• Studies on the effect of chilling on sox genes and protein expression in zebrafish (Danio rerio) embryos

      Desai, Kunjan; University of Bedfordshire (University of BedfordshireUniversity of Bedfordshire, 2012)
      In aquaculture, short term chilled storage has been used to transport brood stock fish embryos for genetic improvement programmes. It is therefore important to understand the effect of chilling on embryos at both developmental and molecular levels. In the present study, gene expression patterns in zebrafish embryos were studied before investigations were carried out on the effect of chilling on gene and protein expression in these embryos. The gene expression results obtained in different developmental stages using conventional PCR showed that, only sox genes were expressed throughout the tested developmental stages from 30% epiboly to 6 somites. Quantitative RT-PCR was then used to investigate sox gene expression patterns during chilling of 50% epiboly stage embryos at 0°C for up to 180 min and also after warming. Significant decreases in sox2 and sox3 expressions were observed when compared to those of controls following chilling whilst significant increases of expressions of the two genes were observed after warming in the embryos chilled for 30 and 60 min. Studies on the impact of cryoprotectant MeOH on sox genes and protein expression showed that 50% epiboly stage zebrafish embryos could tolerate chilling for up to 6 h with or without MeOH. It was observed that expression of all three sox genes were significantly decreased following chilling for 3 h at 0°C. However the degree of decrease was less pronounced in embryos chilled with different concentrations of MeOH. Significant increases in sox genes were observed in hatching stage embryos chilled with 1 M MeOH for 3h but subsequent sox2 and sox19a protein expression was not affected. The effect of long term chilling (18h) on sox gene and protein expression in 50% epiboly stage embryos was also investigated. Improved hatching rates (56% ± 5) were achieved when embryos were chilled with 1 M MeOH + 0.1 M sucrose. Results from gene expression studies showed a stable sox2 gene expression in 18 h chilled embryos in cryoprotectant mixture when compared to that of embryos chilled without cryoprotectant mixture. Similar patterns were observed when the expression of sox2 and sox3 protein was investigated. This is the first study carried out on the effect of chilling in early stage zebrafish embryos at the molecular level. The results obtained from the present study provided useful information on the molecular mechanisms of the effect of chilling on zebrafish embryos and will have important implications in designing chilled storage protocols for fish embryos.