Antifreeze protein gene expression in winter flounder pre-hatch embryos: implications for cryopreservation

Cryopreservation of fish embryos has proven to be an elusive goal. Two reasons for this lack of success are their high chilling sensitivity and the formation of ice crystals while in the frozen state or during the thawing process. Antifreeze proteins (AFP) that protect marine teleost fishes from freezing in subzero waters have been shown to be capable of inhibiting ice recrystallization and protecting cell membranes from cold induced damage. Therefore they have the potential to improve the success of embryo cryopreservation. A recent study demonstrated that vitrified winter flounder embryos continued to show developmental changes following thaw [V. Robles, E. Cabrita, G.L. Fletcher, M.A. Shears, M.J. King, M.P. Herráez, Vitrification assays with embryos from a cold tolerant sub-arctic fish species, Theriogenology 64 (2005) 1633-1646]. Since winter flounder produce AFP it was hypothesized that these proteins, if present in the embryos, could have contributed to this progressive step towards success. Winter flounder produce three species of type 1 AFP: a small liver type, a large "hyperactive" liver type and a skin type. This study was conducted to determine which, if any, of these AFP genes was being expressed in pre-hatch winter flounder embryos. There was no evidence of AFP activity in freshly fertilized embryos. However, low levels of AFP activity were found in embryos at 4, 8, and 11 days post-fertilization. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of the AFP mRNA isolated from the embryos revealed the expression of seven different skin type AFP genes that translated into four distinct AFP. Neither of the liver type AFP genes was expressed in the embryos.