• Use of methanol as cryoprotectant and its effect on sox genes and proteins in chilled zebrafish embryos

      Desai, Kunjan; Spikings, Emma; Zhang, Tiantian (Elsevier, 2015-08)
      Methanol is a widely used cryoprotectant (CPA) in cryopreservation of fish embryos, however little is known about its effect at the molecular level. This study investigated the effect of methanol on sox gene and protein expression in zebrafish embryos (50% epiboly) when they were chilled for 3h and subsequently warmed and cultured to the hatching stages. Initial experiments were carried out to evaluate the chilling tolerance of 50% epiboly embryos which showed no significant differences in hatching rates for up to 6h chilling in methanol (0.2-, 0.5- and 1M). Subsequent experiments in embryos that had been chilled for 3h in 1M methanol and warmed and cultured up to the hatching stages found that sox2 and sox3 gene expression were increased significantly in hatched embryos that had been chilled compared to non-chilled controls. Sox19a gene expression also remained above control levels in the chilled embryos at all developmental stages tested. Whilst stable sox2 protein expression was observed between non-chilled controls and embryos chilled for 3h with or without MeOH, a surge in sox19a protein expression was observed in embryos chilled for 3h in the presence of 1M MeOH compared to non-chilled controls and then returned to control levels by the hatching stage. The protective effect of MeOH was increased with increasing concentrations. Effect of methanol at molecular level during chilling was reported here first time which could add new parameter in selection of cryoprotectant while designing cryopreservation protocol.