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Altering Fish Embryos with Aquaporin-3: An Essential Step Toward Successful Cryopreservation¹

^a Smithsonian Institution, National Zoological Park and Conservation and Research Center, Washington, District of Columbia 20008 ^b Department of Entomology, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910-7500 ^c Department of Physics, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202 ^d Department of Radiology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205 ^e Smithsonian Institution, National Zoological Park, Department of Zoological Research and Molecular Genetics Laboratory, Washington, District of Columbia 20008 ^f Laboratory of Plasma Derivatives, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 ^g Department of Biology, Georgetown University, Washington, District of Columbia 20057

Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.

First decision: 23 January 2002.

¹ This work was supported by grants to D.F. and R.F. from Friends of the National Zoo, to B.P. from the National Institutes of Health (K01 RR00135), and to M.H. from the National Institutes of Health (R01 RR08769), by the Maryland Sea Grant College, and by a private donation from Mrs. S. Sichel.

² Correspondence: M. Hagedorn, Department of Reproductive Sciences, National Zoological Park, Smithsonian Institution, 3001 Connecticut Ave., NW, Washington, DC 20008. FAX: 202-673-4377; hagedornm{at}nzp.si.edu

³ These authors contributed equally to this work

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