Cryoprotectant Production Capacity of the Freeze-Tolerant Wood Frog, Rana Sylvatica

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

JoN P. Cosr,cNzo AND Rlcnano E. LsE, Jn. Department of Zoology, Miami University, Oxford, OH 45056, U.S.A. ReceivedJune 8. 1992 AcceptedAugust 17, 1992

CosmNzo, J. P., and LBp, R. E., Jn. 1993. Cryoprotectantproduction capacityof the freeze-tolerantwood frog, Rana sylvatica.Can. J. Zool. 7I: 7l-75. Freezingsurvival of the wood frog (Rana sylvatica)is enhancedby the synthesisof the cryoprotectantglucose, via liver glycogenolysis.Because the quantityof glucosemobilized during freezingbears significantly on the limit of freezetolerance, we investigated the relationship between the quantity of liver glycogen and the capacity for cryoprotectant synthesis. We successfullyaugmented natural levels of liver glycogenby injecting cold-conditionedwood frogs with glucose.Groups of 8 frogs having mean liver glycogenconcentrations of 554 + 57 (SE), 940 + 57, and 1264 + 66 pmollg catabolized98.7, 83.4, and 52.8%, respectively,of their glycogenreserves during 24 h of freezingto -2.5'C. Glucoseconcentrations con- comitantly increased,reaching 2l + 3, 102 + 23, and 119 + 14 pmollg, respectively,in the liver, and 15 * 3,42 + 5, and 6l * 5 pcmol/ml-, respectively, in the blood. Becausethe capacity for cryoprotectant synthesisdepends on the amount of liver glycogen,the greatestrisk of freezinginjury likely occursduring spring,when glycogenreserves are minimal. Non- glucoseosmolites were important in the wood frog's cryoprotectantsystem, especially in frogs having low glycogenlevels. Presumably the natural variation in cryoprotectant synthesis capacity among individuals and populations of R. sylvatica chiefly reflectsdifferences in glycogenreserves; however, environmental,physiological, and geneticfactors likely are also involved.

CosmNzo, J. P., et LEs, R. E., Jn. i993. Cryoprotectantproduction capacity of the freeze-tolerantwood frog, Rana sylvatica.Can. J. Zool. 7l : 71-75. La survie au gel chez la Grenouille des bois (Rana sylvatica) est favoris6e par la synthbsede glucose, aux propri6t6s cryo- protectrices,via la glycog6nolysedans le foie. Comme la quantit6de glucosemobilis6e durant le gel a une grandeinfluence sur le seuil de tol6ranceau gel, nous avons examin6 la relation entre la quantit6 de glycogdnedu foie et la capacit6de synthbse de la substancecryoprotectrice. Nous avons r6ussi d augmenterles concentrationsnaturelles de glycogdneh6patique en injec- tant du glucoseir des grenouillesacclimat6es au froid. Des groupesde 8 grenouillesayant des concentrationsmoyennes de 554 + 57 (erreur type), 940 + 57 et 1264 * 66 pmol glycogdneont catabolys6respectivemenl 98,7 , 83,4 et 52,8% de leurs r6servesde glycogBneau coursd'un gel de 24 h d -2,5"C. Les concentrationsde glucoseont augment6de faEoncorrespon- dante,atteignantrespectivement2l+3,102+23etll9+14p.mollgdanslefoieet15*3,42+5et61 +5prmoliml dans le sang. Comme la capacit6de synthdsede la substancecryoprotectrice d6pendde la quantit6 de glycogdne dans le foie, le risquede blessuresdues au gel est maximal au printemps,au momentoir les r6servesde glycogbnesont minimales.Les osmolites autres que le glucose se sont av6r6simportants dans le systbmede cryoprotection de la grenouille, pafticulidrement chez les grenouilles d faible teneur en glycogdne. La variation naturelle de la capacit6de synthbsede la substancecryoprotectrice chez les individus et les populations de R. sylvatica reflbte probablement surtout les variations dans les r6serves de glycogdne;cependant, des facteurs6cologiques, physiologiques et g6n6tiquesentrent probablement aussi en jeu. [Traduit par la r6daction]

Introduction to thawing, cryoprotectant returns to the liver and is recon- The overwintering strategies of certain vertebrate ecto- verted to glycogen(Storey and Storey 1986). therms are intriguing examplesof physiological adaptationsto In wood frogs, the cryoprotective effects of glucose depend t life in extreme environments.In particular, the wood frog on its concentration.For example,higher concentrations offer (Ranasylvatica) is one offive terrestriallyhibernating anurans superiorcryoprotection of erythrocytesfrozen in vitro (Costanzo known to tolerate extensive freezing of their body water. The and Lee 1991)and mitigatethe damageassociated with rapid freeze tolerance of the wood frog dependson the production freezing of intact frogs (Costanzoet al. 1991b). Additionally, of largequantities (e.g., up to 0.5 M) of glucose,a cryoprotec- Layne and Lee (1990) associatedhigh tissueglucose concen- tant that demonstrably reduces freezing injury (Canty et al. trationswith low tissueice contents.One obviousimplication 1986; Costanzoand Lee l99l;' Costanzo et al. 1991a) by ofthis relationshipis that frogs synthesizingmore cryoprotec- exerting both specific and colligative effects (Storey 1990; tant incur less cryoinjury. Accordingly, factors influencing Karow 1991). glucose production capacity indirectly determine freezing Earlier studies elucidated the role of the liver in R. sylva- survival. tica's cryoprotectant system (for a review see Storey 1990). Becauseliver glycogenolysisis the chief sourceof glucose, Within minutes, hepatocytesrespond to the initiation of ice the amount of cryoprotectant produced during freezing may formation within the body by producing glucose via glyco- ultimatelybe limited by liver glycogenstores. Heretofore, this genolysis.This process,possibly mediated by a B-adrenergic, relationship has not been studied, probably becausethe natur- cAMP-dependentmechanism (Mommsen and Storey 1992),is ally high variability in liver glycogenin field-collectedfrogs governed by marked changes in the activity and quantity of (e.g., Storeyand Storey 1986, 1987)is problematic.We cir- key regulatoryenzymes (Storey 1990). Glucoseentering the cumventedthis difficulty by administering exogenousglucose blood is distributedto tissuesthroughout the body. Subsequent to both supplementand standardizeliverglycogen levels. Sub-

Printed in Canada / Imprim€ au Canada 72 cAN.J. ZOOLvol-.7l, t993 1500 TrsI-E l. Analysesof the liver and blood of unfrozen wood frogs oo (Ranasylvatica) assayed 5 -6 d following the administrationof saline or 650 mmol slucose

Liver Plasma 1000

Glycogen Glucose Glucose Osmolality h0 Group fumol/g) Q.cmol/g) (pmol/mL) (mosmol/L) (.) >. Saline 554.1+57.4a ll.2+2.1a 1.6+0.2a 257+2a 500 Glucose| 939.5 + 57.2b 10.6+3.0a 2.5+0.2a 254+4a GlucoseII 1264.1+66.2c 7.5+0.9a 5.0+0.9, 249+4a q) F 34.6 0.9 tl.2 1.7 -l P <0.001 0.440 < 0.001 0.209

NorE: The glucoseI group receiveda singledose, and the glucoseII group received Saline Glucose I Glucose II threedoses at 5-d intervals.Values are given as the mean t SEM; N = 8 frogs/group. Meansin eachcolumn followed by a different letterare statisticallydistinguishable (P < Ftc. 1. Liver glycogenin wood frogs (Ranasylvatica) administered 0.05). Glycogen concentrationis expressedin glucoseunits. salineor 650 mmol glucose(glucose I, singledose; glucose II, three dosesat 5-d intervals),measured before or after freezingto -2.5'C. Column height indicatesthe meanvalue from 8 frogs/group;vertical sequently, by measuring the glucose mobilized during freez- bars representSEM. An asteriskindicates that meansfor unfrozen : ing, we evaluated the relationship between liver glycogen and frozen frogs differed statisticallywithin the saline (F 90.9, (F : glucoseII (f : content and the capacity for cryoprotectant synthesis. P < 0.001),glucose I 71.8,P < 0.001),and 31.4.P<0.001)groups. Material and methods Specimens Glycogenconcentrations (expressed in glucoseunits) in the replicate Male R. sylvaticaweighing 15.9 + 0.3 g (mean + SEM; N : 48) sampleswere averagedto produce a single value for each frog. Glu- were collected in February 1991 from breeding ponds in Adams cose was measuredin a separateportion of the liver homogenizedin County, southernOhio. Freezetolerance of frogs from this popula- 7% (wlv) ice-cold perchloric acid and centrifuged for 5 min at tion was previouslyreported (Layne and Lee 1987).To approximate 2000 x g. A spectrophotometricoxidase procedure (No. 510, Sigma the environmentalconditions during naturalhibernation (e.g., Storey Chemical Co.) was used to measureglucose in liver extracts and 1990),the frogs were fasted,kept for >4 weeksin cagescontaining plasma. Water content of liver tissue, expressedas percent wet damp moss, and exposedto 4"C in total darkness. weight, was determinedby drying the remaining portion at 65"C. Analyses of variance were used to statistically compare mean values Glucose loading among treatment groups and between unfrozen and frozen samples. Frogs were randomlyassigned to one of threegroups and adminis- Multiple comparisonsinvolved Fisher's least significantdifferences tered phosphate-bufferedsaline (115 mmol, pH 7.4) or salinecon- test (P < 0.05). taining 650 mmol glucose,by injecting the fluid (volume, 6.7% of body mass) into the dorsal lymph pad. A control group received only Results saline, whereastwo experimental groups received either a single dose of glucose(glucose I) or three dosesdelivered at 5-d intervals(glu- Effect of glucose loading on unfrozenfrogs coseII). All frogs remainedin their cagesfor 5 -6 d at 4"C prior to Tissues of unfrozen frogs were analyzedto determine the further use. fate of the injected glucoseand establishbase-line levels of livers of the frogs receiving glucosecon- Freezing protocol carbohydrates.The receiving Half the frogs in each group were placed inside plastic tubes and tained significantly more glycogen than did those cooledin an ethanolbath maintainedar ca. -2.5" C. A thermocouple only saline, with the highestconcentrations measured in the probepositioned against each frog's abdomenprovided a continuous glucoseII group. Mean glucoseconcentrations in the liver temperaturerecording on a multichanneldata logger (OM500, Omega were statisticallyindistinguishable among the groups(Table 1). -1.0"C, Engineering). After each frog supercooledto ca. ice Although meanplasma glucose levels differed statistically,the nucleation (verified by a recorded exotherm) was initiated by apply- differences were not large enough to influence osmolality ing aerosol coolant to the tube's exterior. During freezing the frogs (Table 1). Consequently,plasma glucose and osmolality lacked -0.07 + 0.002"C ' h ' (N : 24), :ultimately reaching cooled at significantcorrelation (r2 :0.03; F :0.2'7, P > 0.5). -2.5"C afler 24 h. Subsequentlythey were thawedfor 2.5 h at 4"C and used in tissueanalyses. Glycogenolysisand cryoprotectantproduction during freezing present prior to freezing Tissueanalyses The quantity of liver glycogen Thawed frogs, togetherwith the remaining unfrozen frogs, were (assumedto approximatethat measuredin the unfrozen counter- double-pithedand rapidly dissectedon ice. Blood collectedfrom the parts) determinedthe amountremaining after freezing: mean severedaorta in heparinized microcapillary tubes was centrifuged at concentrationsdiffered significantly(F : 18.7, P < 0.001) 2000 x g for 5 min. The plasma was separatedand used in deter- among the treatment groups (Fig. l). Calculatedfrom the minationsof glucose(see below) and osmolality(Wescor 5500 vapor difference between means for corresponding unfrozen and pressureosmometer). Sodium and potassiumlevels in the plasmaof frozen frogs, the portion of the glycogenreserves consumed photometer saline-injected frogs were measured using a flame during freezingwas 98.8% in the salinegroup, 83.4% inthe (Instrumentation Laboratory 943). glucoseI group, and 52.8% in the glucoseII group. An excisedlobe of the liver was cut into four portions (ca. 25 mg) The glucose synthesized during freezing was detected in that were blottedto removesurface moisture and weighedto 0.1 mg. (Fig. Liver glucosewas sub- Glycogen in two portions was determinedby the method of Kemp and both the liver and the blood 2). Kits van Heijningen (1954); methanol extraction (two washings) stantially higher in frozen relative to unfrozen frogs in the : : removed glucoseand ensuredthat only glycogen would be quantified. saline(F :9.2, P:0.009), glucoseI (f A.9, P COSTANZO AND LEE 73

t4tl 340 '.\ O saline , 120 330 A GlucoseI I GlucoseII bo q -.- 100 320 Glucose II >. -1 80 JIU

q Glucose I 960 -h 300 .a Saline 290 o40 q N

q .tr ,)n !--J 280 I q 270 20 40 60 80 100 600 800 1000 1200 1400 (Pmol/ml) Prefreeze Glycogen (pmol/g) Plasma Glucose glucose Frc. 2. Glucoseconcentration in the liver andplasma of wood FIc. 3. Relationshipbetween plasma osmolality and con- -2.5"C. The frogs(Rana sylvatica) frozen to -2.5"C asa functionof liver glyco- centrationin ryoodfrogs (Rana sylvatica) frozen to : :21.5, gen prior to freezing.Data points indicate the meanvalues correlationwas significant (r2 0.51,F P < 0.001).The content : from 8 frogs/group;vertical and horizontalbars representSEM. line of bestfit was X 0.32X + 289.1. Differentupper case and lower casecharacters indicate that liver glucoseand plasma glucose means, respectively, differ statistically. a principal determinantof cryoprotectantproduction, other factors are also involved. 0.002),and glucose II (f : 64.5,P < 0.001)groups;plasma Injecting R. sylvaticawith 650 mmol glucoseelevates glu- glucosewas also higher in the frozen frogs (saline:F : 19.6, coselevels in theliver (from 11.4* 1.9to 26.8 + 3.9 p.mollg) P < 0.001;glucosel:F:61.3, P < 0.001;glucosell:F: and plasma(from 1.4 + 0.3 to 53.9 + 7.8 pmollmL) within I4l.l, P < 0.001). Glucoselevels in the frozenfrogs differed 2.5 h (Costanzoet al. l99la). By allowing a longer (5-6 d) significantly among the treatment groups for both the liver incubationperiod, glucoselevels returnedto normal in liver (F: 10.9,P < 0.001)andplasma (F:29.2, P < 0.001), and near-normalin the blood. Glycogenesisin the glucose- and dependedon the amountof liver glycogenpresent before Ioadedfrogs was evidencedby their markedly elevatedlevels freezing(Fig. 2). Comparisonsof liver glucoseconcentrations of liver glycogen. expressedin micromoles per gram wet weight were judged Hibernating wood frogs are aphagic and must catabolize valid becausethe mean values for liver water content in frozen storednutrients to meet metabolicdemands. Although energy frogs in the saline(11.4 + 1.6%), glucoseI (74.7 + 1.6%), flow during winter is largely negative,our resultssuggest that and glucoseII (13.5 + l.l%) groupswere statisticallyindis- hibernating R. sylvatica retain the ability to convert blood tinguishable(f : 1.3, P : 0.285). sugar into glycogen. Glycogenesisduring thawing not only Freezingsignificantly elevated plasma osmolality, as mean relievesosmotic stress, but becauseexcess glucose is excreted valuesfor frozen frogs in the saline(293 + 3 mosmol), glu- by kidney (Layneet al. 1992),it may also reduceenergy loss. coseI (302 * 2 mosmol), and glucoseII (311 * 3 mosmol) This anabolicprocess, therefore, seems critical for recovering groupswere significantly(F : 105.2,P < 0.001; F : 86.7, the cryoprotectant mobilized during freezing. P < 0.001 and F: 421.9,P < 0.001, respectively)higher than correspondingvalues for the unfrozen frogs (Table 1). Control of liver glycogenolysis : Plasmaosmolalities of frozen frogs differed significantly (F Cryoprotectantproduction in wood frogs is supportedpri- 11.4, P < 0.001) among the treatment groups and were marily by liver glycogenolysis.In routine laboratorytests, the : :21.5, stronglycorrelated with glucoselevels (r2 0.51; F glycogenreserves are partially consumedduring freezing in P < 0.001; Fig. 3). Analysesof plasma from frogs in the frogs collected in autumn and winter (Storey and Storey saline group revealedthat freezing increasedconcentrations of 1986),whereas in spring-collectedfrogs, which are relatively : sodiumions (from 106.3+ l.3to 120.7* 2.1 mmol/L; F glycogenpoor, they are nearly exhausted(Storey and Storey (from 33.2, P < 0.001) and potassiumions 2.9 * 0.1 to 1987).In the presentinvestigation the size of the liver glyco- : 8.4 + 0.5 mmol/L; F 111.6,P < 0.001). gen reservesalso determinedthe amount depleted. The differ- ential utilization suggeststhat substrateavailability is not the Discussion sole factor limiting cryoprotectantproduction. Glycogencatabo- Cryoprotectant synthesis is a vital freezing responsecom- lism ultimatelymay be haltedby the progressiveaccumulation mon amongfreeze-tolerant anurans. Previous study suggested of ice andassociated osmotic imbalance within the liver. Addi- that the quantity of the cryoprotectant glucose present during tionally, high concentrations of glucose may interfere with freezinginfluences survival (Costanzo and Lee 1991;Costanzo glycogenolysis.Environmental constraints may alsobe impor- etaI. l99la), but the factorsgoverning R. sylvatica'scryopro- tant. For example, rapid (but not slow) freezing interferes with tectantsynthesis capacity were poorly understood.Our present glucoseproduction and mobilization, probably by hastening data suggestthat althoughthe size of the glycogenreserve is cardiovascularfailure (Costanzo et aI. 1992). 74 cAN. J. ZOOL.VOL.71. 1993

Glucose synthesisand plasma osmolality protectant synthesis and, hence, freeze tolerance. Other fac- Carbohydratecryoprotectants function colligatively to reduce tors, such as acclimatization, freezing regimen (e.g., cooling freezing injury to cells and tissues by regulating osmotic rate, exposure temperature, and duration), body size, and shrinkageand limiting the amount of ice forming at thermo- population differences may also be important. For example, equilibrium (Karow 1991). In our wood frogs, the glucose wood frogs collected in spring from Ontario (Storey and mobilized during freezing elevated blood osmolality, but Storey 1987) and southern Ohio (this study) achieved similar accountedfor only a portion of the total osmoticincrease. Data cryoprotectant concentrations (13 and 2l p.mollg liver, respec- in Fig. 3 suggestthat even in the absenceof glucoseproduc- tively) during freezrngto -2.5"C, although the former reduced tion, plasmaosmolality would haveincreased from25l mosmol their liver glycogen levels by only 171 pmollg, whereas the (in unfrozen, saline-injectedfrogs) to about 289 mosmol latter consumed 547 p"mollg. Although this discrepancy doubt- (y-interceptof the regressionequation correlating blood glu- less reflects differences in acclimatization, body compartment coseand osmolality).Thus, freezingalone increasedosmolal- size, and experimental protocol, its magnitude nevertheless ity by ca. 32 mosmol. Of this amount, about 20 mosmol suggestsan increase in the efficiency ofcryoprotectant synthe- reflectedincreases in Na+ and K+, which jointly accounted sis, favoring the more northerly conspecifics. Given the pro- for 42-44% of total osmolalityboth beforeand after freezing. nounced genotypic variability among R. sylvatica populations Lactate and alanine, which accumulatein frozen, ischemic (e.g., Weigt 1985), the prospect of genetically enhanced cryo- tissues (Storey and Storey 1986), likely contributed to the protectant production warrants further study. remaining12-mosmol increase. Because the relatively modest increasein plasma glucose, 13.3 pmollml., representsan Acknowledgments (13.3 equally modest increase mosmol) in blood osmolality, We thank T. Easterling, R. Knauff, and M. Wright for non-glucoseosmolites apparentlyplay an important role in aiding with the frog collections. P. Lortz provided technical particularly R. sylvatica'scryoprotectant system, in frogs hav- assistance and C. Chabot critically read the manuscript. ing low glucose-synthesiscapacities. Research was supported by a Charles A. Lindbergh grant to J.P.C. and National Institutes of Health grant No. 1 Rl5 Ecological implications DK-43958-01 and a grant from the Ohio Board of Regents Wood frogs having larger glycogen reservessynthesized Research Challenge to R.E.L. more cryoprotectant during freezing. This result has poten- tially significantimplications for freezingsurvival because the Canty, A., Driedzic, W. R., and Storey, K. B. 1986. Freezetoler- efficacy of glucose apparently dependson its concentration. ance of isolated ventricle strips of the wood frog, Rana sylvatica. For example,the quantityof ice forming in tissues,a primary Cryo-Lett.7:81-86. determinantof freezing survival (Karow 1991), is inversely Costanzo,J. P., and Lee, R. E. 1991. Freeze-thaw injury in related to glucose concentration (Layne and Lee 1990). erythrocytesof the freeze-tolerantwood frog, Rana sylvatica. Am. Furthermore, the level of cryoinjury can be decreasedby J. Physiol.261: Rl346-R1350. experimentally elevating the quantity of glucose present Costanzo,J. P., Lee, R. E., andWright, M. F. 1991a.Glucose load- (Costanzoand Lee l99l; Costanzoet al. 1991a). In wood ing preventsfreezing injury in rapidly cooled wood frogs. Am. frogs, therefore,larger glycogenreserves may confer greater J. Physiol.261: R1549-R1553. freeze tolerance. Costanzo,J. P., Lee, R. E., and Wright, M. F. 1991b.Effect of cooling rate on the survival of frozen wood frogs, Rana sylvatica. In someanurans liver glycogencontent varies geographically, J. Comp. Physiol.B, 161: 225-229. more northerly populationshaving relatively greaterreserves Costanzo,J. P., Lee, R. E., and Wright, M. F. 1992. Cooling rate prior to hibernation(Pasanen and Koskela 7974; Farrar and influences cryoprotectant distribution and organ dehydration in Frye 1973). Whether a similar latitudinalcline exists among freezingwood frogs. J. Exp. Zool. 26I: 3'73-378. R. sylvatica populations is unknown. Becausewood frogs Farrar, E. S., and Frye, B. E. l9'/3. Comparisonof blood glucose inhabitinghigh latitudesmust endure severe winters, this pros- and liver glycogen of larval and adult frogs (Rana pipiens). Gen. pect accordswith the likelihood of their need for a greater Comp. Endocrin.2l: 513-516. glucose-synthesiscapacity. Nevertheless, levels of glycogen, Jungreis,A. M., and Hooper, A. B. 1970. The effectsof long-term a primary metabolic fuel during hibernation, vary seasonally starvation and acclimation temperature on glucose regulation and pipiens. (e.g., Jungreisand Hooper 19101,Koskela and Pasanen1975; nitrogenanabolism in the frog, Rana L Winter animals. Comp. Biochem.Physiol. 32: 417-432. Smith 1950), and the reduced cryoprotectantproduction in Karow, A. M. 1991. Chemical cryoprotectionof metazoancells. (e.g., R. sylvatica in spring relative to autumn Storey and BioScience.41: 155-160. Storey 1987)likely reflectsdiminished glycogen stores. Because Kemp, A., and Kits van Heijningen,A. J. M. 1954. A colorimetric wood frogs encounterfreezing temperatureseven after emerg- micromethod for the determination of glycogen in tissues. ing from hibernation(e.g., Layne and Lee 1987),the greatest Biochem. l. 56: 646-648. risk of freezing injury may occur during spring, particularly Koskela,P., and Pasanen,S. 1975.Effect of thermalacclimation on while the frogs are abroadand en route to breedingponds. seasonalliver and muscle glycogencontent in the common frog, High interindividual variation in liver glycogen levels is Rana temporariaL. Comp. Biochem. Physiol. A, 50: 723-'72"1. characteristicof R. sylvatica, especiallyin spring. In frogs Layne, J. R., Jr., and Lee, R. 8., Jr. 198'7.Freeze tolerance and the from Ontario, Canada,for example,concentrations may range dynamics of ice formation in wood frogs (Rana sylvatica) from southernOhio. Can. J. Zool.65:2062-2065. from 13 to 643 pmollg (Storeyand Storey 1987).Such varia- Layne, J. R., and Lee, R. E. 1990.Glucose may reduceice forma- reflect in energy consumption tion may differences during tion in the freeze tolerant frog (Rana sylvatica). FASEB J. 4: winter or, perhaps,nutrient acquisitionduring the preceding ,4.552.(Abstr.) activeseason. Gender may alsoinfluence the level of glycogen Layne,J. R., Lee, R. E., andCutwa, M. W . 1992.Urine composi- reserves (Pasanenand Koskela 1974). Yariable glycogen tion of the wood frog Rana sylvatica following nonlethal freezing. reservesmay partly explain interindividualvariations in cryo- FASEBJ. 6: 41748. (Abstr.) r

COSTANZOAND LEE IJ

Mommsen, T. P., and Storey, K. B. 1992. Hormonal effects on Storey, K. 8., and Storey, J. M. 1986. Freezetolerant frogs: cryo- glycogen inetabolism in isolated hepatocytesof a freeze-tolerant protectants and tissue metabolism during freeze-thaw cycles. frog. Gen. Comp. Endocrin. ST: 44-53. Can.J. Zool.64;49-56. Pasanen,S., and Koskela, P. 1974. Seasonaland age variation ih the Storey, K. B., and Storey, J. M. 1987. Persistenceof freezetoler- metabolism of the common frog, Rana temporaria L. in northern ance in terrestrially hibernating frogs after spring emergence. Finland. Comp. Biochem. Physiol. A,47: 635-654. Copeia. 1987:720-726. Smith, C. L. 1950. Seasonalchanges in blood sugar,fat body, liver Weigt, L. A. 1985. Geographic variation in isozyme patterns in the gfycogen, and gonads in the common frog, Rana temporaria. wood frog, Rana sylvatica. M.S. thesis, Department of Zoology, J. Exp. Biol.26: 412-429. Miami University, Oxford, Ohio. Storey, K. B. 1990. Life in a frozen state: adaptive strategies for natural freeze tolerance in amphibians and reptiles. Am. J. Physiol.258: R559-R568.