Wood frog (Lithobates sylvaticus) and its cryoprotection mechanism

One of the most fascinating survival strategies that some individuals in the animal kingdom have developed, is without a shadow of doubt, freezing tolerance. Among vertebrates, the ability to withstand full body freezing is extremely rare, we can partially observe this ability in some species of amphibians, such as wood frog (Lithobates sylvaticus), an animal widely distributed in North America and considered the main experimental model for studying the molecular, biochemical and physiological mechanisms involved in this process (STOREY, 2017). 

From the moment that the extracorporeal temperature decreases, specialized proteins induce the formation of ice crystals in extracellular spaces, preventing the abrupt freezing of cells, and avoiding the rupture of membranes (LARSON et al., 2014). During freezing, the animal’s nervous system signals the liver to turn its glycogen reserves into a large amount of glucose, which will be transported to all tissues of the body, increasing the plasma level and regulating the loss of fluid from cells (STOREY and STOREY, 1986). 

Vital signs are interrupted, such as heartbeat, breathing and brain activity, entering a state of anoxia supported by the drastic reduction in metabolism. Some enzymes are modulated to operate at low temperatures, minimizing the consumption of ATP and avoiding the accumulation of toxic metabolites (COSTANZO et al., 2013; LARSON et al., 2014), also observed a significant increase in molecular chaperones (proteins) and antioxidant enzymes, acting uniquely in the stabilization and neutralization of free radicals during the freezing and thawing process, as well as the anoxia itself (STOREY and STOREY, 2013).

Figure 1:(A) Lithobates sylvaticus, frozen and thawed. (B) The graph shows how the concentration of glucose in various tissues (blood, liver, skeletal muscle, heart and kidney) changes over a period of freezing and thawing. Source: J. M. Storey. 

Urea is one of the main cryoprotectors in the freezing of the wood frog, it accumulates in the intracellular spaces, just before the extracorporeal temperature falls, mostly in winter periods. This accumulation acts significantly in the elevation of osmolarity of body fluids, highlighting the depression of the freezing point and restricting the nucleation of ice crystals inside the cells, which contributes to the preservation of membrane integrity, an effect analogous to that conferred by compounds such as glucose and glycerol (STOREY, et al., 2021).

Thawing begins from the periphery to the body center of the animal, occurring in a short time. It is observed, in this period, the return of heart rate and systemic tissue reperfusion (Layne & First, 1991). This process, which occurs gradually and in a coordinated manner, is essential to prevent ischemic lesions and ensure the physiological integrity of tissues. Mitochondrial recovery is also essential at this time, with rapid reactivation of the respiratory chain and controlled increase in oxygen consumption, both factors that prevent excessive oxidative stress and protect cells from the uncontrolled formation of oxygen-reactive species (STOREY, et al., 2021). 

Given the complexity of the physiological processes involved in the survival of the wood frog to freezing, it becomes evident the high degree of adaptive sophistication of this species facing the extreme conditions of the North American winter. The ability to almost completely interrupt metabolism, preserve cellular structure and function during extracellular ice formation and resume full physiological activity after thawing reveals a highly integrated set of biochemical, molecular and mitochondrial mechanisms. With the understanding of how Lithobates sylvaticus prevents cell damage even in conditions of almost total metabolic stop, science approaches to replicate such strategies in cryopreservation in clinical and technological contexts in the conservation of tissues at extreme cold, a transformative potential for human health and biological conservation.

Author: Marcelo Oliveira Loiola - GEAS Brazil Deputy Director of Dissemination.

 August/2025 Wild Panel.

BIBLIOGRAPHICAL REFERENCES: 

1. STOREY, J. M.; WU, S.; STOREY, K. B. Mitochondria and the frozen frog. Antioxidants, v. 10, p. 543, 2021. DOI: 10.3390/antiox10040543.

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5. LARSON, D. J.; MIDDLE, L.; VU, H.; ZHANG, W.; SERIANNI, A. S.; DUMAN, J.; BARNES, B. M. Wood frog adaptations to overwintering in Alaska: new limits to freezing tolerance. The Journal of Experimental Biology, v. 217, Pt 12, p. 2193–2200, 2014. DOI: 10.1242/jeb.101931  .

6. COSTANZO, J.; DO AMARAL, M. C.; ROSENDALE, A.; LEE, R. H. Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog. The Journal of Experimental Biology, v. 216, Pt 18, p. 3461–3473, set. 2013. DOI: 10.1242/jeb.089342  .

7. LAYNE, J. R. Jr.; FIRST, M. C. Resumption of physiological functions in the wood frog (Rana sylvatica) after freezing. American Journal of Physiology, v. 261, n. 1 Pt 2, p. R134–R137, jul. 1991. DOI: 10.1152/ajpregu.1991.261.1.R134.