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

1991 ◽  
Vol 261 (6) ◽  
pp. R1346-R1350 ◽  
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.

Physiological responses of freeze-tolerant and -intolerant frogs: clues to evolution of anuran freeze tolerance

1993 ◽  
Vol 265 (4) ◽  
pp. R721-R725 ◽  
Author(s):  
J. P. Costanzo ◽  
R. E. Lee ◽  
P. H. Lortz
Keyword(s):  
Stress Responses ◽  
Physiological Stress ◽  
Physiological Responses ◽  
Rana Sylvatica ◽  
Freeze Tolerance ◽  
Leopard Frog ◽  
In Vitro Tests ◽  
Body Tissues ◽  
Freeze Tolerant

Freeze tolerance in the wood frog, Rana sylvatica, is promoted by multiple, integrated physiological responses to ice forming within body tissues. By analyzing the freezing responses of the sympatric, but freeze intolerant, leopard frog (R. pipiens), we sought clues to the evolution of anuran freeze tolerance. Physiological responses critical to R. sylvatica's freeze tolerance, such as the synthesis and distribution of the cryoprotectant glucose, protective dehydration of organs, and deferred cardiac failure, were present, but comparatively less pronounced, in R. pipiens. Both species were innately tolerant of hyperglycemia. Glucose supplements did not enhance the freezing viability of R. pipiens, although in vitro tests of cryoprotectant efficacy revealed that glucose and glycerol provided comparable protection to erythrocytes of both species. We conclude that the evolution of freeze tolerance in R. sylvatica is not only promoted by its desiccation tolerance and the fortuitous biophysical consequences of freezing (e.g., exothermic induction of cardioacceleration and moderation of cooling rate) but also involves a progressive enhancement of fundamental physiological stress responses.

Osmotic and freezing tolerance in spermatozoa of freeze-tolerant and -intolerant frogs

1998 ◽  
Vol 275 (3) ◽  
pp. R713-R719 ◽  
Author(s):  
Jon P. Costanzo ◽  
John A. Mugnano ◽  
Heidi M. Wehrheim ◽  
Richard E. Lee
Keyword(s):  
Plasma Membrane ◽  
Rana Sylvatica ◽  
Wood Frog ◽  
Leopard Frog ◽  
Subzero Temperatures ◽  
In Vitro Exposure ◽  
Freeze Tolerant ◽  
Motility Rate ◽  
Winter Breeding

The wood frog ( Rana sylvatica) is a freeze-tolerant species that encounters subzero temperatures during its winter breeding season, whereas the leopard frog ( R. pipiens) is freeze intolerant and breeds in spring. Osmotic and freezing tolerances of spermatozoa from these species were inferred from spermolysis rate, integrity of the plasma membrane as judged using vital dye assay, and motility rate. Sperm of R. sylvatica became motile in hypotonic media (≤220 mosmol/kg) and tolerated in vitro exposure to osmotic concentrations spanning nearly three orders of magnitude. Relative to sperm from R. sylvatica, which were unaffected by freezing at temperatures of −4°C or greater, R. pipiens sperm were more susceptible to osmotic damage and cryoinjury. These differences likely reflect cellular adaptations to somatic freezing in R. sylvatica. Unprotected sperm from both species were extensively damaged by freezing at −8°C, but the presence of glucose, the cryoprotectant used by R. sylvatica, or the permeant glycerol markedly diminished cryoinjury. These data suggest the feasibility of developing gamete cryopreservation protocols to aid efforts in conserving amphibian populations.

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

2020 ◽  
Vol 98 (2) ◽  
pp. 145-153 ◽  
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.

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

2000 ◽  
Vol 78 (6) ◽  
pp. 1085-1091 ◽  
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.

scholarly journals Seasonality of Freeze Tolerance in a Subarctic Population of the Wood Frog,Rana sylvatica

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Jon P. Costanzo ◽  
M. Clara F. do Amaral ◽  
Andrew J. Rosendale ◽  
Richard E. Lee
Keyword(s):  
Cell Damage ◽  
Rana Sylvatica ◽  
Freeze Tolerance ◽  
Wood Frog ◽  
Freezing And Thawing ◽  
Tissue Concentrations ◽  
Hepatic Reserve ◽  
Interior Alaska ◽  
Low Levels ◽  
Marked Cell

We compared physiological characteristics and responses to experimental freezing and thawing in winter and spring samples of the wood frog,Rana sylvatica, indigenous to Interior Alaska, USA. Whereas winter frogs can survive freezing at temperatures at least as low as −16°C, the lower limit of tolerance for spring frogs was between −2.5°C and −5°C. Spring frogs had comparatively low levels of the urea in blood plasma, liver, heart, brain, and skeletal muscle, as well as a smaller hepatic reserve of glycogen, which is converted to glucose after freezing begins. Consequently, following freezing (−2.5°C, 48 h) tissue concentrations of these cryoprotective osmolytes were 44–88% lower than those measured in winter frogs. Spring frogs formed much more ice and incurred extensive cryohemolysis and lactate accrual, indicating that they had suffered marked cell damage and hypoxic stress during freezing. Multiple, interactive stresses, in addition to diminished cryoprotectant levels, contribute to the reduced capacity for freeze tolerance in posthibernal frogs.

Adaptations of plasma membrane glucose transport facilitate cryoprotectant distribution in freeze-tolerant frogs

1993 ◽  
Vol 265 (5) ◽  
pp. R1036-R1042 ◽  
Author(s):  
P. A. King ◽  
M. N. Rosholt ◽  
K. B. Storey
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Rana Sylvatica ◽  
Glucose Transporters ◽  
Freeze Tolerance ◽  
Leopard Frog ◽  
Freeze Tolerant ◽  
High Concentrations

Natural freeze tolerance in several anuran species involves the accumulation of high concentrations of glucose as a cryoprotectant in body fluids and tissues. The present study identifies an important new molecular mechanism supporting freeze tolerance, an adaptive increase in the capacity for facilitated transport of cryoprotectant across plasma membranes by increasing the numbers and/or activity of plasma membrane glucose transporters. Glucose transport by membranes isolated from liver and skeletal muscle was analyzed in two species, the freeze-tolerant wood frog Rana sylvatica and the freeze-intolerant leopard frog Rana pipiens. Membranes from both liver and muscle of R. sylvatica displayed much higher rates of carrier-mediated glucose transport, measured by a rapid filtration technique, compared with corresponding rates for R. pipiens membranes. For the liver Vmax values for glucose transport by membrane vesicles were 69 +/- 18 and 8.4 +/- 2.3 nmol.mg protein-1.s-1 at 10 degrees C for R. sylvatica and R. pipiens, respectively. This difference was due primarily to a greater number of glucose transporters in R. sylvatica liver membranes; the total number of transporter sites, determined by cytochalasin B binding, was 4.7-fold higher in the freeze-tolerant species. For muscle membranes, the Vmax for glucose transport was 4.9 +/- 1 and 0.6 +/- 0.16 nmol.mg-1 x s-1 at 22 degrees C for R. sylvatica and R. pipiens, respectively. However, in muscle there were no differences in the number of membrane transporters between species.(ABSTRACT TRUNCATED AT 250 WORDS)

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

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1834 ◽  
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.

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

1993 ◽  
Vol 71 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Jon P. Costanzo ◽  
Richard E. Lee Jr.
Keyword(s):  
Production Capacity ◽  
Rana Sylvatica ◽  
Freeze Tolerance ◽  
Liver Glycogen ◽  
Wood Frog ◽  
Freezing Injury ◽  
Wood Frogs ◽  
Cryoprotectant Synthesis ◽  
Freeze Tolerant ◽  
The Relationship

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.

Sign in / Sign up

Export Citation Format

Share Document