Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: In vivo and in vitro studies

The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7 d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7 d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation.     

Urea loading enhances postfreeze performance of frog skeletal muscle

The wood frog (Rana sylvatica) is a terrestrial hibernator that can accumulate urea as an osmoprotectant in autumn and winter. This study tested the hypothesis that elevated urea can also function as a cryoprotectant in this freeze-tolerant species. Performance characteristics (threshold stimulus voltage, maximal isometric twitch and tetanic contraction forces, and (1/2) fatigue time) of isolated gastrocnemius muscles were measured before and after experimental freezing at -1.5 degrees C for 18 h, followed by thawing. Frozen/thawed muscles exhibited reduced function relative to baseline (prefreeze) levels; however, muscles preincubated in a saline solution containing urea (80 mmol l(-1)) performed substantially better in some tests than muscles incubated without urea. Concentrations of urea in these treated muscles, approximately 65 mmol l(-1), were within the physiological range in winter R. sylvatica. Reducing tissue urea levels to approximately 33 mmol l(-1) resulted in a similar pattern of response, although the differences between urea-incubated and saline-incubated muscles were not statistically significant. Tests of cryoprotective efficacy were also performed on gastrocnemius muscles from R. pipiens, a closely related, but freeze-intolerant species that hibernates aquatically and thus has little need to accumulate urea. Urea-treated muscles from this species performed no better than muscles incubated in saline, attesting that freeze tolerance cannot be conferred simply by augmenting cryoprotectant levels. Overall, these results bolster an earlier report that urea accumulated in response to low moisture availability can serve a cryoprotective role in freeze-tolerant ectotherms.     

Skin ice nucleators and glycerol in the freezing-tolerant frog <Emphasis Type="Italic">Litoria ewingii</Emphasis>

The brown tree frog (Litoria ewingii) is the only known Southern Hemisphere vertebrate that can survive full-body freezing. Freezing challenges living organisms in many ways, with ice formation in the body producing a suite of physical and metabolic stresses which can damage cells and tissues. The present study looked at two mechanisms that address some of these stresses: cryoprotectants and ice nucleating agents (INAs). Skin secretions from L. ewingii were sampled along with microhabitat substrate and tested for the presence of INAs, which help control ice formation in the body. L. ewingii plasma was tested for seasonal and freezing-induced changes in both glucose and glycerol, which may have a cryoprotective role in freezing-tolerant frogs. Glycerol levels increased on freezing and decreased on thawing, while glucose levels did not change on freezing but increased on thawing. This suggests that glycerol may be acting as a cryoprotectant, although levels are low compared to other frogs. A clear seasonal change was seen in INA activity, with greater activity in winter than in summer. While potent INAs came from the microhabitat substrate, this work has shown for the first time that skin secretions also contain active INAs.     

Infective Juveniles of the Entomopathogenic Nematode,Steinernema feltiae Produce Cryoprotectants in Response to Freezing and Cold Acclimation

Steinernema feltiae is a moderately freeze-tolerant entomopathogenic nematode which survives intracellular freezing. We have detected by gas chromatography that infective juveniles of S. feltiae produce cryoprotectants in response to cold acclimation and to freezing. Since the survival of this nematode varies with temperature, we analyzed their cryoprotectant profiles under different acclimation and freezing regimes. The principal cryoprotectants detected were trehalose and glycerol with glucose being the minor component. The amount of cryoprotectants varied with the temperature and duration of exposure. Trehalose was accumulated in higher concentrations when nematodes were acclimated at 5°C for two weeks whereas glycerol level decreased from that of the non-acclimated controls. Nematodes were seeded with a small ice crystal and held at -1°C, a regime that does not produce freezing of the nematodes but their bodies lose water to the surrounding ice (cryoprotective dehydration). This increased the levels of both trehalose and glycerol, with glycerol reaching a higher concentration than trehalose. Nematodes frozen at -3°C, a regime that produces freezing of the nematodes and results in intracellular ice formation, had elevated glycerol levels while trehalose levels did not change. Steinernema feltiae thus has two strategies of cryoprotectant accumulation: one is an acclimation response to low temperature when the body fluids are in a cooled or supercooled state and the infective juveniles produce trehalose before freezing. During this process a portion of the glycerol is converted to trehalose. The second strategy is a rapid response to freezing which induces the production of glycerol but trehalose levels do not change. These low molecular weight compounds are surmised to act as cryoprotectants for this species and to play an important role in its freezing tolerance.     

Liver glycogen,glucose mobilization and freezing survival in chorus frogs,Pseudacris triseriata

We compared liver glycogen stores and glucose mobilization during freezing among winters in chorus frogs, Pseudacris triseriata, where populations varied in freezing survival. We also characterized tissue glycogen levels across the annual cycle. Frogs with low liver glycogen stores mobilized low amounts of glucose during freezing, and these were correlated with population variation in freezing survival. Moreover, liver glycogen stores were significantly and positively related to body mass. These data suggest that chorus frogs store liver glycogen in preparation for hibernation and that body size and glycogen stores must reach threshold levels for successful survival of freezing bouts during the winter.     

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.     

Cryoprotectants and Extreme Freeze Tolerance in a Subarctic Population of the Wood Frog

Wood frogs (Rana sylvatica) exhibit marked geographic variation in freeze tolerance, with subarctic populations tolerating experimental freezing to temperatures at least 10-13 degrees Celsius below the lethal limits for conspecifics from more temperate locales. We determined how seasonal responses enhance the cryoprotectant system in these northern frogs, and also investigated their physiological responses to somatic freezing at extreme temperatures. Alaskan frogs collected in late summer had plasma urea levels near 10 μmol ml^-1, but this level rose during preparation for winter to 85.5 ± 2.9 μmol ml^-1 (mean ± SEM) in frogs that remained fully hydrated, and to 186.9 ± 12.4 μmol ml^-1 in frogs held under a restricted moisture regime. An osmolality gap indicated that the plasma of winter-conditioned frogs contained an as yet unidentified osmolyte(s) that contributed about 75 mOsmol kg^-1 to total osmotic pressure. Experimental freezing to –8°C, either directly or following three cycles of freezing/thawing between –4 and 0°C, or –16°C increased the liver’s synthesis of glucose and, to a lesser extent, urea. Concomitantly, organs shed up to one-half (skeletal muscle) or two-thirds (liver) of their water, with cryoprotectant in the remaining fluid reaching concentrations as high as 0.2 and 2.1 M, respectively. Freeze/thaw cycling, which was readily survived by winter-conditioned frogs, greatly increased hepatic glycogenolysis and delivery of glucose (but not urea) to skeletal muscle. We conclude that cryoprotectant accrual in anticipation of and in response to freezing have been greatly enhanced and contribute to extreme freeze tolerance in northern R. sylvatica.     

Freeze tolerance in the gray treefrog: cryoprotectant mobilization and organ dehydration.

Freeze tolerance in the frog Rana sylvatica is supported by nonanticipatory mobilization of cryoprotectant (glucose) and redistribution of organ water. Other freeze-tolerant frogs may manifest these responses but differences exist. For example, the gray treefrog (Hyla versicolor) accumulates mostly glycerol as opposed to glucose. The current study reports additional novel features about cryoprotection in H. versicolor. Frogs were acclimated to low temperature for 12 weeks and frozen for 3 days at -2.4 degrees C. Some frogs were then thawed at 3 degrees C for 4 hr. Calorimetry revealed that frozen frogs had 53.9% +/- 11.1% of their body water in ice, and all frogs recovered following this procedure. Plasma glucose was low prior to the onset of freezing (1.1 +/- 0.9 micromol/ml) and it was 20x higher in postfreeze frogs. Constituting nearly 30% of plasma solute, glycerol was 117.2 +/- 13.6 micromol/ml prior to freezing and it remained equally high in postfreeze frogs. Liver water content was moderately lower in frozen frogs when compared to controls (62.9% +/- 3.7% vs. 68.6% +/- 1.7%), whereas postfreeze frogs excessively hydrated their livers (75.7% +/- 2.1%). Less-pronounced changes were seen in muscle water content. H. versicolor can mobilize its major cryoprotectant, glycerol, in response to extended cold acclimation, which is unique in comparison to other freeze-tolerant frogs, and it experiences only moderate organ dehydration during freezing. This species conforms with other freeze-tolerant frogs, however, by mobilizing glucose as a direct response to tissue freezing.     

The Effect of Freezing Rate on the Survival of Cryopreserved African Sharptooth Catfish (Clarias gariepinus) Spermatozoa

Cryoprotective agents were evaluated to find the optimal concentration of the cryoprotectant and most suitable combination of solution and cryoprotectant. A cryoprotective agent composed of 4% glucose and 9% glycerol yielded the best results. It was established that the optimal freezing rate is dependent on the composition of the cryoprotective agent. Maximal survival of catfish spermatozoa (60%) occurs at 5°C min^-1 and faster and slower freezing rates result in poor survival or no survival at all. Incorporation of an isothermal holding period into the freezing rate led to remarkable increase (20-30%) in sperm survival when Me₂SO was present in the cryoprotective agent. Cryoprotective agents containing glucose also showed improved survival when a three phase freezing rate was used. These results lead to the conclusion that the presence of an isothermal holding period in the freezing rate is beneficial for the cryoprotective action of Me₂SO and glucose.     

Endothelial retinoblastoma protein reduces abdominal aortic aneurysm development via promoting DHFR/NO pathway-mediated vasoprotection

Carbamoyl phosphate synthetase I (CPS1) represents an important regulatory enzyme of the urea cycle that mediates the ATP-driven reaction ligating ammonium, carbonate, and phosphate to form carbamoyl phosphate. The freeze-tolerant wood frog (Rana sylvatica or Lithobates sylvaticus) accumulates high concentrations of urea during bouts of freezing to detoxify any ammonia generated and to contribute as a cryoprotectant thereby helping to avoid freeze damage to cells. Purification of CPS1 to homogeneity from wood frog liver was performed in control and frozen wood frogs by a three-step chromatographic process. The affinity of CPS1 for its three substrates was tested in the purified control and freeze-exposed enzyme under a variety of conditions including the presence and absence of the natural cryoprotectants urea and glucose. The results demonstrated that affinity for ammonium was higher in the freeze-exposed CPS1 (1.26-fold) and that with the addition of 400 mM glucose it displayed higher affinity for ATP (1.30-fold) and the obligate activator N-acetylglutamate (1.24-fold). Denaturation studies demonstrated the freeze-exposed enzyme was less thermally stable than the control with an unfolding temperature approximately 1.5 °C lower (52.9 °C for frozen and 54.4 °C for control). The control form of CPS1 had a significantly higher degree of glutarylated lysine residues (1.42-fold increase) relative to the frozen. The results suggest that CPS1 activation and maintenance of urea cycle activity despite the hypometabolic conditions associated with freezing are important aspects in the metabolic survival strategies of the wood frog.

     

Stress-induced antioxidant defense and protein chaperone response in the freeze-tolerant wood frog <Emphasis Type="Italic">Rana sylvatica</Emphasis>

Freeze tolerance is an adaptive response utilized by the wood frog Rana sylvatica to endure the sub-zero temperatures of winter. Survival of whole body freezing requires wood frogs to trigger cryoprotective mechanisms to deal with potential injuries associated with conversion of 65–70% of total body water into ice, including multiple consequences of ice formation such as cessation of blood flow and cell dehydration caused by water loss into ice masses. To understand how wood frogs defend against these stressors, we measured the expression of proteins known to be involved in the antioxidant defense and protein chaperone stress responses in brain and heart of wood frogs comparing freezing, anoxia, and dehydration stress. Our results showed that most stress proteins were regulated in a tissue- and stress-specific manner. Notably, protein levels of the cytosolic superoxide dismutase (SOD1) were upregulated by 1.37?±?0.11-fold in frozen brain, whereas the mitochondrial SOD2 isoform rose by 1.38?±?0.37-fold in the heart during freezing. Catalase protein levels were upregulated by 3.01?±?0.47-fold in the brain under anoxia stress, but remained unchanged in the heart. Similar context-specific regulatory patterns were also observed for the heat shock protein (Hsp) molecular chaperones. Hsp27 protein was down-regulated in the brain across the three stress conditions, whereas the mitochondrial Hsp60 was upregulated in anoxic brain by 1.73?±?0.38-fold and by 2.13?±?0.57-fold in the frozen heart. Overall, our study provides a snapshot of the regulatory expression of stress proteins in wood frogs under harsh environment conditions and shows that they are controlled in a tissue- and stress-specific manner.     

Strategies for exploration of freeze responsive gene expression: advances in vertebrate freeze tolerance

Winter survival for many cold-blooded species involves freeze tolerance, the capacity to endure the freezing of a high percentage of total body water as extracellular ice. The wood frog (Rana sylvatica) is the primary model animal used for studies of vertebrate freeze tolerance and current studies in my lab are focused on the freeze-induced changes in gene expression that support freezing survival. Using cDNA library screening, we have documented the freeze-induced up-regulation of a number of genes in wood frogs including both identifiable genes (fibrinogen, ATP/ADP translocase, and mitochondrial inorganic phosphate carrier) and novel proteins (FR10, FR47, and Li16). All three novel proteins share in common the presence of hydrophobic regions that may indicate that they have an association with membranes, but apart from that each shows unique tissue distribution patterns, stimulation by different signal transduction pathways and responses to two of the component stresses of freezing, anoxia, and dehydration. The new application of cDNA array screening technology is opening up a whole new world of possibilities in the search for molecular mechanisms that underlie freezing survival. Array screening of hearts from control versus frozen frogs hints at the up-regulation of adenosine receptor signaling for the possible mediation of metabolic rate suppression, hypoxia inducible factor mediated adjustments of anaerobic metabolism, natriuretic peptide regulation of fluid dynamics, enhanced glucose transporter capacity for cryoprotectant accumulation, defenses against the accumulation of advanced glycation end products, and improved antioxidant defenses as novel parts of natural freeze tolerance that remain to be explored.     

Time course for cryoprotectant synthesis in the freeze-tolerant chorus frog, Pseudacris triseriata

Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5- and 30-min and 2- and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5-12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.     

Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae.

The freeze-thaw tolerance of Saccharomyces cerevisiae was examined throughout growth in aerobic batch culture. Minimum tolerance to rapid freezing (immersion in liquid nitrogen; cooling rate, approximately 200 degrees C min-1) was associated with respirofermentative (exponential) growth on glucose. However, maximum tolerance occurred not during the stationary phase but during active respiratory growth on ethanol accumulated during respirofermentative growth on glucose. The peak in tolerance occurred several hours after entry into the respiratory growth phase and did not correspond to a transient accumulation of trehalose which occurred at the point of glucose exhaustion. Substitution of ethanol with other carbon sources which permit high levels of respiration (acetate and galactose) also induced high freeze-thaw tolerance, and the peak did not occur in cells shifted directly from fermentative growth to starvation conditions or in two respiratorily incompetent mutants. These results imply a direct link with respiration, rather than exhaustion of glucose. The role of ethanol as a cryoprotectant per se was also investigated, and under conditions of rapid freezing (cooling rate, approximately 200 degrees C min-1), ethanol demonstrated a significant cryoprotective effect. Under the same freezing conditions, glycerol had little effect at high concentrations and acted as a cryosensitizer at low concentrations. Conversely, under slow-freezing conditions (step freezing at -20, -70, and then -196 degrees C; initial cooling rate, approximately 3 degrees C min-1), glycerol acted as a cryoprotectant while ethanol lost this ability. Ethanol may thus have two effects on the cryotolerance of baker's yeast, as a respirable carbon source and as a cryoprotectant under rapid-freezing conditions.     

Post-freeze recovery of peripheral nerve function in the freeze-tolerant wood frog,Rana sylvatica

We investigated the restoration of peripheral nerve function and simple neurobehavioral reflexes in the freeze-tolerant wood frog (Rana sylvatica). Thirty-two specimens, allowed to freeze for 39 h and ultimately cooled to-2.2°C, were sampled at various time intervals up to 60 h after thawing at 5°C was initiated. The sciatic nerves of treated frogs were initially unresponsive to stimulation, but usually regained excitability within 5 h. Except for a slight reduction in nerve excitability characteristics of the compound action potentials of treated frogs were indistinguishable from those of control frogs. Recovery times for the hindlimb retraction and righting reflexes were 8 h and 14 h, respectively. Concentrations of the cryoprotectant glucose increased 8.2-fold in the sciatic nerve and 10.5-fold in the underlying semimembranosis muscle of treated frogs, and remained elevated for at least 60 h after thawing was initiated. These organs lost 47.2% and 15.9%, respectively, of their water during freezing, but were rehydrated within 2 h of the onset of thawing. The accumulation of glucose and the withdrawal of tissue water apparently are cryoprotective responses which enable this species to survive freezing.     

Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog,Rana sylvatica

The wood frog (Rana sylvatica) can survive the winter in a frozen state, in which the frog’s tissues are also exposed to dehydration, ischemia, and anoxia. Critical to wood frog survival under these conditions is a global metabolic rate depression, the accumulation of glucose as a cryoprotectant, and a reliance on anaerobic glycolysis for energy production. Pyruvate kinase (PK) catalyzes the final reaction of aerobic glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. This study investigated the effect of each stress condition experienced by R. sylvatica during freezing, including dehydration and anoxia, on PK regulation. PK from muscle of frozen and dehydrated frogs exhibited a lower affinity for PEP (K_m = 0.098 ± 0.003 and K_m = 0.092 ± 0.008) than PK from control and anoxic conditions (K_m = 0.065 ± 0.003 and K_m = 0.073 ± 0.002). Immunoblotting showed greater serine phosphorylation on muscle PK from frozen and dehydrated frogs relative to control and anoxic states, suggesting a reversible phosphorylation regulatory mechanism for PK activity during freezing stress. Furthermore, PK from frozen animals exhibited greater stability under thermal and urea-induced denaturing conditions than PK from control animals. Phosphorylation of PK during freezing may contribute to mediating energy conservation and maintaining intracellular cryoprotectant levels, as well as increase enzyme stability during stress.     

Annual variation in glycerol mobilization and effect of freeze rigor on post-thaw locomotion in the freeze-tolerant frog <Emphasis Type="Italic">Hyla versicolor</Emphasis>

This study documents post-thaw recovery of jump distance and cryoprotectant mobilization in the freeze-tolerant frog Hyla versicolor over two successive years. Cold acclimated frogs had plasma glycerol levels near 1.0 mM in 2004 but it was nearly 70× higher during 2005. Freezing of frogs induced nearly identical levels of plasma glycerol (ca. 177 mM) during 2004 and 2005. Plasma glucose was only mobilized upon somatic freezing, with averages ranging between 21 and 36 mM. Control jump distance showed no difference between the two years of the study. The post-thaw jump response was identical during the first 2 years despite large differences in glycerol mobilization between these 2 years. Recovery proceeded much faster in 2005 when frogs mobilized glycerol prior to freeze exposure. Frogs were more impaired in their locomotion performance during the initial stages of recovery period when they were frozen at a lower temperature (−3 vs. −1.5°C) but they eventually recovered. Moderate lengthening of the freeze duration (3 vs. 7 days) with the 2004 collection group did not affect recovery of jump distance when frogs were frozen at −1.5°C. Hence, postfreeze impairment of locomotion is dependent of the intensity of the freeze temperature but it is a reversible process that is mitigated when glycerol is more freely distributed to body tissues.