within the AQP family. Three-dimensional analysis of AQP’s depict the interaction of the NPA motif in loop B with the NPA motif in loop E , thereby creating a pore through which water molecules can pass. In the cell membrane AQPs exist as homotetramers containing four pores for water and/or solutes to pass through down their concentration gradient (Figure

2b; Sui et al. 2001; Krane and Kishore 2003; Krane and Goldstein 2007; Kitchen et al.

2015). Water molecules can pass through the pore single file by making and breaking hydrogen bonds within the molecule and with amino acid residues in the constriction region of the AQP (Sui et al. 2001). In the NPA motif, the asparagine residue is suggested to play the major role in forming hydrogen bonds with water molecules

(Eriksson et al. 2013; Kitchen et al. 2015). AQPs and GLPs are similar to a variety of other proteins in that they are inhibited by mercury, HgCl2. Near the NPA motif on loop

E is a cysteine residue which has a high binding affinity for HgCl2, when bound, HgCl2 inhibits permeability of the AQP (Preston et al. 1993; Zhang et al. 1993; Krane and

Kishore 2003; Krane and Goldstein 2007). The side profile of an AQP is a conical shaped extracellular portion which narrows in the transmembrane region and widens out again in

10 the intracellular space (Sui et al. 2001). In general, these proteins exist as tetramers when inserted in the plasma membrane and have a dumbbell or hourglass shape.

Figure 2. Structure of aquaporins. A) Diagram of an aquaporin in its primary structure within a plasma membrane. Intra- and extracellular loops are labeled A-E. The five amino acid sites that vary between AQPs and GLPs are labeled P1-5. Green, red, and purple circles are the NPA motif. Figure from Krane and Kishore 2003. B) An aquaporin structure as a homotetramer generated using Swiss Institute of Bioinformatics. This folding allows for four pores to facilitate the transmembrane flux of water and/or solutes.

There are some structural differences between AQPs and GLPs that give rise to the difference in permeability of water and small solutes. Previous research identified five amino acid positions (P1-P5) that consistently differ between AQPs and GLPs: P1 is located on transmembrane helix 3, P2 and P3 are on extracellular loop E, P4 and P5 are on transmembrane helix 6 (Figure 2b; Heymann and Engle 2000; Krane and Goldstein

2007). The specific amino acid residues in GLPs increase the diameter of the pore and make the constriction region of the protein more polar (Sui et al. 2001). These changes allow glycerol, a larger molecule than water, to more easily pass through the pore. The polar interior of the pore is more attractive to the hydrophobic backbone of glycerol.

Aquaporins in Mammals

CHIP28, or AQP1, was first discovered in mammalian RBCs (Preston et al. 1992) but there are now thirteen known mammalian AQPs/GLPs (AQP0-AQP12; Table 2;

Krane and Goldstein 2007; Kitchen et al. 2015). In contrast to AQP0,1, 2, 4, 5, 6, and 8, which are classic AQPs, AQP3, 7, 9, 10, and 11 are GLPs (Gonen and Walz 2006;

Gorelick et al. 2006; Zardoya 2005; Kitchen et al. 2015). AQP12 is currently under investigation to determine if it falls under the GLP subfamily.

As previously discussed, GLPs are permeable to small solutes such as glycerol.

The functional significance of AQPs and GLPs in facilitating the transmembrane flux of water and/or solutes is commonly studied by phenotypic examination of knockout mice.

AQP3 is involved in wound healing and elasticity of the skin (Hara-Chikuma and

Verkman 2005; Hara-Chikuma and Verkman 2008). AQP7 deficient mice suffered from adipocyte hypertrophy and obesity (Hara-Chikuma et al. 2005). AQP7 also functions in renal proximal tubules and in the liver (Sohara et al. 2005). GLP 3, 7, 9, and 10 are also known to be permeable to urea (Kitchen et al. 2015). Work with AQPs and GLPs and the role they have in human disease is a continually growing field.

Transport Mechanisms of AQP / Cell Signaling

In order for AQPs, and GLPs, to facilitate the transmembrane flux of water and/or solutes, the protein must be embedded within the cell membrane. Increasing overall protein abundance of AQPs via transcription and translation takes longer than the translocation of AQPs to the plasma membrane that occurs within milliseconds via cytoplasmic vesicles (Brown 2003). A well-studied example of membrane localization of

AQPs involves AQP2 in the principal cells of the collecting duct in the kidneys. Arginine vasopressin is an anti-diuretic hormone that binds to the vasopressin type 2 receptor on the basolateral surface of the principal cells. Once bound, the G protein-coupled receptor activates adenylate cyclase which triggers protein kinase A to phosphorylate AQP2.

Phosphorylation results in translocation of AQP2 from intracellular vesicles to the apical membrane. This process increases water reabsorption (Krane and Kishore 2003). In human Caco-3 cells, AQP3 membrane localization is induced by epinephrine, also known as adrenaline, and this process involves protein kinase C (Yasui et al. 2008). Epinephrine

14 has also been shown to be involved in membrane localization of AQP5 (Ishikawa et al.

1999). In some of the above examples of AQP membrane localization, the AQPs are phosphorylated. In addition to hormones, changes in localization of mammalian AQPs also may be induced by osmotic stress (reviewed in Conner et al. 2013).

Aquaporins in D. chrysoscelis

Four members of the AQP/GLP family (HC-1, HC-2, HC-3, and HC-9) have been sequenced and characterized in D. chrysoscelis (Zimmerman et al. 2007; Stogsdill et al. 2017). Analyzing amino acid sequences with a focus on positions 1-5 that vary between AQPs and GLPs resulted in classifying HC-1 and -2 as AQPs and HC-3 as an

GLP (Krane and Goldstein, 2007). These classifications are supported by functional characterization of HC-1, -2, -3, and -9 that was done by injecting the cRNA of each aquaporin into Xenopus oocytes. These oocytes have low inherent water permeability; therefore, when injected oocytes are placed in hypo-osmotic solutions the rate of swelling via water permeability can be described as a function of the specific AQP whose expression has been induced. Oocytes with HC1 or HC-2 had similar osmotic swelling as oocytes with AQP1, whereas oocytes with HC-3 or HC-9 had minimal water uptake but were permeable to glycerol (Zimmerman et al. 2007; Stogsdill et al. 2017). Due to these results, HC-1 and HC-2 are considered AQPs and HC-3 and HC-9 are GLPs. Classifying the proteins as AQPs or GLPs provides insight on their physiological function.

Messenger RNA of all four AQPs/GLPs was detected in the skin, kidney, and bladder. HC-1, -3, and -9 mRNA were also detected in gut, liver, lung, brain, muscle, fat tissue, and RBCs (Zimmerman et al. 2007; Goldstein et al. 2010; Stogsdill et al. 2017).

Tissues known to express all four proteins are limited to skin and kidney. HC-1, -3, and -

9 proteins were also detected in gut, liver, and muscle (Pandey et al. 2010; Stogsdill et al.

2017). Of these AQPs and GLPs, HC-3 is the only protein to be expressed in RBCs of D. chrysoscelis (Mutyam et al. 2011). There is potential for additional AQPs to be discovered in D. chrysoscelis.

Aquaporin Expression Based on Thermal State of D. chrysoscelis

If GLPs are involved in the freeze tolerance of D. chrysoscelis by facilitating the transmembrane flux of solutes that potentially function as cryoprotectants, then fluctuation in GLP abundance and/or subcellular localization should be in sync with the changes in solute concentration. Because glycerol and urea concentrations increase as frogs are cold-acclimated and upon freezing, AQP/GLP expression should also be correlated to the thermal state of the frogs. HC-9 protein levels, both native and glycosylated, were enhanced in liver from frozen or thawed treefrogs compared to warm- acclimated treefrogs (Stogsdill et al. 2017).

Utilizing RBCs to study HC-3 is advantageous because RBCs can be obtained without having to sacrifice the frogs. In most of the tissues that HC-3 mRNA was detected in, the mRNA levels were elevated in tissues from cold-acclimated tree frogs compared to the corresponding tissues from a warm-acclimated frog (Zimmerman et al.

2007; Goldstein et al. 2010). HC-3 protein abundance was also greater in erythrocytes from cold-acclimated frogs compared with warm-acclimated frogs. This trend was consistent regardless of analyzing HC-3 in the native or glycosylated form (Goldstein et al. 2010; Mutyam et al. 2011). The correlation between the changes in temperature, glycerol and urea concentrations, and abundance of HC-3 is suggestive that HC-3 is involved in transmembrane flux of such solutes. Not only is the abundance of HC-3

16 important for facilitating transmembrane flux of solutes, but subcellular localization of

HC-3 is also a factor that may be regulated. The increase in HC-3 abundance in RBCs of

D. chrysoscelis determined via Western blotting does not indicate where, subcellularly, the new protein is located.

RBCs that were imaged under confocal microscopy depict HC-3 evenly distributed throughout the cytoplasm in cells obtained from a warm-acclimated frog, but in cells obtained from a cold-acclimated frog HC-3 expression is pronounced at the in and near the plasma membrane (Goldstein et al. 2010; Mutyam et al. 2011). For HC-3 to facilitate transmembrane flux of solutes, it must be inserted into the plasma membrane rather than being in the nucleus or cytoplasm.

Glycerol Induced HC-3 Expression

RBCs from D. chrysoscelis are nucleated and metabolically active. This allows cells to be cultured under varying conditions and for the cells to respond to such conditions. A cell culture system was established in which erythrocytes from D. chrysoscelis maintained over 90% viability for up to 96 hours (Mutyam et al. 2011). To elucidate the relationship between plasma glycerol accumulation, which occurs as treefrogs are cold-acclimated or upon freezing, and enhanced HC-3 abundance and membrane localization, which also occurs during cold acclimation, cells can be cultured in media with or without glycerol to determine if glycerol alone regulates HC-3 expression. When erythrocytes were obtained from a warm-acclimated treefrog and cultured in media containing approximately 150 mM glycerol, HC-3 protein abundance and membrane localization mimics that of HC-3 expression in erythrocytes from cold- acclimated frogs. Specifically, cells cultured with glycerol have increased abundance of

17 glycosylated HC-3 and increased membrane localization of HC-3 (Mutyam et al. 2011).

More drastic elevations in HC-3 abundance and membrane localization occur when erythrocytes obtained from a cold-acclimated frog were cultured in media with glycerol compared to cells cultured in media without glycerol (Mutyam et al. 2011). So far, correlations have been made between three different factors: 1) increase in plasma glycerol and urea in cold-acclimated frogs, 2) increase in HC-3 abundance and membrane localization in cold-acclimated frogs, and 3) elevated HC-3 abundance and membrane localization in cells when cultured in media that contains glycerol. Research in this field is still lacking the connection between the three correlations above and post-freeze viability of RBCs from D. chrysoscelis.

Post-Freeze Viability of Erythrocytes

Like D. chrysoscelis, R. sylvatica is a freeze-tolerant frog, but factors involved in the freeze tolerance of these two frogs vary. Cold acclimation is not necessary for the freeze tolerance of R. sylvatica, whereas cold acclimation is necessary for the freeze tolerance of D. chrysoscelis. When erythrocytes from warm and cold-acclimated R. sylvatica were frozen and thawed there was no difference is survivability (Costanzo and

Lee 1991). Furthermore, erythrocyte survival after a freeze/thaw cycle was the same regardless if erythrocytes were from humans or freeze-tolerant R. sylvatica (Costanzo and

Lee 1991). These data suggest that there is nothing inherent about RBCs from wood frogs that enable the cells to be freeze-tolerant. Rather, the cells are likely made freeze-tolerant when cryoprotectants are distributed throughout the animal upon freezing. Glucose, a solute that is naturally accumulated in the plasma of R. sylvatica upon freezing, functions as a cryoprotectant to R. sylvatica. Increasing glucose concentration from 150 mM to

1500 mM only increased the post-freeze viability of erythrocytes from wood frogs, not humans (Costanzo and Lee 1991). The difference in post-freeze viability of erythrocytes from wood frog and humans likely stems from the permeability of glucose to each cell type (Guarner and Alvarez-Buylla 1989).

Hypothesis

Freeze tolerance is the remarkable ability of an organism to survive after being frozen, and D. chrysoscelis is one such organism with this ability. Four broad conclusions about the freeze tolerance of D. chrysoscelis are: 1) glycerol, glucose, and urea are accumulated during cold acclimation or at the onset of freezing, 2) HC-3 is permeable to glycerol and presumably permeable to urea, 3) HC-3 protein is expressed in a variety of cell types, specifically RBCs, and 4) HC-3 abundance and membrane localization in

RBCs is enhanced in cold-acclimated treefrogs. To further understand the mechanism of freeze tolerance and cryoprotection of D. chrysoscelis classical tests of freeze tolerance and cryoprotection can be adapted to examine the post-freeze viability of RBCs from D. chrysoscelis (Lovelock 1953; Lovelock 1954; Costanzo and Lee 1991). It is hypothesized that RBCs from cold-acclimated treefrogs will have greater post-freeze viability than

RBCs from warm-acclimated frogs and that glycerol and urea function as cryoprotectants by mitigating osmotic stress during freezing and thawing of D. chrysoscelis. Determining if glycerol and urea function as cryoprotectants has not yet been examined, but this can be investigated in experiments similar to Costanzo’s work with RBCs from R. sylvatica

(Costanzo and Lee 1991). The Krogh principle is a cornerstone for comparative physiology which states, ‘For a large number of problems there will be some animal of choice, or a few such animals on, which it can be most conveniently studied’ (Krogh

1929). One such problem that exists in the field of human medicine is organ transplantation. If the mechanisms of freeze tolerance utilized by D. chrysoscelis are understood, they can be applied to human organ transplantations. The potential benefit of freezing organs would be to have organs readily available to thaw and transplant into patients in need.

References

Borgnia MJ, Agre P (2001) Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli. PNAS 5:2888- 2893

Brown D (2003) The ins and outs of aquaporin-2 trafficking. Am J Physiol Renal Physiol 5:F893-F901

Churchill TA, Storey KB (1991a) Freezing survival of the garter snake Thamnophis sirtalis. Can J Zool 70:99-105

Churchill TA, Storey KB (1991b) Natural freezing survival by painted turtles Chrysemys pieta marginata and C. p. bellii. Am J Physiol 262:530-537

Conner AC, Bill RM, Conner MT (2013) An emerging consensus on aquaporin translocation as a regulatory mechanism. Mol Membr Biol 1:1-12

Costanzo JP, Claussen DL (1990) Natural freeze tolerance in the terrestrial turtle, Terrapene carolina. J Exp Zool 254:228-32

Costanzo JP, Claussen DL, Lee RE (1988) Natural freeze tolerance in a reptile. Cryo- Lett. 9:380-85

Costanzo JP, Lee RE (1991) Freeze-thaw injury in erythrocytes of the freeze-tolerant wood frog, Rana sylvatica. Am J Physiol 6:R1346-R1350

Costanzo JP, Lee RE, Devries AL, Wang TC, Layne JR (1995) Survival mechanisms of vertebrate ectotherms at subfreezing temperatures - applications in cryomedicine. FASEB JOURNAL 5:351-358

Costanzo JP, Lee RE, Lortz PH (1993) Glucose concentration regulates freeze tolerance in the wood frog Rana sylvatica. J Exp Biol 181:245-255

Costanzo JP, Lee RE (2005) Cryoprotection by urea in a terrestrially hibernating frog. J Exp Biol 208:4079-4089

Costanzo JP, Wright MF, Lee RE (1992) Freeze tolerance as an overwintering adaptation in Cope's grey treefrog (Hyla chrysoscelis). Copeia 2:565-569 do Amaral MCF, Frisbie J, Goldstein DL, Krane CM (2018) The cryoprotectant system of Cope's gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation, freezing, and thawing. J Comp Physiol B 188:611-621

Eriksson UK, Fischer G, Friemann R, Neutze R, Enkavi G, Tajkhorshid E (2013) Subangstrom resolution x-ray structure details aquaporin-water interactions. Science 6138:1346-1349

Goldstein DL, Frisbie J, Diller A, Pandey RN, Krane CM (2010) Glycerol uptake by erythrocytes from warm- and cold-acclimated Cope's gray treefrogs. J Comp Physiol B 8:1257-1265

Gonen T, Walz T (2006) The structure of aquaporins. Q Rev Biophys 4:361-396

Gorelick DA, Praetorius J, Tsunenari T, Nielsen S, Agre P (2006) Aquaporin-11: a channel protein lacking apparent transport function expressed in brain. BMC Biochem 7:14

Guarner V, Alvarez-Buylla R (1989) Erythrocytes and glucose homeostasis in rats. Diabetes 4:410-415

Hara-Chikuma M, Sasaki S, Uchida S, Verkman AS, Okabe M, Sohara E, Rai T, Ikawa M (2005) Progressive adipocyte hypertrophy in aquaporin-7-deficient mice: adipocyte glycerol permeability as a novel regulator of fat accumulation. J Biol Chem 16:15493-15496

Hara-Chikuma M, Verkman AS (2005) Aquaporin-3 functions as a glycerol transporter in mammalian skin. Biol Cell 7:479-486

Hara-Chikuma M, Verkman AS (2006) Physiological roles of glycerol-transporting aquaporins: the aquaglyceroporins. Cell Mol Life Sci 12:1386-1392

Hara-Chikuma M, Verkman AS (2008) Roles of aquaporin-3 in the epidermis. J Invest Dermatol 9:2145-2151

Heymann JB, Engel A (2000) Structural clues in the sequences of the aquaporins. J Mol Biol 295:1039-1053

Hirota A, Sakamoto J, Shiojiri N, Suzuki M, Tanaka S, Okada R, Takiya Y (2015) Molecular cloning of cDNA encoding an aquaglyceroporin, AQP-h9, in the Japanese tree frog, Hyla japonica: possible roles of AQP-h9 in freeze tolerance. Zool Sci 3:296-306

Irwin JT, Lee RE (2003) Geographic variation in energy storage and physiological responses to freezing in the gray treefrogs Hyla versicolor and H. chrysoscelis. J Exp Biol 206:2859-2867

Ishikawa Y, Skowronski MT, Inoue N, Ishida H (1999) Alpha(1)-adrenoceptor-induced trafficking of aquaporin-5 to the apical plasma membrane of rat parotid cells. Biochem Biophys Res Commun 1:94-100

Kitchen P, Conner AC, Conner MT, Salman MM, Day RE, Bill RM (2015) Beyond water homeostasis: diverse functional roles of mammalian aquaporins. BBA - General Subjects 1850:2410-2421

Krane CM, Goldstein DL (2007) Comparative functional analysis of aquaporins/glyceroporins in mammals and anurans. Mamm Genome 6:452-462

Krane CM, Kishore BK (2003) Aquaporins: the membrane water channels of the biological world. Biologist 2:81-86

Krogh A (1929) The progress of physiology. Am J Physiol 90:243-251

Laforenza U, Bottino C, Gastaldi G (2015) Mammalian aquaglyceroporin function in metabolism. Bioch Bioph Acta. 2016:1-11

Layne JR (1991) External ice triggers freezing in freeze-tolerant frogs at temperatures above their supercooling point. J Herpetol 1:129-130

Layne JR, Huang JL, Lee REJ (1990) Inoculation triggers freezing at high subzero temperatures in a freeze-tolerant frog (Rana sylvatica) and insect (Eurosta solidaginis). Can J Zool 3:506-510

Layne JR, Jones AL (2001) Freeze tolerance in the gray treefrog: cryoprotectant mobilization and organ dehydration. J Exp Zool 1:1-5

Layne JR, Lee RE (1989) Seasonal variation in freeze tolerance and ice content of the tree frog Hyla versicolor. J Exp Zool 249:133

Layne JR, Stapleton MG (2009) Annual variation in glycerol mobilization and effect of freeze rigor on post-thaw locomotion in the freeze-tolerant frog Hyla versicolor. J Comp Physiol B 2:215-221

Lovelock JE (1953) The haemolysis of human red blood-cells by freezing and thawing. Biochim. Biophys. Acta 10:414-426

Lovelock JE (1954) The protective action of neutral solutes against hemolysis by freezing and thawing. Biochim. J. 56:265-270

Lutoslawska G (2014) Aquaporins in physiology and pathology. TSS 21:185-194

Mazur P (1984) Freezing of living cells: mechanisms and implications. Am J Physiol 3:C125-C142

Mutyam V, Puccetti MV, Frisbie J, Goldstein DL, Krane CM (2011) Dynamic regulation of aquaglyceroporin expression in erythrocyte cultures from cold- and warm- acclimated Cope's gray treefrog, Hyla chrysoscelis. J Exp Zool A Ecol Genet Physiol 7:424-437

Pandey RN, Yaganti S, Coffey S, Frisbie J, Alnajjar K, Goldstein D (2010) Expression and immunolocalization of aquaporins HC-1, -2, and -3 in Cope's gray treefrog, Hyla chrysoscelis. Comp Biochem Physiol A Mol Integr Physiol 1:86-94

Preston GM, Carroll TP, Guggino WB, Agre P (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 5055:385

Preston GM, Jung JS, Guggino WB, Agre P (1993) The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel. J Biol Chem 1:17-20

Rubinsky B, Storey K (1994) H-1 magnetic-resonance-imaging of freezing and thawing in freeze-tolerant frogs. Am J Physiol 6:R1771-R1777

Schmid WD (1982) Survival of frogs in low temperature. Science 215:697-698

Sinclair BJ (1999) Insect cold tolerance: how many kinds of frozen? Eur J Entomol 96:157-164

Sinclair BJ, Addo-Bediako A, Chown SL (2003) Climatic variability and the evolution of insect freeze tolerance. Biol Rev 78:181-195

Sohara E, Rai T, Miyazaki J, Verkman AS, Sasaki S, Uchida S (2005) Defective water and glycerol transport in the proximal tubules of AQP7 knockout mice. Am J Physiol Renal Physiol 6:F1195-F1200

Stogsdill B, Frisbie J, Krane CM, Goldstein DL (2017) Expression of the aquaglyceroporin HC-9 in a freeze-tolerant amphibian that accumulates glycerol seasonally. Physiol Rep 15:e13331

Storey KB, Storey JM (1984) Biochemical adaption for freezing tolerance in the wood frog, Rana sylvatica. J Comp Physiol B 1:29-36

Storey KB, Storey JM (1985) Triggering of cryoprotectant synthesis by the initiation of ice nucleation in the freeze tolerant frog, Rana sylvatica. J Comp Physiol B 2:191- 195

Storey KB, Storey JM (1986) Freeze tolerance and intolerance as strategies of winter survival in terrestrially-hibernating amphibians. Comp Biochem Physiol A Comp Physiol 4:613-617

Storey KB, Storey JM (1988) Freeze tolerance in animals. Physiol Rev 1:27-84

Storey KB, Storey JM (1992) Natural freeze tolerance in ectothermic vertebrates. Annu Rev Physiol 54:619-637

Storey KB, Story JM (1996) Natural freezing survival in animals. Annu Rev Ecol Syst 27:365-386

Storey KB, Storey JM (1997) Adaptations for freezing survival in ectothermic vertebrates. Advances in Molecular and Cell Biology 19:1-32

Storey KB, Storey JM (2004) Physiology, biochemistry, and molecular biology of vertebrate freeze tolerance: the wood frog. In: Fuller BJ, Lane N, Benson EE (ed) Life in the Frozen State. Washington, DC, pp. 243-274

Storey KB, Storey JM (2013) Molecular biology of freezing tolerance. Comp Physiol 3:1283-1308

Sui H, Han BG, Lee JK, Walian P, Jap BK (2001) Structural basis of water-specific transport through the AQP1 water channel. Nature 6866:872-878

Thomas D, Bron P, Ranchy G, Duchesne L, Cavalier A, Rolland J, Raguénès-Nicol C, Hubert J, Haase W, Delamarche C (2002) Aquaglyceroporins, one channel for two molecules. Biochim Biophys Acta 1:181-186

Yancey PH (2005) Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J Exp Biol 208:2819-2830

Yasui H, Kubota M, Iguchi K, Usui S, Kiho T, Hirano K (2008) Membrane trafficking of aquaporin 3 induced by epinephrine. Biochem Biophys Res Commun 4:613-617

Zachariassen KE, Hammel HT (1976) Nucleating agents in the haemolymph of insects tolerant to freezing. Nature 262:285-287

Zardoya R (2005) Phylogeny and evolution of the major intrinsic protein family. Biol Cell 6:397-414

Zhang R, van Hoek A.N, Biwersi J, Verkman AS (1993) A point mutation at cysteine 189 blocks the water permeability of rat kidney water channel CHIP28. Biochemistry 12:2938-2941

Zimmerman SL, Frisbie J, Goldstein DL, West J, Rivera K, Krane CM (2007) Excretion and conservation of glycerol, and expression of aquaporins and glyceroporins, during cold acclimation in Cope's gray tree frog Hyla chrysoscelis. Am J Physiol Regul Integr Comp Physiol 1:R544-R555

CHAPTER 2

POST-FREEZE VIABILITY OF ERYTHROCYTES FROM

DRYOPHYTES CHRYSOSCELIS

Loren Geiss1, M. Clara F. do Amaral2, James Frisbie3, David L. Goldstein3, Carissa M. Krane1

1Department of Biology, University of Dayton, Dayton, OH, 2Department of Biology, Mount St Joseph University, Cincinnati, OH, 3Department of Biological Sciences, Wright State University, Dayton OH

Abstract

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 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

27 data suggest that glycerol and urea are part of a complex cryoprotectant system in D. chrysoscelis.

Introduction

Cope’s gray treefrog, Dryophytes chrysoscelis, is a freeze-tolerant anuran that can survive the freezing and thawing of up to 65% of its extracellular fluids (Schmid

1982; Storey and Storey 1988; Storey and Storey 1992; Layne and Jones 2001). Freeze tolerance allows D. chrysoscelis to survive the subfreezing temperatures that they are naturally exposed to in Canada and the eastern United States (Storey and Storey 1985;

Layne and Lee 1987; Costanzo et al. 1992; Costanzo et al. 1995). Upon freezing, the amount of free water in the extracellular fluid is reduced creating a hypertonic environment, but when ice crystals thaw, increasing free water in the extracellular fluid, the cells are exposed to a hypotonic environment (Mazur 1984; Storey and Storey 1992;

Costanzo et al. 1995; Storey and Storey 2013).

Prior to freezing, D. chrysoscelis goes through a cold acclimation period, during which the frogs acclimate to a gradual decrease in temperature, reduced hours of daylight, and reduced food availability (Zimmerman et al. 2007; Mutyam et al. 2011; do

Amaral et al. 2018). Glycerol and urea are solutes that are naturally accumulated in D. chrysoscelis during cold acclimation or at the onset of freezing. Cold-acclimated frogs accumulate up to ~37 mM of urea and ~50 mM glycerol in the plasma after cold acclimation and up to ~150 mM glycerol in the plasma upon freezing (Zimmerman et al.

2007; do Amaral et al. 2018). Plasma glycerol levels remain elevated after the frog has thawed (do Amaral et al. 2018).

Glycerol and urea are known to function as cryoprotectants in freeze-tolerant anurans (Storey 1997; Costanzo and Lee 2005; Goldstein et al. 2010). Cryoprotectants function by reducing the freezing point, reducing osmotic cell shrinkage, reducing

29 mechanical injury to cell membranes that would occur as cells shrink, and stabilizing intracellular structures and proteins (Mazur 1984; Carpenter and Crowe 1988; Storey

1990; Costanzo and Lee 2005). The present work investigates solutes that potentially function as cryoprotectants to reduce cellular osmotic lysis during freezing and thawing of D. chrysoscelis. Another freeze-tolerant anuran, Rana sylvatica, accumulates glycogen, the source of glucose, and urea as cryoprotectants (Storey and Storey 1985;

Storey and Storey 1986; Storey and Storey 1987; Storey and Storey 1988; Storey and

Storey 1992; Costanzo et al. 1995; Costanzo and Lee 2005; Storey and Storey 2013).

Post-freeze viability of red blood cells (RBCs) from R. sylvatica has previously been investigated and viability is elevated when RBCs are frozen with 150 mM glucose or glycerol (Costanzo and Lee 1991).

The objective of this study is to investigate the cryoprotectant system involved in the freeze tolerance of D. chrysoscelis by adapting classic experiments in tests of freeze tolerance and cryoprotection of RBCs (Lovelock 1953; Lovelock 1954; Costanzo and Lee 1991). We hypothesized that RBCs from D. chrysoscelis would have enhanced freeze tolerance if 1) RBCs are frozen in solutions containing glycerol or urea, 2) RBCs were cultured with glycerol or urea prior to freezing to induce cellular changes that occur during cold acclimation, or 3) the treefrog is cold-acclimated rather than warm- acclimated.

Materials and Methods

Collection and Acclimation of Cope’s Gray Treefrogs

Male Dryophytes chrysoscelis were identified and collected from Greene

County, Ohio as previously described (Zimmerman et al. 2007; Goldstein et al. 2010;

Pandey et al. 2010; Mutyam et al. 2011). Animals were housed individually at Wright

State University, Dayton, OH. Conditions of frog husbandry were kept consistent with previous studies (Zimmerman et al. 2007; Goldstein et al. 2010; do Amaral et al. 2018).

During summer and early fall months, all frogs were kept at 22°C, exposed to 12:12 light: dark photo regime, and fed crickets three times per week. Frogs that were kept in the above conditions throughout late fall and winter were the warm-acclimated frogs; whereas a subset of animals was transferred to a light and temperature-controlled room to be cold-acclimated. These frogs were exposed to a gradual decrease in temperature from

22°C to 5°C. The light cycle was adjusted to 8:16 light-dark cycles to match natural day- night cycles during fall/winter months. Food availability gradually ceased as the animals reduced food intake, but water was consistently available to all frogs. Whole blood was collected into heparinized capillary tubes after puncture of a brachial artery or from the trunk of animals that were sacrificed for other experiments. A total of 14 frogs were used in the present study and minimum of fourteen days elapsed between repeated blood draws from an individual frog. All collection, housing, and experiments involving live animals were approved by the Laboratory Animal Care and Use Committee at Wright

State University.

Freeze Tolerance and Cryoprotection of RBCs when Frozen in 230 mM

PBS

Blood from warm-acclimated frogs was dispensed in phosphate buffered saline

(PBS) at 230 mM (100 mM NaCl, 2 mM KCl, 6 mM Na2HPO4, 1 mM KH2PO4;

Rosendale et al. 2014). An aliquot of cell suspension was mixed 1:1 with Trypan Blue

(Gibco, 15250-061) to determine live versus dead cells and cells were counted with a

31 hemocytometer. Cell suspensions were then centrifuged for 5 minutes, at 2000xg and

22°C and the supernatant was removed. Cell pellets and stock solutions of PBS (230 mM) ± 150 or 1500 mM glycerol (Fisher, BP358-212), glucose (Sigma, G8270-100G), or urea (Fisher, BP169-500) were chilled on ice for 10 minutes to be at matching temperatures. Cells were resuspended in PBS (230 mM) ± glycerol, glucose, urea at 7.8 million cells per ml solution and immediately incubated at 0 for 30 minutes or placed in a water bath at 0°C which was cooled to and held at -6°C for 30 minutes. Ice nucleation was induced in samples at -6°C with Envi-ro-tech Freezing Spray (Thermo Scientific,

6769038). Samples were held at 4°C for 15 minutes to thaw. Complete cell lysis was obtained by freezing cells in liquid nitrogen for 30 minutes.

Freeze Tolerance and Cryoprotection of Cultured RBCs when Frozen in

230 mM PBS

Blood from warm-acclimated frogs was dispensed into Complete Cell Culture

Media (CCCM, medium prepared as described in Mutyam (2011): RPMI 1640; HyClone,

SH30027.01). Within 40 minutes cell suspensions were centrifuged for 10 minutes, at

1000xg and 22°C. Cells were resuspended at 1 million cells/ml of CCCM ± 150 mM glycerol, glucose, or urea and cultured. At the 24-hour timepoint, cells were resuspended in fresh CCCM ± 150 mM glycerol, glucose, or urea and cultured an additional 24 hours.

Cells were centrifuged for 5 minutes, at 2000xg and 22°C. Cell pellets and stock solutions were chilled on ice for 10 minutes to be at matching temperatures. Cells were resuspended in PBS (230 mM, chilled on ice) ± glycerol, glucose, urea at 7.8 million cells per ml solution and immediately incubated at 0 for 30 minutes or placed in a water

32 bath at 0°C which was cooled to and held at -6°C for 30 minutes. Samples were held at

4°C for 15 minutes to thaw.

Freeze Tolerance and Cryoprotection of RBCs from Warm and Cold-

Acclimated D. chrysoscelis Frozen in 280 mM PBS

Blood from warm and cold-acclimated frogs was dispensed in CCCM; CCCM was at the respective temperature at which the frog had been acclimated. Within 40 minutes, the samples were centrifuged for 5 minutes, at 2000xg to pellet out the RBCs, which were then washed in PBS (280 mM, Sigma, 806552) and centrifuged for 5 minutes, at 2000xg at the respective temperature at which the frog had been acclimated.

Cell pellets were chilled on ice for 10 minutes. Cells were resuspended at 7.8 million cells/ml of PBS (280 mM, chilled on ice) ± 150 mM glycerol, glucose, urea, or sorbitol

(Fisher, BP439-500), or 83 mM NaCl (Fisher, PB358-212). Cells were immediately incubated at 0 for 30 minutes or placed in a water bath at 0°C which was cooled to and held at -8°C for 30 minutes. Samples were held at 4°C for 15 minutes to thaw.

Hemoglobin Assay

Cell viability through freezing and thawing was assessed from the release of hemoglobin into solution, indicative of cell lysis, similar to Constanzo and Lee (-1991).

After the 15 minutes at 4°C, cell suspensions were mixed via inverting tubes, then centrifuged for 5 minutes at 2000xg and 4°C. Supernatant, 100μl, from each sample was collected and obtained in individual tubes. The remaining supernatant and cell pellet were briefly vortexed (10 seconds) and erythrocytes were lysed via three freeze-thaw cycles (2 minutes in dry ice, 2 minutes at 37°C). Lysed erythrocyte suspensions were collected,

100μl, and placed in individual tubes. 1ml Hemoglobin Reagent (Pointe Scientific,

H7504-120) was dispensed into each tube containing 100μl supernatant or 100μl lysed erythrocyte suspension. After a 3-minute incubation period, the absorbance was measured at 540nm with a spectrophotometer (Thermo Electron Corporation, BioMate 3). Whole blood hemoglobin standard (Pointe Scientific, H7506-STD) was used to calculate the hemoglobin concentration in the aliquots of RBC supernatant and lysed RBCs as described by the manufacture’s protocol (Hemoglobin Reagent Set, Pointe Scientific).

The hemoglobin concentration of both supernatant and cellular fractions were calculated by dividing the absorbance of the treatment sample by the absorbance of the hemoglobin standard. Percent hemolysis was calculated as the hemoglobin concentration of the supernatant fraction over the hemoglobin concentrations of supernatant and cellular fractions combined. Viability of erythrocytes is inversely related to hemolysis; the cells that were frozen in liquid nitrogen for 30 minutes provided a 100% hemolysis standard which was converted to 0% post-freeze viability.

Data Analysis

Mean viability ± standard error of the mean (SEM) are the descriptive summaries used for the variables measured. Comparisons of variables between control and treatment groups were performed using a repeated-measures analysis of variance

(ANOVA) followed by a Tukey’s HSD test. Analyses were performed using R software version 3.4.2. Statistical significance was accepted at p<0.05.

Results

Freeze Tolerance and Cryoprotection of RBCs when Frozen in 230 mM

PBS

To determine if solutes function as cryoprotectants and improve post-freeze viability, the baseline post-freeze viability was established. Cells from warm-acclimated frogs that were suspended in 230 mM PBS and frozen to -6°C for 30 minutes had

31.6±4.9% post-freeze viability (Fig.1). Post-freeze viability of frog RBCs frozen to -6°C was greater when cells were frozen with 150 mM glycerol (59.4±8.7%, p<0.05) or urea

(72.9±4.3%, p<0.001, Fig.1). In contrast, freezing cells with 150 mM glucose did not enhance post-freeze viability of RBCs (46.7±2.6%, p>0.05, Fig.1).

Solute concentration was raised from 150 mM to 1500 mM to determine if increasing the amount of solute would improve post-freeze viability of D. chrysoscelis

RBCs. Post-freeze viability of RBCs did not change when cells were frozen with 150 mM or 1500 mM glycerol (59.4±8.7% vs. 84.9±3%, p>0.05), glucose (46.7±2.6% vs.

43.3±8.8%, p>0.05) or urea (72.9±4.3% vs. 64.3±7.7%, p>0.05; Fig.2).

Freeze Tolerance and Cryoprotection of Cultured RBCs when frozen in 230

mM PBS

D. chrysoscelis undergo cold acclimation during which plasma glycerol and urea levels are elevated. To investigate if increasing the length of exposure of RBCs from D. chrysoscelis to solutes results in cellular changes and improved post-freeze viability,

RBCs were cultured for 48 hrs with or without glycerol, glucose, or urea, then frozen in

230 mM PBS. Baseline viability of cells cultured in media without supplemental solutes and then frozen to -6°C in 230 mM PBS was 23.4±3.8% (Fig.3). Culturing RBCs in

35 media augmented with 150 mM glycerol, glucose, or urea prior to freezing cells to -6°C in 230 mM PBS without supplemental solutes did not improve post-freeze viability

(respectively: 28.4±8.1%, p>0.05, Fig.3a; 31.1±7.4%, p>0.05, Fig.3b; 31±6.7%, p>0.05,

Fig.3c). Regardless of whether cells were cultured in media with or without supplemental solutes, post-freeze viability was not enhanced when cells were frozen in PBS without those supplements.

RBCs in vivo that are exposed to accumulated extracellular solutes during cold acclimation are still exposed to those solutes upon freezing. To better mimic the in vivo conditions, RBCs were cultured in media with or without supplemental solutes and then frozen to -6°C in 230 mM PBS with the respective solute. Baseline values of post-freeze viability were obtained from cells cultured in unaugmented media then frozen in PBS with 150 mM glycerol (58.0±9%, Fig.3a), glucose (38.7±7.5%, Fig.3b), or urea

(65.6±6.9%, Fig.3c). Viability of RBCs was not different when cells were cultured in media with a solute and then frozen with the corresponding solute (glycerol: 62.6±5.7%, p>0.05, Fig3a; glucose: 51.8±5.4%, p>0.05, Fig3b; or urea: 64.2±2.4%, p>0.05, Fig3c).

These data suggest that culturing cells in media with or without a solute does not alter post-freeze viability if cells are frozen with the solute.

Cells that were cultured in media augmented with glycerol or urea and frozen in to -6°C in 230 mM PBS with the respective solute had greater post-freeze viability than cells that were cultured in media augmented with glycerol or urea but frozen in PBS without supplemental solutes. Glycerol at the time of freezing increased viability from

28.4±8.1% to 58.0±9% (p<0.05, Fig.3a). Supplemental urea in PBS increased cellular viability from 31±6.7% to 64.2±2.4% (p<0.01, Fig.3c). These data show the importance

36 of cryoprotectants at the time of freezing. Cells that were cultured in media augmented with glucose did not vary in post-freeze viability when the cells were frozen in PBS alone or in PBS with glucose, respectively 31.1±7.4% vs. 51.8±5.4% (p>0.05, Fig.3b).

Freeze Tolerance and Cryoprotection of RBCs from Warm and Cold-

Acclimated D. chrysoscelis when frozen in 280 mM PBS with or without

supplemental solutes

To determine if cold acclimation aids in cellular freeze tolerance, RBCs from warm and cold-acclimated frogs were frozen to -8°C in 280 mM PBS. Erythrocytes from warm-acclimated treefrogs that were left unfrozen, 0°C, had a viability of 85.1±2.6% which is greater than the viability of cells that were frozen to -8°C, 18.9±1.3% (p>0.05,

Fig.4a). Unfrozen cells from cold-acclimated frogs had an 88.9±4.9% viability which was greater than the viability of cells from cold-acclimated frogs after being frozen,

45.8±2.9% (p>0.05, Fig.4b). Erythrocytes from cold-acclimated frogs were 2.4 times more freeze-tolerant than erythrocytes from warm-acclimated frogs (p<0.01, Fig4c).

Post-freeze viability of erythrocytes from both warm and cold-acclimated frogs was enhanced by the addition of glycerol or urea, solutes that function as cryoprotectants.

Supplemental urea improved post-freeze viability of RBCs from warm-acclimated frogs by 2.5-fold (p<0.01, Fig4a). Post-freeze viability of RBCs from warm-acclimated frogs that were frozen in PBS with urea, 47.4±5.2%, was similar to that of cells from cold- acclimated frogs that were frozen without a solute, 45.8±2.9% (p<0.05, Fig.4c).

The greatest post-freeze viability occurred when cells came from cold- acclimated frogs and were frozen in PBS with glycerol, 71.6±8.9%, or urea, 71.9±1.6%.

The viability of cells, from cold-acclimated frogs, after being frozen with glycerol or urea

37 was the same as the viability of those cells in PBS with glycerol or urea that were not frozen (p>0.05, Fig.4b). Glycerol and urea are solutes that function as cryoprotectants because they improve post-freeze viability of cells. To determine if the enhanced post- freeze viability was due to the specific properties of glycerol and urea as opposed to the elevated osmolarity in those solutions, cells were also frozen in PBS with glucose, NaCl, or sorbitol. These solutes had no cryoprotective effect on RBCs from warm or cold- acclimated frogs (Fig.4c).

Discussion

The goal of this study was to investigate freeze tolerance of RBCs that were frozen with or without specific extracellular solutes, cultured with or without those solutes, and derived from warm- and cold-acclimated treefrogs. Previous studies indicate a cold acclimation period is necessary for Cope’s gray treefrogs to become freeze- tolerant. One known response to cold acclimation and the onset of freezing is the elevation of plasma glycerol, urea, and glucose levels (Zimmerman et al. 2007; do

Amaral et al. 2018). RBCs of D. chrysoscelis are used to study the phenomenon of freeze tolerance because RBCs are nucleated, enabling cell composition to be altered when cells are cultured under different conditions in vitro (Mutyam et al. 2011). It is hypothesized that glycerol and urea fluctuate intra- and extracellularly and function as cryoprotectants to reduce osmotic stress that occurs during freezing and thawing; and this hypothesis is supported by results from this study.

What is the inherent freeze-tolerance of anuran RBCs?

RBCs may be exposed to different plasma osmolarities as the frog freezes and thaws, so different osmolarities of PBS were used. RBCs from D. chrysoscelis that were

38 in isotonic 280 mM PBS and frozen to -8°C had 18.9% post-freeze viability. Preliminary data of RBCs from D. chrysoscelis that were frozen in 230 mM PBS at -8°C had such low post-freeze viability that the addition of solutes did not enhance post-freeze viability.

However, when RBCs from D. chrysoscelis were frozen in 230 mM PBS at -6°C, the viability of cells was 31% and improved with the addition of 150 mM glycerol or urea. It seems that at -8°C the freeze tolerance of RBCs in hypotonic environments is reduced.

Without cryoprotectants, up to ~70-81% of cells from warm-acclimated frogs lysed after one freeze/thaw cycle. In comparison, RBCs from another freeze-tolerant anuran, R. sylvatica, had ~60% survival after being frozen to -6°C and 14% survival after being frozen to -8°C, in 230 mM PBS. (Costanzo and Lee 1991). These data suggest that RBCs from freeze-tolerant anurans vary in inherent freeze-tolerance. Rather, freeze-tolerance of

RBCs relies on a complex set of cellular systems that may vary among species.

Do permeating solutes influence the freeze tolerance of RBCs from D.

chrysoscelis?

RBCs from warm-acclimated frogs have been cultured for 48 hrs in CCCM± glycerol or urea to isolate the effects of these specific solutes on cell viability from any other variables that are related to cold acclimation. Cells cultured in CCCM + glycerol or urea prior to freezing cells in PBS with the respective solute had similar viability as cells that were cultured in just CCCM but frozen in PBS + glycerol or urea. These data highlight the importance of glycerol and urea availability to cells at the time of freezing to have cryoprotective effects.

Accumulation of glycerol and urea are not the only changes that occur in D. chrysoscelis during cold acclimation. One other known response to cold acclimation is

39 changes in aquaglyceroporin (GLP) HC-3 protein abundance and membrane localization.

GLPs are transmembrane proteins known to facilitate the intra- and extracellular flux of water and small molecules such as glycerol and urea (Borgnia and Agre 2001; Thomas et al. 2002; Krane and Kishore 2003; Hara-Chikuma and Verkman 2006; Krane and

Goldstein 2007). HC-3 is expressed in RBCs of D. chrysoscelis (Zimmerman et al. 2007;

Pandey et al. 2010; Mutyam et al. 2011) and has been shown to be permeable to glycerol

(Zimmerman et al. 2007; Goldstein et al. 2010). It is proposed that HC-3, like other

GLPs, is permeable to urea, in addition to glycerol, and therefore facilitates the intra- and extracellular flux of these cryoprotectants. It is hypothesized that glycerol and urea enter cells and reduce water loss that would otherwise occur upon freezing when cells are in a hypertonic environment.

Unlike cells frozen with glycerol and urea, the addition of glucose, NaCl, or sorbitol to cells from D. chrysoscelis did not improve post-freeze viability. In consideration of the solutes tested, it is suggestive that there is a solute specific effect on freeze tolerance of RBCs from D. chrysoscelis.

It is known that cold acclimation is necessary for the freeze tolerance of D. chrysoscelis (Storey and Storey 1988; Costanzo et al. 1992; Costanzo et al. 1995;

Goldstein et al. 2010). The present data suggest cold acclimation results in some cellular changes because RBCs from cold-acclimated frogs had a 2.4-fold increase in post-freeze viability compared to cells from warm-acclimated frogs. To our knowledge, this is the first study on freeze-tolerant anurans reporting that cold acclimation improves freeze tolerance at the cellular level.

Under most conditions tested (PBS at 230 or 280 mM, and RBCs from warm or cold-acclimated frogs), the addition of 150 mM glycerol or urea increased post-freeze viability of RBCs. Increasing the glycerol and urea levels from 150 to 1500 mM did not further enhance post-freeze viability of RBCs from warm-acclimated frogs. These data are suggestive that there is a colligative threshold to the cryoprotective effects of glycerol and urea.

The most compelling result obtained from the present study is that RBCs, from cold-acclimated frogs, that were frozen in PBS + glycerol or urea have the same post- freeze viability as cells that were unfrozen. The combination of glycerol and urea accumulation as well as other physiological changes during cold acclimation are all involved in the freeze tolerance and cryoprotection of D. chrysoscelis.

The post-freeze viability of RBCs from R. sylvatica was ~61 and 36% when 150 mM glycerol or glucose, respectively, was added upon freezing at -8°C and when 1500 mM glycerol or glucose was added the post-freeze viability was enhanced to ~95 and

86% respectively (Costanzo and Lee 1991). With RBCs from D. chrysoscelis, increasing the concentration of glycerol 10-fold did not further enhance post-freeze viability as it did with RBCs from R. sylvatica. D. chrysoscelis does not produce as much glucose as other freeze-tolerant anurans, such as R. sylvatica (Storey and Storey 1986; Storey and Storey

1987; Costanzo et al. 1992; Layne and Jones 2001). It is evident that distinct cryoprotect strategies are utilized by the two freeze-tolerant anurans: D. chrysoscelis and R. sylvatica.

What factors influence the viability of RBCs through freezing and thawing?

Overall, our data suggest that RBCs from D. chrysoscelis have a solute specific response in terms of freeze tolerance, which aligns with the natural solute accumulation.

Furthermore, RBCs from cold-acclimated frogs have greater post-freeze viability than cells from warm-acclimated frogs, and cells from cold-acclimated frogs that were frozen with cryoprotectants have the same viability as cells that were not frozen. One limitation in the present study is that it takes ~15 minutes for the temperature in the water bath to drop from 0°C to -6 or -8°C; which is faster than what D. chrysoscelis in the Ohio region would naturally be exposed to. Increasing the rate of temperature change that the cells are exposed to, likely results in a higher proportion of cell lysis to occur. A second limitation is that these frogs are wild caught animals that may vary, in unknown ways, in age and health. These factors may influence the post-freeze viability of the RBCs that are obtained from the frogs. The fluctuation of environmental conditions to which the different cohorts of D. chrysoscelis are exposed prior to collection may further influence results. For example, one cohort of frogs had elevated plasma glycerol levels after cold acclimation, whereas plasma glycerol levels were elevated once the frogs were frozen when the frog cohort was collected a different year (Zimmerman et al. 2007; do Amaral et al. 2018). The timing of glycerol and urea accumulation brings up a third limitation. In the experimental conditions, 150 mM glycerol or urea are added to a solution with RBCs in a single step. This may introduce acute osmotic shock for the RBCs that would not occur to RBCs within the frogs. Controlling for individual and cohort variation of the frogs is beyond the scope of this experiment.

Prior to this study, it was known that glycerol, glucose, and urea function as cryoprotectants to freeze-tolerant anurans and that these three solutes are naturally accumulated in D. chrysoscelis. The present study examines freeze tolerance at the cellular level. The data suggest a solute specific response because glycerol and urea

42 improve post freeze viability whereas glucose, NaCl, and sorbitol do not. Furthermore, there is an inherent difference in RBCs from cold-acclimated frogs that results in greater freeze tolerance compared to warm-acclimated frogs. Insight gained by investigating the freeze tolerance and cryoprotection strategies utilized by D. chrysoscelis, could potentially be compared with other freeze-tolerant anurans. The strategies utilized by freeze-tolerant anurans could be applied to the freezing and cryopreservation of cells, tissues, or organisms that are otherwise not freeze-tolerant.

Acknowledgements

Statistical support for this project was provided by Julia Chapman at the

University of Dayton. This research was supported by National Science Foundation (IOS-

1121457 to CMK and DLG), Schuellein Chair in the Biological Sciences to CMK, and

Schuellein Chair Graduate Research Fellowship to LG.

Author contributions: LG, MCFA, and CMK conceived and designed the study;

LG, MCFA, and JF performed experiments; LG analyzed the data; LG, MCFA, and

CMK interpreted results of experiments; LG prepared the figures and drafted the manuscript; LG, MCFA, JF, DLG, and CMK edited and revised the manuscript; LG,

MCFA, JF, DLG, and CMK approved the final version of the manuscript.

References

Borgnia MJ, Agre P (2001) Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli. PNAS 98:2888- 2893

Carpenter JF, Crowe JH (1988) The mechanism of cryoprotection of proteins by solutes. Cryobiology 25:244-255

Costanzo JP, Lee RE (1991) Freeze-thaw injury in erythrocytes of the freeze-tolerant wood frog, Rana sylvatica. Am J Physiol 261:R1346-R1350

Costanzo JP, Lee RE (2005) Cryoprotection by urea in a terrestrially hibernating frog. J Exp Biol 208:4079-4089

Costanzo JP, Lee RE, Devries AL, Wang TC, Layne JR (1995) Survival mechanisms of vertebrate ectotherms at subfreezing temperatures - applications in cryomedicine. FASEB Journal 9:351-358

Costanzo JP, Wright MF, Lee RE (1992) Freeze tolerance as an overwintering adaptation in Cope's grey treefrog (Hyla chrysoscelis). Copeia 1992:565-569

Costanzo JP, Lee RE (2005) Cryoprotection by urea in a terrestrially hibernating frog. J Exp Biol 208:4079-4089 do Amaral MCF, Frisbie J, Goldstein DL, Krane CM (2018) The cryoprotectant system of Cope's gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation, freezing, and thawing. J Comp Physiol B 188:611-621

Goldstein DL, Frisbie J, Diller A, Pandey RN, Krane CM (2010) Glycerol uptake by erythrocytes from warm- and cold-acclimated Cope's gray treefrogs. J Comp Physiol B 180:1257-1265

Hara-Chikuma M, Verkman AS (2006) Physiological roles of glycerol-transporting aquaporins: the aquaglyceroporins. Cell Mol Life Sci 63:1386-1392

Krane CM, Goldstein DL (2007) Comparative functional analysis of aquaporins/glyceroporins in mammals and anurans. Mamm Genome 18:452-462

Krane CM, Kishore BK (2003) Aquaporins: the membrane water channels of the biological world. Biologist 50:81-86

Layne JR, Jones AL (2001) Freeze tolerance in the gray treefrog: cryoprotectant mobilization and organ dehydration. J Exp Zool 290:1-5

Layne JR, Lee RE (1987) Freeze tolerance and the dynamics of ice formation in wood frogs (Rana sylvatica) from southern Ohio. Canadian J Zoology 65: 2062-2065

Lovelock JE (1953) The haemolysis of human red blood-cells by freezing and thawing. Biochim. Biophys. Acta 10:414-426

Lovelock JE (1954) The protective action of neutral solutes against hemolysis by freezing and thawing. Biochim. J. 56:265-270

Mazur P (1984) Freezing of living cells: mechanisms and implications. Am J Physiol 247:C125-C142

Rosendale AJ, Lee,Richard E. Jr, Costanzo JP (2014) Effect of physiological stress on expression of glucose transporter 2 in liver of the wood frog, Rana sylvatica. J Exp Zool A Ecol Genet Physiol 321:566-576

Schmid WD (1982) Survival of frogs in low temperature. Science 215:697-698

Storey KB (1990) Life in a frozen state: adaptive strategies for natural freeze tolerance in amphibians and reptiles. Am J Physiol 258:R599-568

Storey KB (1997) Organic solutes in freezing tolerance. Comp Biochem Physiol A 117:319-326

Storey KB, Storey JM (1985) Triggering of cryoprotectant synthesis by the initiation of ice nucleation in the freeze tolerant frog, Rana sylvatica. J Comp Physiol B 156:191- 195

Story KB, Storey JM (1986) Freeze tolerance and intolerance as strategies of winter survival in terrestrially-hibernating amphibians. Comp Biochem Physiol 83A:613-617

Story KB, Storey JM (1987) Persistence of freeze tolerance in terrestrially-hibernating frogs after spring emergence. Copeia 1987:720-726

Storey KB, Storey JM (1988) Freeze tolerance in animals. Physiol Rev 68:27-84

Storey KB, Storey JM (1992) Natural freeze tolerance in ectothermic vertebrates. Annu Rev Physiol 54:619-637

Storey KB, Storey JM (2013) Molecular biology of freezing tolerance. Compr Physiol 3:1283-1308

Figure Legends

Fig.1 Cryoprotection of D. chrysoscelis erythrocytes via 150 mM of solute. Post-freeze viability (%) of erythrocytes from warm-acclimated frogs after being frozen to -6C in

230 mM PBS ± 150 mM glycerol, glucose, or urea. Mean ± SEM. N=3-6. Data points labeled with the same letter indicate similar post-freeze viability. p<0.05

Fig.2 Cryoprotection of D. chrysoscelis erythrocytes with 150 or 1500 mM solutes. Post- freeze viability (%) of erythrocytes from warm-acclimated frogs after being frozen to

-6C in 230 mM PBS + glycerol, glucose, or urea. Mean ± SEM. N=3-6. Data points labeled with the same letter indicate similar post-freeze viability. p<0.05

Fig.3 Cryoprotection of D. chrysoscelis erythrocytes with acute and long term exposure to solutes. Post-freeze viability (%) of erythrocytes from warm-acclimated frogs were cultured in CCCM ± A) glycerol, B) glucose, or C) urea for 48 hrs. Cells were frozen to

-6C in 230 mM PBS ± the respective solute. Mean ± SEM. N=3-6. Data points labeled with the same letter indicate similar post-freeze viability. p<0.05

Fig.4 Freeze tolerance and cryoprotection of erythrocytes from warm and cold- acclimated D. chrysoscelis. Post-freeze viability (%) of erythrocytes incubated in PBS ±

150 mM glycerol, glucose, urea, or sorbitol, or 83 mM NaCl at 0 and -8C from A) warm-acclimated or B) cold-acclimated frogs. C) viability of erythrocytes from warm and cold-acclimated treefrogs frozen at -8C in PBS ± glycerol, glucose, urea, NaCl, or sorbitol. Mean ± SEM. N=3-6. Data points labeled with the same letter indicate similar post-freeze viability. p<0.05

Fig.1

Fig.2

Fig.3

Fig.4

CHAPTER 3

FUTURE DIRECTIONS AND BROADER IMPACTS

The aim of the current study was to further understand the strategy utilized by

Dryophytes chrysoscelis that enable it to be freeze-tolerant. Plasma glycerol and urea levels are elevated in frogs that have been cold-acclimated or frozen compared to warm- acclimated frogs (Zimmerman et al. 2007; do Amaral et al. 2018). Compared to RBCs from warm-acclimated frogs, RBCs from cold-acclimated D. chrysoscelis have increased abundance and membrane localization of HC-3, a protein which is permeable to glycerol and presumable permeable to urea (Krane and Goldstein 2007; Zimmerman et al., 2007;

Goldstein et al. 2010; Mutyam et al. 2011). Urea is naturally accumulated during cold acclimation and functions as a cryoprotectant, but it is currently unknown if HC-3 is permeable to urea. An HC-3 ortholog, AQP3, is expressed in mammals and is known to be permeable to urea. Other mammalian aquaglyceroporins, AQP7, -9, and -10, are also known to be permeable to urea (Conner et al. 2013; Lutoslawska 2014; Kitchen et al.

2015; Laforenza et al. 2015). It is hypothesized that HC-3 is also permeable to urea, and that urea and glycerol both fluctuate intra- and extracellularly to reduce osmotically induced cell lysis. To further understand the role of urea as a potential permeating cryoprotectant, a functional assay should be performed. The proposed experiment would involve HC-3 mRNA being injected into Xenopus oocytes and the oocytes would then be

53 placed in solutions containing urea, a technique similar to that described by Goldstein et al. (2010).

To clarify if the enhanced post-freeze viability of erythrocytes was a result of the exposure of cells to extracellular glycerol or urea, or the transmembrane flux of glycerol, and presumably urea, by HC-3, a potential adaption to the present experiment would be to block HC-3. Mercury chloride (HgCl2) is a known inhibitor of AQPs and has been demonstrated to inhibit HC-3 permeability to glycerol in Xenopus oocytes (Preston et al.

1993; Zhang et al. 1993; Krane and Kishore 2003; Krane and Goldstein 2007; Goldstein et al. 2010; Lutoslawska 2014). The present experiment was adapted so that a subset of

RBCs was resuspended in a PBS solution that contained a cryoprotectant and HgCl2 or just a cryoprotectant. It is hypothesized that cryoprotection of cells is partially dependent upon the permeability of solutes. Anticipated post-freeze viability of cells with cryoprotectants without HgCl2 would be greater than cells with HgCl2. Unexpectedly, in preliminary experiments HgCl2 caused a precipitate to form when mixed with hemoglobin and the hemoglobin reagents. Measuring cell viability in terms of hemolysis with a spectrophotometer is not a reliable method when HgCl2 is an added variable. An alternative method to measure cell viability is with a flow cytometer. Preliminary data is suggestive that HgCl2 can be utilized to inhibit HC-3, but further optimization with that protocol would need to be done (data not shown).

In the present study, cellular post-freeze viability is measured quantitatively by a hemolysis assay. However, examination of post-freeze cell morphology could provide additional insight to how cells are responding to freezing and thawing. Confocal microscopy can be utilized for qualitative or quantitative analysis of cell morphology.

Following the hypothesis that glycerol and urea function as cryoprotectants by mitigating osmotic swelling and shrinking during freezing and thawing, cells frozen with glycerol or urea are anticipated to have similar morphology as cells that were not frozen. Preliminary analysis was done with samples of cells before and after freezing. RBCs were obtained from warm- and cold-acclimated frogs and incubated at 0°C or -8°C in PBS (280 mM) for 30 minutes then fixed onto slides. Cells were incubated with Texas Red-X Phalloidin

(Invitrogen T7471, Eugene, OR, USA) to stain the cytoplasm followed by TO-PRO-3

Iodide (Invitrogen T3605, Eugene, OR, USA) to stain the nucleus. Erythrocytes were imaged via confocal microscopy at 100x magnification. The images provided are the center most cross sections of the RBCs. Cells that were left unfrozen are oblong in shape and the nuclei are centered within the 2D surface area of the cytoplasm of each cell. The morphological appearance of cells from warm-acclimated or cold-acclimated frogs that were frozen to -8°C in media containing glycerol and urea is similar to the cells that were never frozen. In contrast, RBCs that were frozen in PBS alone or in PBS containing glucose, NaCl, or sorbitol have nuclei that appear more circular in shape and have a smaller cytoplasmic surface area (Figure 1). These changes in shape and size suggest the membrane integrity has been compromised and reduction in cell volume.

Figure 1. Erythrocytes after incubation at 0C or -8C. A) Confocal microscopy of erythrocytes from warm-acclimated frogs after a 30-minute incubation in PBS at 0C. B) Confocal microscopy of erythrocytes from warm-acclimated frogs after a 30-minute incubation in PBS ± glycerol, glucose, urea, NaCl, or sorbitol at -8C. C) Confocal microscopy of erythrocytes from cold-acclimated frogs after a 30-minute incubation in PBS ± glycerol, glucose, urea, NaCl, or sorbitol at -8C. Nucleus was stained with TO-PRO-3 Iodide (blue) and cytoplasm was stained with Texas Red-X Phalloidin (red). Identifies cells with intact cytoplasm. Identifies cells with reduced cytoplasm. Scale bar = 20 μm. N=1.

Cell morphology is a characteristic that can be used to distinguish osmotically regulated volume changes of cells before and after freezing and the role cryoprotectants have in mitigating such changes. One limitation of confocal microscopy is not being able to observe the ratio of live to dead cells, as only intact cells can be visualized after cytoplasmic and nuclear staining. These images are supplemental to the hemolysis assay data which suggest that glycerol and urea function as cryoprotectants to RBCs from D. chrysoscelis. In order to quantify the degree of potential cell volume changes that occur the post-freeze 2D surface area of the cytoplasm can be measured or as a ratio to the respective nucleus size. These measurements can be done with software such as ImageJ.

With quantitative measurements, statistical analysis of cell volume change can be performed which could aid in understanding the solute specific effects on cellular freeze tolerance.

In the present experiment and in all the above potential experiments, RBCs from

D. chrysoscelis undergo one freeze/thaw cycle and that one dip in temperature is just the beginning step to understand the complex cryoprotectant strategy utilized by treefrogs. In the wild, treefrogs are exposed to subfreezing temperatures throughout the winter months. These frogs can go through multiple freeze/thaw cycles within on season and the time spent frozen can be longer than the 30 minutes that the RBCs were frozen in the present work. In early January 2017, the temperatures in Southwestern Ohio did not rise above -5°C for four consecutive days; this cold stretch was preceded by a stretch of nights in which the temperatures dropped below freezing (Ohio Committee for Severe

Weather Awareness). One way to further understand the freeze tolerance and cryoprotection of D. chrysoscelis would be to put cells through multiple freeze/thaw

57 cycles that would better represent the environmental conditions experienced by wild frogs. In light of the present data, where ~72% of RBCs from cold-acclimated frogs survive one freeze/thaw cycle, it is unlikely the frogs would survive more than a few consecutive freezing events with continuous loss of RBCs, unless the cellular changes occurred during the first freezing event that improve cellular viability for subsequent freezing events.

Freeze tolerance is utilized by six anuran species, and each species has its own specific mechanisms involved in this phenomenon. The present work investigates freeze tolerance and cryoprotection at the cellular level using RBCs from D. chrysoscelis.

Knowledge gained by studying freeze tolerance and cryoprotection at the cellular level can be applied to more complex systems such as organs and whole organisms. First and foremost, insight on how D. chrysoscelis survives the harsh winters is important in understanding and respecting the diversity of all life forms. Secondly, knowledge gained on how a naturally freeze-tolerant anuran survives freezing can be applied to cells and organs of humans, who are not freeze-tolerant. The ability to sustain viability of frozen human organs would substantially improve the number of organs available for organ transplantations. It is estimated that ~20 people die each day while waiting for an organ

(UNOS). Medical urgency and tissue compatibility are two factors that are accounted for when determining which recipient patient gets a donated organ. Another key factor is geography. Currently organs taken from a donor patient have a time limit in which they need to be transplanted into the recipient patient for the organ to still be viable; kidneys last 24-36 hours, livers last 8-12 hours, and heart and lungs last 4-6 hours on ice (UNOS).

If donated organs could be frozen, the time it would take to for the organ to be

58 transported from donor patient to a potential recipient patient that is in a different geographic location would no longer be a limitation. Furthermore, all donated organs could potentially be frozen and thus a bank of all organ types can be stored for future recipient patients. In other words, all organs would be saved for the perfectly matched recipient patient. The Krogh principle states, ‘For a large number of problems there will be some animal of choice, or a few such animals on, which it can be most conveniently studied’ (Krogh 1929). To advance in fields on biology to the point of freezing human organs for cryopreservation and transplantation, the step is to understand physiological mechanisms in organisms that are naturally freeze-tolerant such as D. chrysoscelis.

References

Conner AC, Bill RM, Conner MT (2013) An emerging consensus on aquaporin translocation as a regulatory mechanism. Mol Membr Biol 1:1-12 do Amaral MCF, Frisbie J, Goldstein DL, Krane CM (2018) The cryoprotectant system of Cope's gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation, freezing, and thawing. J Comp Physiol B 188:611-621

Goldstein DL, Frisbie J, Diller A, Pandey RN, Krane CM (2010) Glycerol uptake by erythrocytes from warm- and cold-acclimated Cope's gray treefrogs. J Comp Physiol B 8:1257-1265

Kitchen P, Conner AC, Conner MT, Salman MM, Day RE, Bill RM (2015) Beyond water homeostasis: Diverse functional roles of mammalian aquaporins. BBA - General Subjects 1850:2410-2421

Krane CM, Goldstein DL (2007) Comparative functional analysis of aquaporins/glyceroporins in mammals and anurans. Mamm Genome 6:452-462

Krane CM, Kishore BK (2003) Aquaporins: the membrane water channels of the biological world. Biologist 2:81-86

Krogh A (1929) The progress of physiology. Am J Physiol 90:243-251

Laforenza U, Bottino C, Gastaldi G (2015) Mammalian aquaglyceroporin function in metabolism. Bioch Bioph Acta. 2016:1-11

Lutoslawska G (2014) Aquaporins in physiology and pathology. TSS 21:185-194

Ohio Committee for Severe Weather Awareness (2017) Ohio Winter Summary 2016- 2017. http://www.weathersafety.ohio.gov/OhioWinterSummary.aspx. Accessed 2 July 2018

Preston GM, Jung JS, Guggino WB, Agre P. (1993) The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel. J Biol Chem 1:17-20

UNOS (2018) Matching Organs, Saving Lives. https://unos.org/. Accessed 2 July 2018

Zhang R, van Hoek ,A.N., Biwersi J, Verkman AS (1993) A point mutation at cysteine 189 blocks the water permeability of rat kidney water channel CHIP28. Biochemistry 12:2938-2941