Life in a frozen state: adaptive strategies for natural freeze tolerance in amphibians and reptiles.

Life in a frozen state: adaptive freeze tolerance in amphibians

strategies for natural and reptiles

KENNETH B. STOREY Institute of Biochemistry and Department of Biology, Carleton University, Ottawa, Ontario KlS 5B6, Canada

STOREY, KENNETH B. Life in a frozen state: adaptive strategies for natural freeze tolerance in amphibians and reptiles. Am. J. Physiol. 258 (Regulatory Integrative Comp. Physiol. 27): R559-R568, 1990.-Winter survival for various species of amphibians and reptiles that hibernate on land depends on freeze tolerance, the ability to survive for long periods of time with up to 65% of total body water as extracellular ice. Freeze tolerance has been described for four species of frogs, one salamander, and hatchlings of the painted turtle. A very limited tolerance also occurs in garter snakes. Studies of freeze tolerance in vertebrates have primarily focused on the wood frog Rana sylvatica and have assessed the regulation of cryoprotectant synthesis, cryoprotectant action in freezing preservation of isolated cells and tissues, metabolism and energetics under the ischemic conditions imposed by freezing, and the role of ice-nucleating agents in blood. The adaptations that preserve life at subzero temperatures for these animals illustrate the principles of vertebrate organ cryopreservation and may have important applications in the development of technology for the freezing preservation of transplantable human organs.

cryobiology; winter hibernation of ectothermic wood frog; painted turtle; garter snake

Frogs of various colours are numerous in those parts as far North as the latitude 61’. . . . I have frequently seen them dug up with the moss, (when pitching tents in Winter) frozen as hard as ice; in which state the legs are as easily broken off as a pipe-stem . . . but by wrapping them in warm skins, and exposing them to a slow fire, they soon recover life. Journal of Samuel Hearne, relating his explorations of Canada’s Arctic in the years 1769-1772 TEMPERATURES over vast areas of our planet drop far below the freezing point of water. The formation of ice crystals within cells is uniformly lethal throughout the animal and plant kingdom, and organisms that inhabit seasonally cold areas of the Earth must have welldeveloped mechanisms for enduring or avoiding longterm exposures to temperatures below OOC.The biology of cold tolerance has a long history of scientific study, including pioneering studies in centuries past by Boyle, Reamur, and Dumeril, among others (4, 13, 39). Today the research has diversified into many fields, including cold acclimation, plant and animal cold hardiness, hibernation, and cryomedicine (reviews include 3, 14, 22, 26, WINTER

0363-6119/90

$1.50

Copyright

vertebrates;

cryoprotectants;

53, 55). Indeed, among the many contributions to comparative physiology made by Per Frederik Scholander (see acknowledgements) were extensive studies of the cold hardiness adaptations of polar animals (40, 41). My focus in the present article is cold hardiness in terrestrially hibernating vertebrate ectotherms and, in particular, the phenomenon of natural freeze tolerance displayed by selected amphibian and reptile species. Recent advances in understanding the biochemical and physiological adaptations that underlie natural freezing survival by vertebrate animals are not only fascinating in themselves but offer to cryomedicine the model systems with which to seek mechanisms for mammalian organ cryopreservation. WINTER SURVIVAL VERTEBRATES

STRATEGIES

OF

ECTOTMERMIC

Few amphibian and reptile species are found at high latitudes, and the northern limits of the range of individual species often appear to be determined by two factors, summer seasons that are too short for egg development or juvenile growth and/or the lack of suitable winter

0 1990 the American

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hardiness strategies (17, 37). For species that survive in cold climates, the options for winter hardiness are three: 1) use behavioral tactics to elude exposure to freezing temperatures; 2) endure subzero temperatures but maintain a liquid state by a depression of the supercooling and freezing points of body fluids; or 3) tolerate the formation of ice in extracellular fluid spaces (53). Those ectothermic vertebrates that are successful in cold climates primarily rely on options 1 and 3 for winter hardiness. The capacity for freeze avoidance, that is so well developed in arthropods (57), is of limited use to amphibians and reptiles, for the animals are susceptible to inoculative freezing by environmental ice propagating across the skin. The majority of reptiles and amphibians in cold climates elude subzero exposure by choosing subterranean or aquatic hibernation sites. Toads may dig as much as one meter down into the earth to avoid the frost line, salamanders follow rodent burrows deep underground, and snakes choose warm underground dens (17, 37). Many frogs and turtles hibernate at the bottom of ponds and streams (36). For freshwater turtles, this choice has lead to the development of an extreme anoxia tolerance that permits 3-4 mo of underwater survival without breathing (19). A selected group of species has developed the third option, that of freeze tolerance. These have in common the choice of terrestrial hibernation but using sites (e.g., under forest leaf litter, in shallow nests) that have limited insulation value. To date, the list of freeze-tolerant vertebrates includes four species of frogs, a Siberian salamander, and hatchlings of the painted turtle (Table 1). Limited freezing survival can also be demonstrated for garter snakes but, as discussed later, the physiological relevance of this is debatable. Modern recognition and analysis of the mechanisms of freeze tolerance in vertebrates began only recently. Natural freeze tolerance was first reported by Schmid in 1982 for two species of frogs, Rana syluatica and Hyla uersicolor (43, and was soon confirmed and extended to other frog species by studies in my laboratory (43, 49, 50). Freezing survival by reptiles was first reported in TABLE

REVIEW

1988, for hatchling painted turtles by my laboratory (54) and for garter snakes by Costanzo et al. (8). Although only these few vertebrate species experience natural freezing during winter hibernation, the phenomenon occurs widely among plants (26) and invertebrate animals, including many terrestrial insect species as well as selected intertidal marine invertebrates such as bivalves, gastropods, and barnacles (1, 33, 53). Studies of these systems, as well as empirical research during the development of medical cryopreservation techniques, have elucidated the mechanisms of freezing damage to cells as well as many of the principles of cryoprotection. I begin by outlining the principles of freeze tolerance, then examine the specific strategies of vertebrate ectotherms for freeze tolerance, and finally analyze the applications of model studies with these animals to the development of mammalian organ-preservation techniques. FREEZE

TOLERANCE:

RISKS

VS. BENEFITS

Freezing has many dangers for the unprotected cell, organ, or animal, and most of these are lethal (14, 31, 53). Ice crystals within a body cause physical damage. When propagating through extracellular spaces,they can sever cell-to-cell connections and damage capillaries. Within the cell, ice destroys the internal structure and microcompartmentation that is vital to metabolic function. Structural damage is also intensified by recrystallization, the ability of small, thermodynamically unstable ice crystals to regroup into larger crystals over time. Ice formation also has serious osmotic consequences for cells, since the removal of pure water into ice greatly concentrates the solutes in remaining liquid compartments. When freezing is extracellular, this causes osmotic shock, dehydrating and collapsing cells and, if a critical minimum cell volume is passed, irreversibly damaging cell membranes. Freezing also causes metabolic damage, since ice in extracellular spaces stops the intertissue transport of oxygen, fuels, and metabolic wastes. All of these injurious effects of ice are well known to cryobiologists working toward the goal of freezing pres-

1. Freeze- tolerant amphibians and reptiles Common

Amphibians Frogs Rana sylvatica

Wood

Hyla versicolor Hyla crucifer Pseudacris triseriata Salamanders Hynobius keyserlingi Reptiles Turtles Chrysemys Snakes Thamnophis

picta

sirtalis

Name

frog

Grey tree frog Spring peeper Chorus frog

Hibernation

Site

Under forest leaf litter, logs, tree roots Same Same Same

Low

Temperature Tolerated

Glucose

24, 42, 45, 49, 52

-3 to -6°C At least -3°C At least -3°C

Glycerol Glucose Glucose

25, 42, 43 50,52 50,52

Glycerol

2

Glycerol, glucose, taurine?

54

None

8; T. Churchill unpublished

salamander

Forest litter, crevices in rocks, tundra

-35°C

Painted

turtle

Shallow nests on exposed banks

-4

Adults in underground communal dens

-0.75 to -1.5”C Briefly at -2.5”C

snake

Ref. No.

-6 to -8°C

Siberian

Garter

Cryoprotectant

to -8°C

detected

and K. Storey, observations


EDITORIAL

ervation of human tissues and organs. Indeed, the complexity of the problems involved (which also include the toxicity of the unnatural chemicals added as cryoprotectants) are the reason that successful cryopreservation is still limited to selected single cell and simple tissue systems (e.g., blood, sperm, embryos, cornea, skin, pancreatic islets), although 40 years have passed since Polge et al. (38) first employed glycerol for the freezing preservation of sperm. At a first glance, then, natural freeze tolerance would appear to necessitate a complicated set of adaptations, including control over the site and rate of ice formation, regulation of cell volume, mechanisms for protecting proteins and subcellular structures from denaturation, and a well-developed ischemia tolerance. It seems surprising, perhaps, that freeze tolerance has arisen independently in so many animal groups. However, in reality, the physiological and biochemical mechanisms of freeze tolerance are (with the exception of ice-nucleating proteins) well-developed strategies used frequently by animals to adapt to other environmental stresses. Freeze tolerance in vertebrates combines elements of desiccation tolerance, response to hyperosmotic stress, anoxia tolerance, and the catecholamine-mediated control of liver glycogenolysis that are well known in other systems. Given these capacities, and limited by skin that is no barrier to the propagation of ice crystals, it becomes logical, then, that terrestrially hibernating amphibians and reptiles should accept freezing when environmental temperatures drop below the equilibrium freezing point of body fluids (typically about -0.5 to -0.8OC for vertebrates). These animals opt, therefore, for the long-term metabolic stability that the frozen state can guarantee as opposed to the ever-present risk of instantaneous, lethal freezing attached to a supercooling strategy. ADAPTIVE

STRATEGIES

OF

FREEZE

TOLERANCE

The known adaptive mechanisms of freeze tolerance include the following (53). Control of extracellular ice. Specific ice-nucleating proteins in extracellular compartments induce a regulated freezing of extracellular fluids at high subzero temperatures (above -10°C) and by their actions permit a slow freeze concentration of cells, leaving no supercooled compartments and eliminating the risk of intracellular nucleation (12). Thermal hysteresis proteins limit physical damage by extracellular ice by inhibiting recrystallization when freezing is long term (23). Cell volume regulation. High concentrations of lowmolecular-weight carbohydrate cryoprotectants (most commonly polyhydric alcohols such as glycerol or sorbitol) are synthesized, and these provide colligative action to prevent cell volume from dropping below a critical minimum during freezing (14). Stabilization of subcellular organization. Low-molecular-weight protectants (e.g., trehalose, proline) are employed to stabilize membrane bilayer structure (e.g., by inhibiting phase transitions) against the physical stresses imposed by the reduction of cell volume (11). Carbohydrate cryoprotectants, in addition to colligative actions, also physically stabilize protein-enzyme structure and

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function against low temperature, dehydration, or highsalt stresses (11). The content of unfreezable water in cells is also enhanced by increasing the amount of cellular water ordered (or “bound”) by both macromolecules and low-molecular-weight protectants (53). Ischemic tolerance. Long-term survival in the ischemic state imposed by the presence of ice in extracellular compartments is served by a well-developed anoxia tolerance, mechanisms for reestablishing homeostatic control in the frozen state, and an inducible metabolic rate depression during hibernation (53). FREEZE

TOLERANCE

IN

FROGS

Reports of experimental freezing of frogs date well back into the last century, but these deal only with aquatic frogs exposed to very brief freezing at very mild temperatures (e.g., l-2 h at -1 to -2°C) (6, 13, 22). Exploration of the natural phenomenon among terrestrially hibernating frogs has begun only recently. The wood frog R. sylvatica, the grey tree frog H. uersicolor, the spring peeper H. crucifer, and the chorus frog Pseudacris triseriata all hibernate on the forest floor, insulated from the full force of winter air temperatures by layers of leaf litter and snow. Nonetheless, microhabitat temperatures may drop as low as -8°C and, since freezing is initiated between -0.5OC (if seeded by environmental ice) and -2 to -3°C (the supercooling point), the animals may spend several weeks of any winter in a frozen state (27, 29). Estimates of ice content range from 35 to 65% of total body water, with the lethal limit being substantially reduced in summer compared with winter (24, 25, 42, 53). The maximal amount of ice, 65% of total body water, is a figure that is also generally the lethal limit for other freeze-tolerant animals as well as cryopreserved cells (31, 53). This represents a reduction in cell volume to the critical minimum volume; shrinkage beyond this amount appears to irreparably damage membrane bilayer structure (32). The development of freeze tolerance in frogs was probably aided by the general tolerance of amphibians for wide variations in body water and ion contents (20, 37), for, as long as ice formation is extracellular, freezing has the same osmotic consequences for cells as does dehydration or hyperosmotic stress. Anurans, for example, appear unaffected by a rise in electrolyte concentrations (Na’ in particular) in the cerebrospinal fluid that in mammals would lead to severe nervous system dysfunction (20). The model for most of our studies has been the wood frog. This species is the most northerly distributed amphibian in North America with a range that extends well above the Arctic circle, following the tree line throughout the Northwest Territories, the Yukon, and Alaska (9). This is the species that was the subject of Samuel Hearne’s 18th century journal entry (18). In experimental tests, we have documented freezing survival ranging to at least 2 wk; for example, adult female R. syluatica survived 13 days of freezing at -2.5”C, whereas 50% of immature adults survived -6°C for 11 days (45). Nucleation occurs in the body extremities and, as freezing progresses, ice crystals appear under the skin, interspersed with skeletal muscles. and fill the abdominal


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cavity surrounding all organs. Breathing, heart beat, and blood flow gradually slow and then stop. When fully frozen, a large mass of blood is found pooled in distended sinuses above the heart, suggesting that blood is drained from organs during the freezing process. Freezing is a relatively slow event; for Z-g wood frogs, the freezing exotherm persists for at least 3 h (Fig. 1; 44), whereas maximum ice formation in 14-g frogs requires -24 h at -3°C with a half-time of 6.5 h and a linear rate of 2.9%/h (24). Such slow rates provide ample time for controlled freeze concentration of cells, synthesis and distribution of cryoprotectant, and a regulated transition to an ischemic state. We have investigated several of the adaptive mechanisms involved in freeze tolerance in frogs. These are summarized below. Blood proteins. The presence of ice-nucleating proteins and thermal hysteresis proteins is key to inducing and regulating the growth of ice crystals in extracellular fluid compartments of freeze-tolerant insects (12, 23). Predictably, similar proteins should serve the same function in freeze-tolerant vertebrates. Using the technique of differential scanning calorimetry, we tested the blood of R. syluatica for these activities (56). There was no evidence for the presence of a proteinaceous antifreeze, but blood contained nucleating activity that induced crystallization at -6 to -7°C (Table 2). Ice nucleators were present in cell-free blood, serum, and plasma from frogs, suggesting that the compounds are soluble plasma components in vivo, possibly plasma proteins. Nucleating activity is present in both autumn- and spring-collected frogs, but it is not yet clear whether these are specifically

REVIEW

synthesized nucleating proteins or normal plasma proteins that have this activity. However, the nucleating activity is transferrable to human plasma; as little as 0.5% vol/vol of frog blood raised the crystallization temperature of human plasma from -16.1 (alone) to -7.l”C with the addition of frog nucleators. Although clearly present in frog blood, the role of these ice-nucleating agents needs further clarification, since natural freezing is initiated between -0.5 and -3OC, substantially above the temperature at which plasma ice nucleators act under experimental conditions. Perhaps these agents require “activation” in some way to alter their effective crystallization temperature in vivo or perhaps they act as centers for ice-crystal growth in extracellular fluid compartments but do not themselves actually seed the process. Cryoprotectants and regulation of their synthesis. Frogs accumulate low-molecular-weight carbohydrate cryoprotectants. In R. syluatica, H. crucifer, and P. triseriata the protectant is glucose, whereas in H. uersicolor it is glycerol (43, 49, 50). Measured levels of glucose in the blood of freezing-exposed R. syluatica range up to 550 pmol/ml or 9.9 g/100 ml (the average is 200-250 pmol/ml) compared with levels of l-5 pmol/ml in control, unfrozen frogs. Freezing-exposed frogs are extremely hyperglycemic, therefore, and the controls on blood and organ glucose content must be modified somewhat from the usual vertebrate mechanisms that closely regulate glucose. Cryoprotectant is synthesized in the liver from large glycogen stores (up to 180 mg/g or 1,000 pmol/g as glucose) that are built up before hibernation; amounts are 2- to lo-fold higher than in hibernating aquatic

90

FIG. 1. Triggering of cryoprotectant glucose production by ice nucleation in freeze-tolerant wood frog Rana syluatica. Top trace, typical freezing curve for Z-g wood frogs placed in a -3°C constanttemperature incubator. Ice nucleation results in a pronounced jump in body temperature (freezing exotherm). Arrows indicate sampling times relative to appearance of exotherm. Bottom bar graphs show metabolic parameters at these times with units on the y-axis of pmol/ml for blood glucose, pmol/g wet wt for liver glucose, and 5% enzyme activity in the a form for glycogen phosphorylase. Note that glucose production is initiated within 5 min of the appearance of freezing exotherm. [Modified from Storey and Storey (U).]

180

Minutes blood glucose liver glucose EI % phosphorylase

q n

60-

3

,a .


EDITORIAL

2. Ice-nucleating activity in blood of Rana sylvatica, identified by differential scanning calorimetry TABLE

Crystallization Temperature,

‘C

-7.4-c-0.3 -6.6t0.2 -6.3t0.3

Frog blood Frog serum Frog plasma Phosphate-buffered Human plasma Human plasma

-21.1t2.0 -16.ltl.5 -7.lTO.2

saline + frog blood

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Values are means k SE. Frog blood is cell-free blood; heparintreated blood was frozen, then thawed and centrifuged to remove red cell ghosts. Serum was also heparin treated. Mixture of human plasma and frog cell-free blood was 99.505%. [Data from Wolanczyk et al. (56).1

species. Novel to freeze-tolerant frogs is the mechanism of triggering cryoprotectant production, which occurs only in response to the initiation of ice formation in peripheral tissues. Unlike the situation in cold-hardy arthropods, there is no anticipatory accumulation of cryoprotectant over the autumn months. Instead, glucose in R. sylvatica is produced at high rates (20 prnol. g-l h-l at -2.5”C) during the initial minutes and hours of freezing (Fig. 1) and is rapidly distributed via the blood to other organs of the body (44, 51). An organ-specific pattern of cryoprotectant distribution results, with glucose levels highest in central organs (liver, heart, brain) and lower in peripheral tissues (Fig. 2) (46). This distribution apparently results from the progressive restriction of blood flow to peripheral organs as freezing progresses. When thawed, glucose is returned from all organs and restored as liver glycogen, but subsequent cycles of freezl

ing and thawing repeat the same pattern (Fig. 2; 46, 51). H. versicolor appears to regulate its cryoprotectant pool somewhat differently for, although glycerol production is triggered by freezing exposure, the cryoprotectant persists in body fluids for several weeks after thawing (43). The biochemical regulation of cryoprotectant synthesis has been thoroughly studied in R. sylvatica. Primary control over glucose synthesis by liver rests with the enzyme glycogen phosphorylase, which is rapidly activated in response to the initiation of ice formation (Figs. 1 and 3) (44). Analysis of metabolite changes in liver during the early minutes indicate the additional participation of an inhibitory block on glycolysis occurring at phosphofructokinase that serves to divert flux into glucose output from the liver (47). Glucose synthesis from glycogen requires only three enzymes (phosphorylase, phosphoglucomutase, and glucose-6-phosphatase), and the simplicity of this pathway plus the natural mechanisms for rapidly activating liver glycogenolysis in vertebrates are probably the primary reasons for the choice of glucose as the cryoprotectant in frogs. Indeed, the cryoprotectant response in frogs may be an exaggeration of the vertebrate “fight or flight” response, the catecholamine-mediated activation of glucose output from the liver in response to stress. However, whereas epinephrine action on mammalian liver might elicit a short-term twoto fivefold rise in blood glucose, freezing triggers a lOOto ZOO-fold increase in blood glucose in frogs that persists throughout a freezing episode (49, 51). As yet we have been unable to detect changes in blood catecholamine content of frogs as a response to the initiation of freezing, but administration of the ,&adrenergic blocker propran-

ALANINE

fl -t-T-CT

h

'=TFT

c‘F

I

w

CFTFT

CIF'T'F'T -

z

m

i

CFTFT

1 1 I

CFTFT

GLUCOSE

I

1

CFTFT

Liver

I

-F-E-E

Heart

c'

Kidney

c'

m

Brain

Muscle

CFTFT

Lung

FIG. 2. Levels of glucose, lactate, and alanine in organs of R. syhztica over consecutive freezing (at -25°C) and thawing (at 3°C) exposures, each 2 days in duration. C, 3°C controls; F, frozen; T, thawed. Muscle is leg skeletal muscle, skin is from ventral abdomen. Values for liver glycogen cycles oppositely to glucose levels and were 980 pmol/g in controls and 285, 760, 327, and 724 pmol/g measured as glucosyl units over the succeeding freeze-thaw exposures [Modified from Storey (46).]

c

Skin


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EDITORIAL

/ a

0

,,,,yy

I

C25N min

hours FREEZING

1123

iA J-Y&

1 3 1234 hours days THAWING

FIG. 3. Modulation of glycogen phosphorylase activity in liver of R. syluatica over a course of freezing at -3°C (timed from appearance of freezing exotherm) and thawing at 3°C. Enzyme activities are in U/g wet wt, glucose levels are in pmol/ml for blood, and pmol/g wet wt for liver. [From Storey and Storey (53).]

0101blocks the freezing-induced output of glucose from liver (K. Storey, J. Storey and S. Perry, unpublished observations). It appears likely, therefore, that the link between the freezing trigger (ice formation at the skin) and the cryoprotectant response (glucose output from liver) is a nervous-hormonal one, probably involving catecholamine stimulation of liver glycogenolysis via the mediation of adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinase action. The mechanism of phosphorylase control in R. syluatica liver is also unusual. Activation has two parts, an immediate rise in the percentage of total enzyme in the active a form and a slower rise in the total activity of enzyme expressed. This is illustrated in Fig. 3. The immediate response occurs within minutes and underlies the initial activation of glucose output after the appearance of the freezing exotherm (Fig. 1; 44). Over a longer time course, however, the total activity of phosphorylase rises 4- to 6-fold, and in combination these two mechanisms result in the 7- to 13-fold increase in active enzyme that supports the massive glycogenolysis and glucose output of the early hours of freezing (53). The same response by phosphorylase is seen during freezing exposure in both H. crucifer and H. versicolor (T. Churchill and K. Storey, unpublished observation). An electrophoretic and immunological study has further investigated the control of phosphorylase in R. syhatica liver and found some distinct changes from the situation typically described for mammals (10). Freezing did not change the titre of phosphorylase protein in R. syluatica liver, indicating that the observed rise in total

REVIEW

phosphorylase activity was not due to enzyme synthesis. A comparison of gels stained for enzyme activity vs. cross-reactivity to rabbit phosphorylase antibody revealed, however, that only the phosphorylated, active a form of the frog liver enzyme could be detected with the activity stain even in the presence of large amounts of activators (AMP, sulfate) of the b form (10). Thus it appears that the biphasic activation of phosphorylase, described above, should be interpreted in a different way. The slow rise in total phosphorylase activity during freezing probably actually represents the b-to-a conversion, the b form of phosphorylase in R. syluatica liver being inactive and not detectable by the conventional use of allosteric activators. What, then, do we make of the rapid rise in what appeared to be the percent phosphorylase a ? As yet we don’t know, but obviously one part of the activation mechanism involves a change in the sensitivity of phosphorylase a to AMP activation. Metabolism in frozen state. Circulatory changes during the early hours of freezing may preserve oxygen supplies to the brain and other sensitive organs for as long as possible, but ultimately, when freezing is complete, there is no breathing, no heart beat, and no blood flow (45). Individual organs must survive autonomously using endogenous fuel reserves, preserving cellular energetics, and tolerating the accumulation of waste products. Survival of freezing for periods of up to 2 wk has been documented (longer times have not been tested), so that frog organs are obviously well equipped for survival under ischemic conditions. All organs of R. syluatica contain substantial glycogen reserves (but only in liver are these used for cryoprotectant synthesis), and these are depleted over days of freezing (51). Fermentative glycolysis fuels metabolism with both lactate and alanine accumulating as end products (Fig. 2). Both the net accumulation of products and the proportions of lactate and alanine vary between organs. For example, total product accumulation was IO-fold higher in heart than in skeletal muscle, and heart produced predominantly lactate, whereas the ratio of lactate to alanine accumulated was 2.5:l in kidney, and k4.5 in skeletal muscle (51). The dominance of alanine as a product in skeletal muscle suggested that amino acid fermentation may also contribute to energy needs in this organ during freezing and, indeed, levels of fermentable amino acids, aspartate, glutamate, and glutamine all declined in muscle in the frozen state (51). Freezing also has organ-specific effects on cellular energetics. Energy charge and total adenylate levels remained high and constant in skeletal muscle over several days of freezing supported by anaerobic glycolysis and creatine phosphate hydrolysis (46, 51). In liver, however, ATP content declined over time, possibly because of the lack of phosphagen reserves, but energy charge was maintained at a high level by a progressive fall in the total adenylate content. In both organs, however, any energy stress observed was reversed after animals thawed. Long-term survival of freezing is, of course, aided by the effects of subzero temperature on metabolic rates and by the cessation of physical activities (e.g., movement, heart beat) while frozen. For many animals, survival under environmental extremes is also potentiated


EDITORIAL

by entry into a dormant state. For example, submerged, hibernating turtles at 3°C have a metabolic rate that is only 10% of that of the same animals in air at 3°C (19). Hibernating small mammals also employ specific metabolic rate depression (in addition to a drop in body temperature) to reduce metabolic demands over the winter (16). Whether such processes also aid survival during freezing in frogs is not yet clear. Metabolic depression does not appear to be a factor in the overwintering survival of aquatic frogs (36), and we have not found the widespread protein-enzyme phosphorylation in tissues from frozen frogs that has characterized the transition to a depressed state in other systems that we have analyzed (47, 48). Glucose cryoprotection. Recent studies have examined the actions of glucose as a cryoprotectant for R. syluatica cells and organs in vitro, and these have indicated that glucose may have key advantages for the cryopreservation of vertebrate organs. Ventricle strips from R. syluatica readily survive freezing at -5°C for 1 h and regain contractility on thawing when frozen in the presence of 250 mM glucose as a cryoprotectant (7). However, when frozen without cryoprotectant or with the substitution of 250 mM glycerol (a commonly used protectant in cryomedicine), muscle strips do not regain contractility after thawing. Obviously, then, the natural cryoprotectant offers benefits for freezing protection over and above simple colligative actions in cell volume regulation. These could include 1) a fuel supply for metabolism during the ischemic, frozen period, and 2) an effect as a metabolic depressant. This last action is supported by some recent studies with isolated hepatocytes. For protection of the structural integrity of the cell during freezing (measured as a function of lactate dehydrogenase leakage after thawing), we found that glucose offered cryoprotection that was just as good as that provided by more commonly used cryoprotectants such as glycerol or dimethyl sulfoxide (DMSO) (K. Storey and T. Mommsen, unpublished observations). However, structural integrity, as measured by enzyme leakage, is not necessarily a very rigorous test of freezing survival and so we added to our analysis a test of metabolic integrity, the measurement of rates of urea biosynthesis by hepatocytes. This activity requires the integrated functioning of enzymatic pathways in both the cytoplasmic and mitochondrial compartments of the cell as well as intact transport processes across the mitochondrial membrane. When freezing survival was assessed as the ability to synthesize urea after thawing, the apparent percent survival was lower than that assessed by enzyme leakage for all the protectants tested. What was intriguing, however, was the effect of cryoprotectant washout on urea-synthesizing capacity. For all cryoprotectants used, except glucose, the removal of these compounds was highly damaging, and the rate of urea biosynthesis after the washout dropped to ~50% of the control value (K. Storey and T. Mommsen, unpublished observations). Glucose behaved oppositely, however. The presence of high glucose strongly depressed the rates of urea synthesis in both unfrozen and freezingexposed cells, but when glucose was washed out of the

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cells, rates of urea biosynthesis rebounded to normal values. It appears possible, then, that high glucose helps to arrest metabolism in frog hepatocytes and in doing so contributes to a reduction in energy expenditures during freezing that would prolong survival time in the frozen state. FREEZE

TOLERANCE

IN TURTLES

Until the spring of 1988, we believed that frogs were the only vertebrate animals that naturally survived freezing. Then a conversation with Dr. R. J. Brooks of the University of Guelph, Ontario, Canada alerted us to an intriguing problem in the reproductive biology of the painted turtle (Chrysemys picta). Eggs of this species are laid in June or July and hatch in late summer. However, the hatchlings remain in the nest over the first winter and emerge in the spring when environmental conditions are favourable for rapid juvenile development (15). In northern climates this means that hatchlings spend the winter in shallow nests (Cl0 cm deep) on exposed sandy banks with little chance for physical insulation from winter weather. Nest temperatures of -6 or -8°C were recorded by Dr. Brooks in January and February 1988 at our study site in Algonquin Park, Ontario (54). Subzero temperatures have been measured previously in painted turtle nests, but the assumption by other authors had always been that the hatchlings survived by supercooling (5). In April we exhumed these nests and transported the live hatchlings to our lab for experimental tests of cold hardiness. We monitored cooling behavior of individual turtles via thermistors taped to the plastron. Animals supercooled to -3.3 t 0.24”C and then froze. Turtles readily survived 24 h of freezing at -4°C with an average of 53.4 & 2.0% of total body water as ice (54). These animals, then, proved to be the first example of natural freezing survival by a reptile. The tolerance of turtles for freezing proved, as for frogs, to be closely matched to the natural range of winter nest temperatures. Thus 24 h of freezing at -10.9”C raised ice content to 67% ice and proved to be lethal. To assessthe adaptations supporting freeze tolerance in hatchling turtles, we began by surveying the levels of putative cryoprotectants in the freezing-exposed animals. Of the common carbohydrate cryoprotectants, freezing stimulated the accumulation of glucose and glycerol in turtle blood and organs (Table 3). Turtles also accumulated large amounts of lactate during the 24-h freezing exposure as well as some amino acids (taurine being the largest contributor to the increase in free amino TABLE 3. Metabolite levels in blood and liver of freeze- tolerant turtles Blood,

Glucose Glycerol Lactate Taurine Total amino Values al. (54).]

acids

are means

pmol/g

wet wt

Liver,

pmol/g

wet wt

Control

Frozen

Control

Frozen

7.6t0.9 0.25t0.03 14.0-c-2.5 0.7t0.12 2.1t0.34

15.9t1.5 0.75kO.11 38.2t7.2 2.0t0.27 4.7t0.43

3.9t0.6 ND 1.4t0.4 4.2t0.11 7.4kO.53

13.7kO.9 ND 20.4k2.5 4.5t0.52 7.9kO.45

-+ SE: ND,

not determined.

[Data

from

Storey

et


R566

EDITORIAL

acid pool size). The levels of these compounds in springcollected turtles were not high enough, however, to provide significant colligative effects in freezing protection, but the responsiveness of these metabolites to freezing exposure suggested that they might prove to be the winter-active cryoprotectants. We have found previously for freeze-tolerant frogs that the cryoprotectant response to freezing declines very rapidly after spring emergence. Freezing conditions that would stimulate the accumulation of ZOO-300 mM glucose in R. syluatica organs during the winter months produce only 20-50 mM in the spring (52). The reason appears to be the commitment of glycogen reserves to other uses in the spring; in frogs this probably supports the physical activity of the animals at the breeding ponds. The same winter vs. spring response to freezing may occur in turtles and, indeed, our first experiments with hatchlings collected in the autumn suggest that this is probably the case. When painted turtle hatchlings removed from nests in September were exposed to the same conditions of freezing as were used on the spring-collected animals (-4°C 24 h), the metabolite response was substantially different, a sevenfold rise in liver glycerol content plus twofold increases in liver glucose and lactate (T. Churchill and K. Storey, unpublished observations). Again, the level of cryoprotectants accumulated were not very high, but the capacity for their synthesis was clearly evident in autumn-collected turtle hatchlings. Future research will need to profile the natural levels of cryoprotectants in turtles over a winter season as well as experimentally analyze the triggering and regulation of biosynthesis, which clearly has additional components compared with cryoprotectant production in freeze-tolerant frogs. Freshwater turtles of the Chrysemys and Pseudemys genera are the premier facultative anaerobes among vertebrates. Adult turtles may spend 3-4 mo submerged in ponds while overwintering without breathing and with a very limited level of extrapulmonary gas exchange. Anoxia induces a metabolic rate depression that lowers whole body heat output to -10% of the normoxic value (19). Nonetheless, blood lactate content soars to as much as 100 mM and, despite compensatory changes in blood ions that contribute to buffering, blood pH drops by -0.7 U. A balanced metabolism is maintained, however. Membrane potential difference falls within minutes when mammalian brain is exposed to anoxia, because glycolytic ATP output can not keep pace with the energy demand of ATP-driven membrane ion pumps. Turtles, however, show no disturbance of membrane potential difference with the transition to anoxia (28), indicating that they can automatically coordinate the activities of ATPdriven ion pumps with those of the opposing ion channels as ATP output falls. The mechanisms of metabolic rate depression that serve the hibernating adult turtle would also serve long-term freezing survival by the hatchlings, and the combination of natural anoxia-induced metabolic rate depression with freeze tolerance may provide hatchling turtles with the greatest capacity for long-term freezing survival of any of freeze-tolerant vertebrates. Indeed, since the development of methods for inducible metabolic depression is proving to be a critical compo-

REVIEW

nent of mammalian organ preservation technology, hatchling painted turtles may become the premier model animal for studies of organ cryopreservation. FREEZE

TOLERANCE

IN

GARTER

SNAKES

Compared with the hatchling painted turtles, the evidence is less compelling that freeze tolerance forms a natural part of the cold hardiness strategy of garter snakes. Garter snakes, Thamnophis sirtalis, are the most northerly distributed reptile in North America (9). They hibernate underground, adults from northern populations frequently massing in communal dens and migrating considerable distances from summer ranges to winter denning sites (17). Macartney et al. (30) have provided recordings of both den air temperature and snake body temperatures from a communal den in northern Alberta. Neither fell below 0°C [snake body temperature (Tb) ranged from 1.8 to 6.5”C] at any time over the winter, whereas ambient temperature outside the den was below -10°C and frequently below -25”C, for the five midwinter months. Air temperature in a reference den not used by snakes ranged from -10 to -20°C over the same time. These data suggest that the choice of den sites is key to the winter survival of garter snakes in northern regions and that natural winter exposures to temperatures below 0°C are probably rare. To directly examine the supercooling and freezing behavior of the species, we chose garter snakes collected from the Interlake area of Manitoba. Animals migrate to the communal den sites in late August and can be collected in large numbers at this time. These snakes, when chilled slowly in a low-temperature incubator, showed good supercooling capacity with the average whole animal supercooling point being -5.5OC (T. Churchill and K. Storey, unpublished observations). Snakes cooled in contact with damp moss had a reduced supercooling capacity with nucleation occurring at -1.2”C. This indicates that ice can propagate across some area of the skin surface and suggests that the water content of the hibernation site may be a critical component in the choice of den. The length of time that snakes can survive freezing, however, is low. When we seeded animals at -2.5”C, survival was high only when freezing exposures were brief (~3 h). After 10 h of freezing, the survival rate was only 50%, and no snakes survived freezing for 24 or 48 h when body ice contents rose to >70% (T. Churchill and K. Storey, unpublished observations). Costanzo et al. (8) similarly reported high survival of brief freezing for a Wisconsin population of T. sirtalis; snakes survived 6 h of freezing exposure at -3.3”C, which resulted in ice contents that ranged from 18 to 36%. These authors found that extended freezing survival (48 h) could be achieved only by raising the temperature of the surrounding bath to -0.75”C after nucleation had occurred, so that the equilibrium ice content reached only -34% of total body water. These data indicate, therefore, that freeze tolerance of garter snakes is poor. Survival is limited to very short freezing exposures or to very mild freezing temperatures where the resulting equilibrium ice content is kept low.


EDITORIAL

Such tolerance is not well suited to the environmental conditions of the winter dens. Temperature change is slow in these sites, and if temperature fell as low as the supercooling point (or even to any value much below the freezing point of body fluids), it would be difficult to imagine circumstances in which ice formation would not proceed to an equilibrium ice content that was lethal. Even if inoculative freezing occurred, temperatures would have to remain very mild (surely higher than -l”C), so as not to exceed a lethal ice content. Such limited freeze tolerance might, however, be adaptive in dealing with infrequent and short-term nocturnal frosts during the autumn or spring.

This review was presented as the keynote address in the Scholander Award session of the Comparative Physiology section, American Physiological Society, at the Federation of American Societies for Experimental Biology Meeting, March 19-23, 1989, New Orleans, LA. REFERENCES 1. AARSET, review). 1982.

2.

3. 4.

APPLICATIONS

TO

CRYOMEDICINE

Studies with freeze tolerant vertebrates are particularly relevant to the field of cryomedicine, for freezetolerant frogs and turtles have obviously developed natural solutions to the numerous problems that plague the development of freezing technology for transplantable organs. Some of these problems include 1) difficulties in cell-volume regulation both as the result of extracellular ice formation and in the infusion and removal of cryoprotectants, 2) the metabolic toxicity to mammalian organs of the commonly used cryoprotectants such as DMSO and glycerol, 3) the problems of evenly cooling, freezing, or rewarming a large organ mass to prevent physical damage by ice, and 4) the metabolic decay associated with ischemia and hypothermia. The need for intense empirical experimentation to develop separate freezing protocols for each individual cell type has meant that progress has been slow and is still limited to single cell suspensions (e.g., blood, sperm) and simple tissues (e.g., embryos, corneas, skin). Freeze-tolerant animals have solved the problems of how to regulate the penetration of ice into extracellular fluid spaces, distribute a compatible cryoprotectant in organ-specific amounts, and control for differing organ sensitivities to the freezing process (including differences in ischemia tolerance or in the tolerance for cell-volume changes). Freeze tolerance is obviously a real physiological capability of the organs of lower vertebrates and, as such, identification of the critical elements of molecular adaptation in these animals should lead the way to applied technologies for mammalian organ cryopreservation. Many lessons could be learned from the animal model systems. The choice of glucose as the cryoprotectant in frogs is intriguing, since vertebrates normally regulate glucose levels within strict limits. However, as discussed earlier, glucose appears to give superior cryoprotection to vertebrate organs compared with glycerol or DMSO and apparently has additional actions besides the colligative effects in cellvolume regulation. Much remains to be explored to fully understand the molecular mechanisms involved in natural freeze tolerance.

5. 6. 7. 8. 9. 10.

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A. V. Freezing Comp. Biochem.

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D. I., A. N. LEIRIKH, AND E. I. MIKHAILOVA. Winter hibernation of the Siberian salamander Hynobius heyserlingi. J. EuoZ. Biochem. Physiol. 1984 (3): 323-327, 1984 [in Russian, English summary]. BOWLER, K., AND B. J. FULLER (Editors). Temperature and animaZ cells. Cambridge, UK: Sot. Exp. Biol. Symp, 1987, vol. 41. BOYLE, R. New Experiments and Observations Touching Cold. London, 1683. BREITENBACH, G. L., J. D. CONGDON, AND R. C. VAN LOBEN SELS. Winter temperatures of Chrysemys picta nests in Michigan: effects on hatchling survival. Herpetologica 40: 76-81, 1984. CAMERON, A. T., AND T. I. BROWNLEE. The effect of low temperature on the frog. Trans. R. Sot. Can., series 3, vol 7, sect. 4: 107124, 1913. CANTY, A., W. R. DRIEDZIC, AND K. B. STOREY. Freeze tolerance of isolated ventricle strips of the wood frog, Rana syluatica. Cryo Lett. 7: 81-86, 1986. COSTANZO, J. P., D. L. CLAUSSEN, AND R. E. LEE. Natural freeze tolerance in a reptile. Cry0 Lett. 9: 380-385, 1988. COOK, F. R. Introduction to Canadian Amphibians and Reptiles. Ottawa: National Museums of Canada, 1984. CRERAR, M. M., E. S. DAVID, AND K. B. STOREY. Electrophoretic analysis of liver glycogen phosphorylase activation in the freezetolerant wood frog. Biochim. Biophys. Acta 971: 72-84, 1988. CROWE, J. H., L. M. CROWE, J. F. CARPENTER, AND C. A. WISTROM. Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem. J. 242: l-10, 1987. DUMAN, J. G. Insect antifreezes and ice-nucleating agents. Cryobiology 19: 613-627, 1982. DUM~RIL, M. A. A l’action du froid sur les grenouilles. Ann. Sci. Nat. Zool. 12: 346, 1849. FRANK, F. Biophysics and Biochemistry at Low Temperatures. Cambridge, UK: Cambridge Univ. Press, 1985. GIBBONS, J. W., AND D. H. NELSON. The evolutionary significance of delayed emergence from the nest by hatchling turtles. EuoZution 32: 297-303, 1978. GEISER, F. Reduction of metabolism during hibernation and daily torpor in mammals and birds: temperature effect or physiological inhibition? J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. BERMAN,

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20. 21. 22. 23.

Thanks to J. M. Storey for critical reading of the manuscript and to T. A. Churchill for preliminary data on freeze tolerance of turtles and snakes. Thanks also to Dr. R. J. Brooks, University of Guelph, Ontario, Canada for collecting hatchling turtles and providing data on turtle nest temperatures, and to Drs. D. Crews and M. Mendonca, University of Texas at Austin for providing garter snakes.

R567

REVIEW

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EDITORIAL

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REVIEW

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