Characterization of a Major Permeability Barrier in the Zebrafish Embryo¹

^a Reproductive Physiology Program, National Zoological Park, Smithsonian Institution, Washington, District of Columbia 20008 ^b Physics Department, IUPUI, Indianapolis, Indiana 46202 ^c Radiology Department, Johns Hopkins School of Medicine, Baltimore, Maryland 21205

Fish embryos represent a class of multicompartmental biological systems that have not been successfully cryopreserved, primarily because of the lack of understanding of how water and cryoprotectants permeate the compartments. We are using the zebrafish embryo as a model to understand these kinetics. Zebrafish embryos have two major compartments, the blastoderm and the yolk, which is surrounded by the multinucleated yolk syncytial layer (YSL). We determined the water and cryoprotectant permeability in these compartments using two methods. First, we measured shrink/swell dynamics in optical volumetric experiments. Zebrafish embryos shrank over time and did not re-expand while immersed in dimethyl sulfoxide (DMSO) or propylene glycol. Second, we measured DMSO uptake with diffusion-weighted nuclear magnetic resonance spectroscopy. DMSO uptake was rapid during the first few minutes, then gradual thereafter. We used one- and two-compartment models to analyze the data and to determine the permeability parameters. We found that the two-compartment model provided a better fit to the data. On the basis of this model and in the presence of DMSO, the yolk and blastoderm had very similar water permeabilities (i.e., 0.01 and 0.005 µm x min^-1atm^-1, respectively), but they had different DMSO permeabilities separated by three orders of magnitude (i.e., 5 x 10^-6 and 1.5 x 10^-3 cm/min, respectively). The low solute permeability of the yolk predicted that the yolk/YSL compartment should be more susceptible to cryodamage. To test this, the yolk, blastoderm, and YSL were examined at the ultrastructural level after vitrification. Only the YSL incurred significant damage after freezing and thawing (p 0.05).

¹ Supported by grants from the National Institutes of Health (R29 RR08769) (M.H.), and Maryland Sea Grant College (M.H.), and from IUPUI for a Sabbatical Year Award (F.W.K.), and the Smithsonian Institution for a Smithsonian Short-Term Visitor Award (F.W.K.).

² Correspondence: M. Hagedorn, National Zoological Park, Smithsonian Institution, 3001 Conn. Ave., NW, Washington, DC 20008. FAX: 202 673 4733; mhagedorn{at}nzp.si.edu

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