Sequencing and Gene Expression of HC‐9, an Aquaporin‐9 Homolog, in Freeze‐tolerant Cope's Gray Treefrog

Abstract

Cope's gray treefrogs, Hyla chrysoscelis, accumulate glycerol in response to cold as part of their freeze tolerance mechanism. The glycerol is suspected to derive from glucose that is stored in the liver before it is exported to the bloodstream for distribution to other tissues. We therefore hypothesized that tissues from H. chrysoscelis, including hepatocytes, would express a homolog of aquaporin 9 (AQP9), a transmembrane protein from the aquaglyceroporin family that facilitates glycerol transport in mammalian liver. We also hypothesized that AQP9 would be up-regulated during cold acclimation to promote glycerol permeability. To test this hypothesis, we designed primers based on published AQP9 sequences, generated cDNA from mRNA extracted from liver, and identified a 945 base pair gene, HC-9, with high homology (69%) to human AQP9. Quantitative PCR analysis revealed HC-9 expression in liver, stomach, bladder, kidney, eye, lung, muscle, and skin (and at low levels in intestine, brain, heart, fat, and red blood cells). In tissues from frogs that had been cold acclimated, HC-9 was not up-regulated in any tissue and was down regulated in muscle and stomach, contrary to our hypothesis. In frozen and thawed animals, expression of HC-9 RNA mRNA was down-regulated in most tissues. Ongoing studies are characterizing the transport properties of HC-9 and the pattern of HC-9 protein expression in cold acclimation and freezing. Supported by NSF IOS-1121457.