Freeze-tolerant species, such as wood frogs (Rana sylvatica), are susceptible to multiple co-occurring stresses that they must overcome to survive. Freezing is accompanied by mechanical stress and dehydration due to ice crystal formation in the extracellular space, ischemia/anoxia due to interruption in blood flood, and hyperglycemia due to cryoprotective measures. Wood frogs can survive dehydration, anoxia, and high glucose stress independently of freezing, thereby creating a multifactorial model for studying freeze-tolerance. Oxidative stress and high glucose levels favors the production of pro-oxidant molecules and advanced glycation end product (AGE) adducts that could cause substantial cellular damage. In this study, the involvement of the high mobility group box 1 (HMGB1)-AGE/RAGE (receptor for AGE) axis and the regulation of ETS1 and EGR1-mediated angiogenic responses were investigated in liver of wood frogs expose to freeze/thaw, anoxia/reoxygenation and dehydration/rehydration treatments. HMGB1 and not AGE-adducts are likely to induce the activation of ETS1 and EGR1 via the RAGE pathway. The increase in nuclear localization of both ETS1 and EGR1, but not DNA binding activity in response to stress hints to a potential spatial and temporal regulation in inducing angiogenic factors. Freeze/thaw and dehydration/rehydration treatments increase the levels of both pro- and anti-angiogenic factors, perhaps to prepare for the distribution of cryoprotectants or enable the repair of damaged capillaries and wounds when needed. Overall, wood frogs appear to anticipate the need for angiogenesis in response to freezing and dehydration but not anoxic treatments, probably due to mechanical stress associated with the two former conditions.

Wood frogs (Rana sylvatica) experience months of whole-body freezing during winter. Anoxia is one of the side stresses along with cell dehydration and hyperglycemia. Among multicellular organisms, communication and coordination is essential between neighbouring cells, particularly under stress conditions. Notch signaling is an effective communication channel between cells and regulates multiple pro-survival pathways. Signaling initiates when membrane-bound ligands Delta-like (DLL) or Jagged (JAG) interact with notch receptors. Activated receptor undergoes cleavage to release intracellular domain (NICD) in the cytoplasm. NICD translocates to the nucleus and forms a transcriptional complex with MAML and RBPJ that interacts with promoter regions and activates stress-specific genes. The role of notch signaling in enduring anoxia was assessed by studying the pathway components using immunoblots, TF-ELISA, and qPCR on treated samples of liver and heart. Bioinformatics tool Pymol was used to prepare structures based on available protein sequences for ligands, NOTCH receptor and the transcriptional complex. The results showed an increase in the levels of both ligands and receptors but decreased levels of RBPJ, suggesting an effective transmission of stress signal but suppressed gene transcription that goes in accordance with lowering energy expense required in energy crisis during anoxia. The study suggests Notch-independent activation of HES1 and HES5. Tissue-specific response of HES1, HES5, and MAML implies energy conservation and myocardial protection. The current study is the first analysis of the regulation of notch signaling in amphibians on encountering anoxic conditions that present multiple future directions.