Cryoprotectant production capacity of the freeze-tolerant wood frog, Rana sylvatica

Freezing survival of the wood frog (Rana sylvatica) is enhanced by the synthesis of the cryoprotectant glucose, via liver glycogenolysis. Because the quantity of glucose mobilized during freezing bears significantly on the limit of freeze tolerance, we investigated the relationship between the quantity of liver glycogen and the capacity for cryoprotectant synthesis. We successfully augmented natural levels of liver glycogen by injecting cold-conditioned wood frogs with glucose. Groups of 8 frogs having mean liver glycogen concentrations of 554 ± 57 (SE), 940 ± 57, and 1264 ± 66 μmol/g catabolized 98.7, 83.4, and 52.8%, respectively, of their glycogen reserves during 24 h of freezing to −2.5 °C. Glucose concentrations concomitantly increased, reaching 21 ± 3, 102 ± 23, and 119 ± 14 μmol/g, respectively, in the liver, and 15 ± 3, 42 ± 5, and 61 ± 5 μmol/mL, respectively, in the blood. Because the capacity for cryoprotectant synthesis depends on the amount of liver glycogen, the greatest risk of freezing injury likely occurs during spring, when glycogen reserves are minimal. Non-glucose osmolites were important in the wood frog's cryoprotectant system, especially in frogs having low glycogen levels. Presumably the natural variation in cryoprotectant synthesis capacity among individuals and populations of R. sylvatica chiefly reflects differences in glycogen reserves; however, environmental, physiological, and genetic factors likely are also involved.

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Freeze-thaw injury in erythrocytes of the freeze-tolerant wood frog, Rana sylvatica

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1991.261.6.r1346 ◽

1991 ◽

Vol 261 (6) ◽

pp. R1346-R1350 ◽

Cited By ~ 1

Author(s):

J. P. Costanzo ◽

R. E. Lee

Keyword(s):

Cell Injury ◽

Osmotic Fragility ◽

Rana Sylvatica ◽

Freeze Tolerance ◽

Wood Frog ◽

In Vitro Tests ◽

Freezing And Thawing ◽

Freeze Tolerant ◽

High Concentrations

Erythrocytes from the freeze-tolerant wood frog (Rana sylvatica) were subjected to in vitro tests of freeze tolerance, cryoprotection, and osmotic fragility. The responses of cells from frogs acclimated to 4 or 15 degrees C were similar. Erythrocytes that were frozen in saline hemolyzed at -4 degrees C or lower. The addition of high concentrations (150 and 1,500 mM) of glucose or glycerol, cryoprotectants produced naturally by freeze-tolerant frogs, significantly reduced cell injury at -8 degrees C, but concentrations of 1.5 or 15 mM were ineffective. Hemolysis was reduced by 94% with 1,500 mM glycerol and by 84% with 1,500 mM glucose; thus glycerol was the more effective cryoprotectant. Mean fragility values for frog erythrocytes incubated in hypertonic and hypotonic saline were 1,938 and 49 mosM, respectively. Survival in freeze tolerance and cryoprotection experiments was comparable for erythrocytes from frogs and humans, suggesting that these cells may respond similarly to freezing-related stresses. However, the breadth of osmotic tolerance, standardized for differences in isotonicity, was greater for frog erythrocytes than for human erythrocytes. Our data suggest that erythrocytes from R. sylvatica are adequately protected by glucose under natural conditions of freezing and thawing.

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DNA methylation and regulation of DNA methyltransferases in a freeze-tolerant vertebrate

Biochemistry and Cell Biology ◽

10.1139/bcb-2019-0091 ◽

2020 ◽

Vol 98 (2) ◽

pp. 145-153 ◽

Cited By ~ 2

Author(s):

Jing Zhang ◽

Liam J. Hawkins ◽

Kenneth B. Storey

Keyword(s):

Dna Methylation ◽

Transcriptional Repression ◽

Freeze Tolerance ◽

Dna Methyltransferases ◽

Wood Frog ◽

Activity Levels ◽

Wood Frogs ◽

Metabolic Role ◽

Freeze Tolerant

The wood frog is one of the few freeze-tolerance vertebrates. This is accomplished in part by the accumulation of cryoprotectant glucose, metabolic rate depression, and stress response activation. These may be achieved by mechanisms such as DNA methylation, which is typically associated with transcriptional repression. Hyperglycemia is also associated with modifications to epigenetic profiles, indicating an additional role that the high levels of glucose play in freeze tolerance. We sought to determine whether DNA methylation is affected during freezing exposure, and whether this is due to the wood frog’s response to hyperglycemia. We examined global DNA methylation and DNA methyltransferases (DNMTs) in the liver and muscle of frozen and glucose-loaded wood frogs. The results showed that levels of 5-methylcytosine (5mC) increased in the muscle, suggesting elevated DNA methylation during freezing. DNMT activities also decreased in muscle during thawing, glucose loading, and in vitro glucose experiments. Liver DNMT activities were similar to muscle; however, a varied response to DNMT levels and a decrease in 5mC highlight the metabolic role the liver plays during freezing. Glucose was also shown to decrease DNMT activity levels in the wood frog, in vitro, elucidating a potentially novel regulatory mechanism. Together these results suggest an interplay between freeze tolerance and hyperglycemic regulation of DNA methylation.

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Glucose concentration regulates freeze tolerance in the wood frog Rana sylvatica

Journal of Experimental Biology ◽

10.1242/jeb.181.1.245 ◽

1993 ◽

Vol 181 (1) ◽

pp. 245-255 ◽

Cited By ~ 3

Author(s):

J. P. Costanzo ◽

R. E. Lee ◽

P. H. Lortz

Keyword(s):

Rana Sylvatica ◽

Freeze Tolerance ◽

Conclusive Evidence ◽

Freezing Injury ◽

Critical Determinant ◽

Wood Frogs ◽

Glucose Levels ◽

Elevated Glucose ◽

Ice Content

In spring, the lowest temperature during freezing that can be survived by wood frogs (Rana sylvatica) from southern Ohio is approximately −3 degrees C. We investigated whether the thermal limit of freeze tolerance in these frogs is regulated by tissue levels of glucose, a putative cryoprotectant that is distributed to tissues during freezing. Frogs receiving exogenous glucose injections prior to freezing showed dose-dependent increases in glucose within the heart, liver, skeletal muscle and blood. Tissue glucose concentrations were further elevated during freezing by the production of endogenous glucose. Most glucose-loaded frogs survived freezing to −5 degrees C, whereas all control (saline-injected) frogs succumbed. Further, we investigated some mechanisms by which glucose might function as a cryoprotectant in R. sylvatica. Organ dehydration, a normal, beneficial response that reduces freezing injury to tissues, occurred independently of tissue glucose concentrations. However, elevated glucose levels reduced both body ice content and in vivo erythrocyte injury. These results not only provided conclusive evidence for glucose's cryoprotective role in R. sylvatica, but also revealed that tissue glucose level is a critical determinant of freeze tolerance capacity in this species.

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Cold hardiness in two helminth parasites of the freeze-tolerant wood frog, Rana sylvatica

Canadian Journal of Zoology ◽

10.1139/z00-034 ◽

2000 ◽

Vol 78 (6) ◽

pp. 1085-1091 ◽

Cited By ~ 7

Author(s):

Douglas C Woodhams ◽

Jon P Costanzo ◽

Jonathan D Kelty ◽

Richard E Lee, Jr.

Keyword(s):

Cold Hardiness ◽

Rana Sylvatica ◽

Wood Frog ◽

Digenetic Trematode ◽

Helminth Parasites ◽

Wood Frogs ◽

Freeze Avoidance ◽

Body Tissues ◽

Freeze Tolerant

Wood frogs, Rana sylvatica, tolerate the freezing of their body tissues as an overwintering adaptation. Various parasites infect wood frogs of northern populations, but nothing is known about their strategies for surviving within a frozen host. We examined winter-conditioned wood frogs that were experimentally exposed to 0°C (nonfrozen) or 4°C (frozen) to determine whether endoparasites survive the freezing of their host. We found no differences in the prevalence or intensity of adult lungworms Rhabdias ranae (Nematoda) or of larvae of an unidentified species of digenetic trematode between these groups. Live individuals of both species were observed in hosts that recovered from experimental freezing at 4°C. Within the host, R. ranae also tolerated exposure to 5°C, a temperature near the lower limit of survival of the wood frog. Cryostage experiments showed that, like its host, R. ranae was highly susceptible to inoculative freezing and tolerant of the freezing of its tissues. Rhabdias ranae frozen in vitro in the presence or absence of 250 mM glucose, the cryoprotectant used by wood frogs, recovered from a 10-h exposure to 4°C. The mechanism of cold tolerance used by larval trematodes was not investigated; however, we hypothesize that freeze avoidance by supercooling may be important in this species. Freeze-tolerant anurans, such as the wood frog, are useful subjects in the study of coevolution of thermal tolerance in parasites and their host.

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Glucose loading prevents freezing injury in rapidly cooled wood frogs

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1991.261.6.r1549 ◽

1991 ◽

Vol 261 (6) ◽

pp. R1549-R1553 ◽

Cited By ~ 4

Author(s):

J. P. Costanzo ◽

R. E. Lee ◽

M. F. Wright

Keyword(s):

Inverse Relationship ◽

Rana Sylvatica ◽

The Body ◽

Freezing Injury ◽

Wood Frogs ◽

Rapid Cooling ◽

Exogenous Glucose ◽

Production And Distribution ◽

Freeze Tolerant ◽

And Control

The wood frog (Rana sylvatica) is the most commonly studied of ten species of freeze-tolerant vertebrates. Under natural (i.e., slow) rates of cooling, freezing initiates the production of the cryoprotectant glucose, which is mobilized from the liver and distributed to tissues throughout the body. Rapid cooling during freezing is injurious to wood frogs, probably because cryoprotectant production and mobilization are inhibited. To test this hypothesis, we investigated whether rapid-cooling injury is reduced if exogenous glucose is experimentally introduced to tissues before freezing. Glucose-loaded and control (saline-injected) wood frogs were rapidly cooled during freezing to -2.5 degrees C and subsequently assayed for injury at both cellular (erythrocyte) and neuromuscular (behavioral reflex) levels. Rapid cooling produced substantial hemolysis in control frogs, but erythrocyte injury was significantly reduced in glucose-loaded frogs. Similarly neuromuscular injury was significantly higher in control frogs than in glucose-loaded frogs. These findings suggest that rapid-cooling injury results from an inadequate production and distribution of endogenous glucose during freezing. Furthermore, the inverse relationship between the degree of freezing injury and the quantity of exogenous glucose present strongly implicates glucose as a cryoprotectant in R. sylvatica.

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Organ-specific metabolism during freezing and thawing in a freeze-tolerant frog

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1987.253.2.r292 ◽

1987 ◽

Vol 253 (2) ◽

pp. R292-R297 ◽

Cited By ~ 11

Author(s):

K. B. Storey

Keyword(s):

Rana Sylvatica ◽

Liver Glycogen ◽

Freezing And Thawing ◽

Wood Frogs ◽

Anaerobic Energy ◽

Organ Specific ◽

Glucose Accumulation ◽

Frozen State ◽

Freeze Tolerant ◽

Product Accumulation

Freeze-tolerant wood frogs, Rana sylvatica, were exposed to three consecutive freeze-thaw cycles. Each 2-day freezing exposure resulted in the breakdown of liver glycogen and an accumulation of high quantities of glucose in all 10 tissues tested; during each 2-day thaw glucose was restored as liver glycogen. The data suggest that frogs do not maintain cryoprotectants throughout the winter in anticipation of freezing but only synthesize and/or maintain glucose during actual freezing episodes. The pattern of glucose accumulation during freezing suggests a peripheral vasoconstriction as freezing progresses, leaving circulation open to central organs (liver, heart, and brain) for as long as possible. Lactate and alanine contents in tissues rose during each freezing exposure and were reduced during each thaw. Based on anaerobic end-product accumulation, organs appeared to vary up to 10-fold in anaerobic energy requirements in the frozen state. Levels of adenylates and fructose-2,6-biphosphate were measured in liver and muscle and provided additional evidence of tissue-specific differences in metabolism in the frozen state.

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Anti-apoptotic response during anoxia and recovery in a freeze-tolerant wood frog (Rana sylvatica)

PeerJ ◽

10.7717/peerj.1834 ◽

2016 ◽

Vol 4 ◽

pp. e1834 ◽

Cited By ~ 20

Author(s):

Victoria E.M. Gerber ◽

Sanoji Wijenayake ◽

Kenneth B. Storey

Keyword(s):

Skeletal Muscle ◽

Rana Sylvatica ◽

Freeze Tolerance ◽

Wood Frog ◽

Apoptotic Pathway ◽

Atp Production ◽

Freeze Tolerant ◽

Specific Regulation ◽

Total Body ◽

Cellular Components

The common wood frog,Rana sylvatica, utilizes freeze tolerance as a means of winter survival. Concealed beneath a layer of leaf litter and blanketed by snow, these frogs withstand subzero temperatures by allowing approximately 65–70% of total body water to freeze. Freezing is generally considered to be an ischemic event in which the blood oxygen supply is impeded and may lead to low levels of ATP production and exposure to oxidative stress. Therefore, it is as important to selectively upregulate cytoprotective mechanisms such as the heat shock protein (HSP) response and expression of antioxidants as it is to shut down majority of ATP consuming processes in the cell. The objective of this study was to investigate another probable cytoprotective mechanism, anti-apoptosis during oxygen deprivation and recovery in the anoxia tolerant wood frog. In particular, relative protein expression levels of two important apoptotic regulator proteins, Bax and p-p53 (S46), and five anti-apoptotic/pro-survival proteins, Bcl-2, p-Bcl-2 (S70), Bcl-xL, x-IAP, and c-IAP in response to normoxic, 24 Hr anoxic exposure, and 4 Hr recovery stages were assessed in the liver and skeletal muscle using western immunoblotting. The results suggest a tissue-specific regulation of the anti-apoptotic pathway in the wood frog, where both liver and skeletal muscle shows an overall decrease in apoptosis and an increase in cell survival. This type of cytoprotective mechanism could be aimed at preserving the existing cellular components during long-term anoxia and oxygen recovery phases in the wood frog.

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