The long range goal is to cryopreserve teleost embryos successfully, specifically zebrafish embryos. The availability of viable embryos after cryopreservation could have a profound influence on the conservation of rare or threatened species and medical research. The zebrafish has become one of the more important vertebrate models for the study of development and genetics. The preservation of their mutant and transgenic lines is essential, because these lines will play an important role in future studies on human health and disease. However, fish embryos have never been cryopreserved successfully. The applicants believe they know why. For successful cryopreservation, water must exit, and an appropriate cryoprotectant enter all the cells. A major permeability barrier in the zebrafish embryo: the YSL, which develops between the yolk and blastodermhas been found by the investigators. Due to its low permeability, the YSL blocks water exit from, and entry of some cryoprotectants into, the yolk. Cryopreservation destroys the YSL, presumably because ice-crystals form in the yolk. Although, this YSL-barrier can be overcomed by directly microinjecting cryoprotectant into the yolk, two problems remain: 1) removing water from the large yolk compartment, and 2) preventing trapped cryoprotectant within the yolk from becoming teratogenic. To cryopreserve fish embryos successfully, water and cryoprotectants must permeate the tissues more readily. It is proposed to increase permeability by introducing pore-forming MIPs into the membranes of zebrafish embryos.
The research Aims are: 1) to examine molecular techniques to increase the zebrafish membranes permeability to water and cryoprotectants by introducing MIPs, specifically by injecting RNA from the water channels (AQP1 and AQP3) and the glycerol facilitator (GlpF); and 2) to develop vitrification methods to cryopreserve somatically modified and normal cryoprotectant-injected embryos. Vitrification is a preferred cryopreservation method because it can circumvent the zebrafish's sensitivity to chilling. Modifying somatic cells in fish embryos with the RNA from MIPs may enhance their permeability. In preliminary studies, somatic cells in zebrafish embryos have been successfully altered with AQP1, increasing the water permeability 67%. Although these preliminary results show great promise, further study is needed on the introduction and optimization of different types of MIPs, their analysis, and use for vitrification. Most importantly, these somatic changes will not affect the germ line, so they are ideal for genomic banking to maintain important genetic stocks for biomedical and endangered fish research.
| Hagedorn, M; Peterson, A; Mazur, P et al. (2004) High ice nucleation temperature of zebrafish embryos: slow-freezing is not an option. Cryobiology 49:181-9 |
| Hagedorn, M; Lance, S L; Fonseca, D M et al. (2002) Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation. Biol Reprod 67:961-6 |
| Janik, M; Kleinhans, F W; Hagedorn, M (2000) Overcoming a permeability barrier by microinjecting cryoprotectants into zebrafish embryos (Brachydanio rerio). Cryobiology 41:25-34 |
