Anti-apoptotic and antioxidant defenses in the freeze tolerant wood frog, Rana sylvatica.
Multiple biochemical adaptations support natural freeze tolerance by wood frogs, Rana sylvatica. The present research explored the role of anti-apoptotic and antioxidant defenses in organ survival of freeze/thaw stresses using PCR and Western blotting to analyze the expression of selected genes and proteins. The STAT family of transcription factors mediate both pro- and anti-apoptotic gene responses. Elevated amounts of phosphorylated (active) Stat5 (Tyr694) and/or phospho-Stat3 (Ser727) in selected frog organs during freeze/thaw suggest activation of anti-apoptotic defenses to help organs recover from metabolic insults caused by freezing. However, levels of phospho-Stat1 (Tyr701), a pro-apoptotic signal, also rose in kidney and muscle during thawing. Increased amounts of anti-apoptotic proteins including Bcl-2 and phospho- Bcl-2 (Ser70) in liver and skeletal muscle and Bcl-xL (and phospho-Bcl-2) in kidney could help counteract freeze-induced apoptotic signals that were evidenced by higher levels of Bad protein (liver, muscle, kidney) and phospho-Bad (Ser112) (kidney) and enhanced DNA laddering. Antioxidant defense via glutathione S-transferase (GST) was evaluated by analyzing the expression of GST isozymes. GST Pi protein rose in four organs during freeze/thaw and GST Pi mRNA was freeze up-regulated in liver. GST M1/2, M5, A3 and A5 were freeze- or thaw- responsive in selected organs. Freeze-induced changes in the transcription factors, Nrf2 and MafG, and elevated MafG in the nucleus suggest that these regulate the freeze up-regulation of antioxidant enzymes by targeting the antioxidant response element of genes. Both anti-apoptotic and antioxidant defenses are important aspects of natural freezing survival.