|dc.description.abstract||Oxidative stress, an imbalance in the cellular redox systems, could be a major influence on organism development. To investigate this possibility I carried out basic research into the antioxidant systems in the zebrafish developmental model. Of interest were ways of measuring stress, ways of inducing stress, the molecular pathways involved, and the developmental consequences for the zebrafish.
Peroxiredoxin 3 (Prdx3), the protein responsible for peroxide reduction in the mitochondia, was investigated as an indicator of oxidative stress in zebrafish. The custom antibody used in this study was capable of detecting recombinant zebrafish Prdx3 in both reduced and oxidized states. In zebrafish protein extracts the oxidized dimer ran at 37 kDa while the reduced monomer ran at 23 kDa. In addition, there was also a 15 kDa small band detected in zebrafish extracts that might represent a Prdx3 cleavage product. Immunodetection of Prdx3 redox state in zebrafish protein extracts has potential as a way of measuring the level of mitochondrial oxidative stress in vivo.
Several approaches were taken to characterise the functional role of Prdx3 in the developing zebrafish embryo. Insight was gained from in situ hybridisation which showed the prdx3 transcript to be expressed in the developing haematopoietic system. Knockdown of Prdx3 in zebrafish could have allowed the function of Prdx3 to be tested in zebrafish. Unfortunately, no knockdown of Prdx3 was observed in zebrafish embryos following morpholino injection. To this end custom TALENs were constructed capable of targeting the prdx3 locus but a mutant line has not yet been established.
The level of oxidative stress during embryogenesis is a largely unexplored influence on adult phenotype and subsequent generations. To induce oxidative stress in the embryo, the chemical auranofin (AFN) was used to inhibit antioxidant defences. Static embryonic exposure to AFN resulted in developmental defects, including cerebral haemorrhaging at high doses and jaw malformation at lower doses. Oxidative stress was increased in embryos exposed to AFN, as evident by the DNA damage resulting from the 5 μM dose and the increased redox state observed with the 1 μM dose. Embryos generated a transcriptional response as early as 1 dpf after AFN exposure beginning at 6 hpf. The most responsive transcripts were prdx1 and gstp1 which in response to 5 μM AFN were increased over 3 and 7 fold by 1 dpf, respectively.
To investigate the long-term consequences of oxidative stress, embryos exposed to the low 0.5 μM AFN dose between 6 and 24 hpf were raised to adulthood. Both cohorts 1 and 2 showed lower odds of breeding success following the AFN treatment. Within this AFN treatment group the breeding success was lower in outcrosses of treated females. Fertilization rate was also decreased in incrosses of treated fish suggesting an effect on both males and females. Males from the treatment arms had a trend towards less motile sperm, potentially contributing to the reduced fertilization rate. There was no difference in the incidence of developmental defects in the offspring from the cohort treatment arms.
Research into the mechanisms of environmentally-induced phenotypic change, and its transmission between generations, requires models in which to do so. This study contributes a basis for undertaking longitudinal cohort studies in the zebrafish. In addition, the findings support the idea that the environmental conditions during development can have long-term effects on adult health. This thesis will serve as a useful reference for hypothesis generation and the design of future studies aiming to investigate environmental effects on development and inheritance in the zebrafish model.||