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Frozen Frogs: Expert Q&A

  • Posted 04.01.05
  • NOVA scienceNOW

For a week in April 2005, Jon Costanzo answered questions about frozen frogs as well as aspects of cryopreservation and its hoped-for uses in medicine.

Jon Costanzo

Jon Costanzo

Jon Costanzo, Ph.D., is a professor of zoology and a researcher at Miami University in Ohio. Full Bio

Photo credit: Courtesy of Dr. Jon Costanzo

Jon Costanzo

Jon Costanzo, Ph.D., is a professor of zoology and a researcher at Miami University in Ohio. His research in Miami's Laboratory for Ecophysiological Cryobiology investigates reptiles and amphibians that live in harsh climates, especially cold ones. Dr. Costanzo's studies of amphibian freeze tolerance recently spawned a project aimed at cryopreserving amphibian germ cells for captive breeding. Another of Dr. Costanzo's recent projects, currently funded by the National Science Foundation, focuses on the role of urea in freeze tolerance and metabolic depression in hibernating wood frogs.

Q: What is the scientific name of the wood frog? I would like to research it. Fascinating! Wes Jensen

Jon Costanzo: This small, handsome frog is Rana sylvatica, so named for its affinity for wooded habitats. It's commonly found in moist forests across the continent and has the distinction of being the most northerly ranging of all North American amphibians. Some populations are found above the Arctic Circle! It's also the most terrestrial of all North American ranids, the large group of "true frogs," which include the bullfrog and other common pond frogs. In addition to surviving extreme cold, this tough little frog tolerates a good measure of dehydration, no doubt an adaptation to its life away from water. For more information about the wood frog's winter ecology and physiology, please see our Web page: http://www.units.muohio.edu/cryolab/projects/VertCryobiology.htm

Q: Do the hearts of frozen frogs beat on the order of "days" or "weeks" while frozen? Or does the heart beat only when the frog is not frozen (or, perhaps, slightly thawed)? We were confused as to whether or not there are absolutely no heartbeat or metabolism while frozen or just a greatly slowed heartbeat. ("Just" is not the right word, as even a weekly or monthly beat is just amazing!) Anonymous

Costanzo: The heart rate of a wood frog, or any ectothermic ("cold-blooded") animal, strongly depends on its body temperature. When the frog's temperature falls to near the freezing mark, the heart pulses slowly (only a few beats per minute), but quite regularly, and blood is circulated throughout the body. In fact, the heart continues to beat for many hours after the frog's tissues have begun to freeze. This is important because the cryoprotectant, glucose, is made in the liver and must be circulated to cells throughout the body.

However, later in the freezing process the heart stops completely. Ice encases the organ and forms inside the muscle and the chambers. There's no need for a working pump now because the blood, too, is frozen. This state of arrested heart function can be tolerated for many days and perhaps months, but upon thawing the contractions spontaneously resume. Imagine our amazement when we witnessed those first few blips on a thawing frog's EKG!

Q: Hi, Jon. I was wondering if the same mechanism is possible in human beings? I mean, we've all seen the movies where people are frozen and preserved, then allowed to "thaw" and carry on normally many years later. But is this possible? Is there something about the frog's anatomy that allows it to do this?

Thanks, Anonymous

Costanzo: Currently it's not possible to revive frozen humans, and the technical difficulties of adding and withdrawing cryoprotectants and of achieving controlled freezing and thawing of a large, intact body could be insurmountable. However, that's not to say that a greater variety of human tissues and organs couldn't be successfully cryopreserved. Cryomedical research has historically relied on mammalian models for experimentation, even though these tissues do not naturally experience cold. The wood frog and several other amphibians and reptiles obviously have "solved" not only the problem of freezing individual tissues and organs, but also that of simultaneously freezing all organ systems. Who knows what might be achieved if we learn more about natural mechanisms of freeze tolerance and use this information to guide efforts to cryopreserve human tissues and organs?

Q: I was watching the segment about frozen frogs and was intrigued to hear that the thawed out little frogs were ready for a lady frog so quickly after their ordeal. You implied that they were mating. I never knew that frogs actually mated. I knew that they practiced "amplexus," in which the male hangs on to the female for dear life until such time as she decides to lay her eggs. He then dismounts and fertilizes them in the water. Is this true with wood frogs? Please explain. Rick

Costanzo: The wood frog breeds earlier than any other frog—typically mid-February in Ohio, but even earlier in the South—so it's possible that they thaw out just hours or days before appearing at their breeding ponds. They begin mating immediately and the congregation disperses just a few days later. Although we casually refer to the reproductive act as "mating" or "breeding," this needn't imply that fertilization is internal. You are absolutely correct in that the pair engages in "amplexus," a clasping embrace that serves to properly align their bodies. However, note that sperm are shed at the same time the female releases her eggs, not afterwards.

Q: How do the cryoprotectants (glucose, ethylene glycol, etc.) keep water from leaving the cells? Jill Vernes

Costanzo: Great question, complex answer! First, realize that when a frog freezes, ice forms only in the spaces outside the cells, never inside them. Second, recall that ice is relatively pure and is formed of water but no solutes. Now, when the solution outside the cells gradually freezes, the salts and other solutes there become quite concentrated. This condition creates an osmotic imbalance across the water-permeable cell membrane and, as you might imagine, the pressure differential is relieved when water inside the cell flows outside.

However, if too much water leaves, the cell shrinks excessively and the plasma membrane may fail. In fact, this dynamic—not mechanical damage from jagged ice crystals—is thought to be a primary mechanism of freezing injury to tissues. Cryoprotectants (glucose and glycerol are used by frogs) help cells retain water by 1) colligatively reducing the quantity of ice that forms; and 2) increasing the intracellular solute concentration and thereby limiting the osmotic pressure differential.

Q: Do the brain or other organs of the wood frog use oxygen when the frog is frozen? Charles Byrd

Costanzo: During the early stages of freezing the blood continues to deliver oxygen to the tissues. However, once freezing progresses and the circulation fails, the cells must survive without oxygen. They do this by using anaerobic glycolysis, an energy-producing metabolic pathway that does not require oxygen. Lactate, the primary end product of anaerobic glycolysis, accumulates in the blood of frozen frogs. Although little ATP is produced this way, the energy demand in frozen frogs is greatly depressed.

Q: Have any genes been identified in the mechanism of this freezing? Anonymous

Costanzo: Freeze tolerance is a complex adaptation that involves a host of responses at multiple levels of biological organization. Recent studies have shown that freezing/thawing is associated with patterns of altered expression of various genes. The protein products of these genes probably enable cells to cope with the various stresses imposed by freezing/thawing, including cell dehydration, membrane perturbation, oxygen deprivation, and reoxygenation. This work has largely relied on cDNA microarray screening and the screening of cDNA libraries. One of our graduate students is using 2-D gel electrophoresis to identify proteins contributing to freeze tolerance in the hatchling painted turtle.

Q: Could I induce a wood frog to freeze for students in my classroom with an ice cube? Anonymous

Costanzo: If your ice cube is an ordinary chunk of frozen water and you remove it from the freezer, it will quickly warm to its melting point, 0°C. Touch the frog with that and you've only made him chilly. Because the frog's tissues contain dissolved salts and other solutes, they won't freeze until the frog has cooled to approximately -0.6°C.You'd need to keep the frog at this temperature, or below, for it to freeze.

But please don't attempt to demonstrate freeze tolerance to your students by placing a frog in a household freezer. The temperature there, typically -18° to -20°C, is much too cold and the frog will die. Instead, you might demonstrate the phenomenon of natural freeze tolerance using larvae of the goldenrod gall fly (Eurosta solidaginis), which can survive exposure to -40°C! We offer a Goldenrod Gall Fly Lab (high school level) and other educational materials on our Web page: http://www.units.muohio.edu/cryolab/education/AntarcticLessons.htm

Q: Are only wood frogs able to undergo freezing or are other kinds of frogs as well, say, Cope's tree frog and the eastern gray tree frog? Also, is there any physical way (aside from sound) to differentiate the two species I named? I have read that the answer is no but I wanted to ask an expert. E. Ulry

Costanzo: Aside from the wood frog, several tree frogs are freeze-tolerant, including the spring peeper (Pseudacris crucifer), the chorus frog (Pseudacris triseriata), and the two species you mentioned. All of these frogs share something in common: they are northern species that hibernate beneath duff on the forest floor, well within the reach of frost. Freeze tolerance in the eastern gray treefrog (Hyla versicolor) was discovered by William D. Schmid in 1982. Our laboratory reported freeze tolerance in Cope's tree frog (Hyla chrysoscelis) a decade later.

Morphologically, the two species are indistinguishable. However, I recall from that project that Cope's tree frog, a diploid species, has much smaller red blood cells than the tetraploid H. versicolor, and we used that characteristic to confirm our species identification.

Q: Is there a difference in the life span of wood frogs that freeze and wood frogs that don't freeze? Jeanne

Costanzo: There isn't much known about the natural life span of wood frogs, but it's likely that they live only a few years. Although freezing undoubtedly stresses the animals, there's no evidence that freezing impacts longevity. Interestingly, we once studied the reproductive behavior of male frogs and found that freezing diminished their mating performance. After thawing, the frogs weren't as vocal or active, and they seemed to have more difficulty recognizing potential mates. Individuals that somehow avoid freezing may have greater mating success.

Q: Is there a minimum temperature from which the frog will not revive? Bob, Hickory, North Carolina

Costanzo: There is a minimum survival temperature for wood frogs and all other freeze-tolerant animals. This temperature depends on several variables, such as season, thermal acclimation, physiological condition, and rate of freezing. For the frogs in our study population in southern Ohio, the critical minimum temperature is approximately -4 to -5°C. Frogs cooled below this temperature do not recover, probably because too much ice has formed within their tissues.

Q: How far has research gone in cryobiology with respect to organ transplant and freezing organs for prolonged time? Ali, Pickering College, Grade 11
Ontario, Canada

Costanzo: Unfortunately, progress in this area has been slow. Currently, human organs harvested for transplants are not frozen but are kept briefly in cold-storage solutions.

Some cryobiologists believe that the study of naturally freeze-tolerant amphibians and reptiles, which share many physiological characteristics with humans, can provide important clues to the development of methods for cryopreserving and banking human organs. Several studies with rat hearts have shown promising results. In those experiments, rat hearts recovered function after being frozen with cryoprotectants and stored frozen at -3.4°C for up to six hours.

Q: Would it be accurate to say that the frog is "dead" during its frozen state—that is, if the same level of nonactivity was observed in a human, would doctors diagnose the patient as deceased? Keith Kerr, Texas A&M University

Costanzo: Clinical death refers to the medical state involving the complete and irreversible cessation of all body functions. Commonly it is equated with brain death, although in emergency medical care, clinical death occurs when heartbeat and respiration have ceased.

Fully frozen wood frogs show no heartbeat or (pulmonary) breathing, so they could be considered "clinically dead." Because there are no published reports concerning the brain function of freezing frogs, we don't know whether or not this criterion is satisfied. Nevertheless, in the strictest sense the designation fails because the cessation of body functions is entirely and spontaneously reversible.

Q: How durable are these frogs in the frozen state? Could they withstand being stepped on and still survive? Paul Hile, Monroe High School, Grade 9
Monroe, Michigan

Costanzo: That's a tough question! Frozen or not, frogs are delicate animals and they can't tolerate much compression. As I'm sure you know, unfrozen frogs are fairly elastic and pliable. When frozen, their bodies become rigid and possibly more prone to mechanical damage. However, whether a frozen frog could survive being stepped on depends on how much force is applied to it. In nature, being crushed in this way is probably not a common problem because the surrounding soil, which is also frozen, absorbs some of the force.

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