Abstract:
Oxygen (O2) is fundamental to the survival of organisms. Exposure to low O2, or hypoxia, causes adverse effects to humans. The brain, in particular, is sensitive to changes in O2 concentration, resulting in harmful conditions such as stroke. Some non-human animals are capable of experiencing extreme hypoxia without injury. Tripterygiidae (NZ triplefin fish) present an interesting study group as some species within this highly related family are extremely hypoxia-tolerant while others are not. Animals utilise different mechanisms to achieve hypoxia-tolerance, one of which is by upregulation of the hypoxia-inducible factors (HIF), in particular, HIF-1α. HIF-1α is a transcription factor that is rapidly degraded in conditions with sufficient O2 by a prolyl hydroxylase enzyme (PHD). The main aim of this thesis was to determine what, if any, differences in HIF-1α regulation exist between two triplefin species disparate in hypoxia-tolerance, Forsterygion varium, a hypoxia-sensitive species, and Bellapiscis medius, a hypoxia-tolerant species. The question was attempted to be answered by detection and quantification of HIF-1α protein, and development of a PHD enzyme assay. Both assays used brain samples from triplefin fish subjected to differing O2 states: normoxia, hypoxia exposure of one hour at 20 % of atmospheric O2, or anoxia exposure until loss of equilibrium. Detection of HIF-1α was achieved by western blotting using a mammalian antibody against triplefin brains which showed positive results. However, no differences in HIF-1α protein were discovered between the two species, under any of the different O2 states utilised in this study. Measurement of PHD involved the detection of a key substrate of the enzyme, 2-oxoglutarate (2OG). 2OG was found to be in higher concentrations following acute anoxia compared to the other O2 states. The rate of change of the substrate over the 30-minute reaction was calculated for PHD enzyme activity, and here, no differences between species or O2 stated was found. These findings indicate that there may be some change in the availability of PHD substrate with a complete lack of O2, however, for both acute anoxia and one hour of hypoxia, HIF-1α protein is not differentially regulated in F. varium and B. medius brains.
