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

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.

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

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1993.265.5.r1036 ◽

1993 ◽

Vol 265 (5) ◽

pp. R1036-R1042 ◽

Cited By ~ 5

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)

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

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1993.265.4.r721 ◽

1993 ◽

Vol 265 (4) ◽

pp. R721-R725 ◽

Cited By ~ 6

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.

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