71 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 (erreurtype), 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 substancecryoprotec- trice 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