I know what you’re thinking. What in the world does a tree frog have to do with humans freezing their organs? And yes, before beginning my undergraduate research project, I too would likely be thinking this exact same thought. But nature allows animals to do a very funny thing: adapt according to their environment. And the gray tree frog that you are itching to read about in the following paragraphs has adapted in one of the most extreme ways found in the animal kingdom: it has the ability to freeze itself and survive.

Credit: John White, University of California, Berkeley

My 2017 summer research project, funded through the American Physiological Society, is focused on Cope’s gray tree frog, Hyla chrysoscelis. H. chrysoscelis has developed a dramatic strategy to combat the cold during the winter months, known as freeze tolerance. Freeze tolerance permits this tree frog to withstand the cold by allowing it to convert 50-65% of its total fluid body water into ice. During its time spent frozen, Cope’s gray tree frog experiences fundamental changes in its physiology and biochemistry, including the abandonment of:

• Blood circulation
• Breathing
• Nerve conduction
• Brain activity
• A heartbeat

Yes, you read that right. It has no heartbeat while it is frozen.

However, as wintertime comes to a close and external temperatures rise, the thawing process begins, vital signs reactivate, and normal life resumes for this frog. My research project for the summer is aimed at understanding a specific cellular process involved in the whole body freezing of H. chrysoscelis, which may provide future scientists with the capacity to not only freeze human organs in the future, but also freeze our entire bodies.

H. chrysoscelis experiences a variety of stresses during freezing and thawing, most of them being osmotic stresses. This is because the majority of the fluid surrounding the frog’s cells freezes, leaving a higher concentration of solutes outside the cell than prior to freezing. To combat this disruption of fluid homeostasis, H. chrysoscelis has evolved a transmembrane protein known as aquaglyceroporin HC-3 that acts as a channel for water and solutes to flow through, which helps to re-establish a fluid homeostatic equilibrium. In non-science terms, this protein, HC-3, helps to keep the cells of this frog from shriveling up like raisins or bursting open like a balloon with too much air.

My hypothesis demands a week long experiment, involving a variety of techniques that enable me to begin with the blood of this frog, fresh from its brachial artery, and shrink all the way down to the molecular level to analyze HC-3, the protein that I suspect to be a vital player in the freezing process. The first part of my experiment requires culturing red blood cells from Hyla chrysoscelis over a period of 48 hours, which in non-science terms translates to: keep 90% of these cells alive over a period of two days. Sounds simple, right? Well, after the first two weeks of failed attempts in maintaining proper viability of the cells in culture, I was left devastated, demoralized, and with numerous flasks of cells contaminated with who-knows-what. Science, without mercy, and in just a period of two weeks, showed me just how difficult it would be to successfully complete my week long experiment by halting me after just the first two days.

As the summer has progressed, I have finally achieved viable cell cultures for the full 48 hours through troubleshooting and controlling for one variable at a time, leading me to the conclusion that a certain reagent I was adding to the media my cells were kept in had been contaminated earlier in the summer. Currently, I am now focusing on more demanding techniques, such as western blotting and immunocytochemistry, while still always encountering new obstacles to overcome and problems to solve. New to research, and even newer to researching full time, I have come to enjoy the independence and autonomy that I’ve found in the laboratory. Every day is a new opportunity, and a new challenge, demanding my full attention and effort to successfully complete my experiments for the day. Through the few successes and many failures of my summer of research thus far, I have gained a deeper appreciation for science and continue to aspire to engage in scientific research throughout the rest of my professional career.

References

1. White, John. “Hyla chrysoscelis, Cope’s Gray tree frog.” University of California, Berkley Regents (2006).

Dante Pezzutti is a rising senior majoring in Pre-Medicine at the University of Dayton in Dayton, Ohio. He is a 2017 Undergraduate Summer Research Fellow (UGSRF) working in Dr. Carissa Krane’s laboratory at U.D. over the summer. Dante’s fellowship is funded through the American Physiological Society. After graduation, Dante plans to attend medical school to become a physician, and also aspires to engage in bench-to-bedside translational research as a medical doctor.