Although north Australian winters are extremely mild by any standards(around 20°C), Australian crocodiles still have to adjust to these chilly conditions relative to their scorching summers. The ability of ectothermic animals to remain functional and active when exposed to cold environmental temperatures requires a suite of typical physiological adjustments, known as the cold acclimation response. Among these adjustments, mitochondrial levels increase in order to maintain energy production when reduced temperatures slow biochemical reactions. This led a University of Sydney team, Tonia Schwartz,Shauna Murray and Frank Seebacher, to investigate whether the improved metabolic capacity resulting from cold acclimation is associated with protective mechanisms against reactive oxygen species (ROS), a possibly damaging by-product of increased metabolic capacity. The group first hypothesised that mitochondrial uncoupling proteins (UCPs) found in other vertebrates are also present in reptiles, given their suggested role in ROS protection. Second, the authors proposed that the expression of the genes coding for these proteins would increase when crocodiles are exposed to winter temperatures.
Initially, the researchers had to find out whether UCPs are expressed in crocodiles, as they had not been reported in reptiles previously. Using UCP gene sequence information from various vertebrates, the team designed primers that would target and amplify all UCP genes and found that crocodiles apparently express three UCP genes. Next the team began to study the evolution of this gene family by performing a phylogenetic analysis of the crocodile UCP genes relative to UCP gene sequences from organisms ranging from plants to fish, amphibians, birds and mammals. They found that one of the three crocodile UCP genes is homologous to the vertebrate UCP2 gene, and two cloned genes are homologous to vertebrate UCP3. Like birds,crocodiles do not appear to have UCP1 homologues; however, the two UCP3 genes group with the birds' single UCP gene, agreeing with their shared common ancestor.
While it is unclear what the functional roles of UCP2 and UCP3 are, the cold acclimation experiment provides a good starting point to explore their potential protective role against ROS. After acclimating crocodiles to temperatures mimicking winter and summer conditions for a month, the team collected liver and skeletal muscle samples to measure the level of the reptile's UCP genes using quantitative real-time PCR. When exposed to colder winter conditions, crocodiles up-regulate the expression of UCP2 and UCP3 in their liver, but not their skeletal muscles. The authors'previous work also showed that the activity of some Australian crocodile mitochondrial enzymes increases in the liver during cold acclimation, in agreement with the increase in mitochondrial levels.
In accordance with their hypotheses, the team have shown that UCP gene expression increases when ectothermic reptiles are acclimated to the cold. The correlated changes in mitochondrial oxidative capacity, which would lead to increased ROS production in liver, also suggest the potential role of UCPs in protection against ROS. The functional basis of these observations remains to be clarified. Nevertheless, the increase in UCP gene expression in response to cold might be another component of the cold acclimation response in ectotherms.
