WATER DISTRIBUTION AND PERMEABILITY OF ZEBRAFISH EMBRYOS, BRACHYDANIO-RERIO

Teleost embryos have not been successfully cryopreserved. To formulate successful cryopreservation protocols, the distribution and cellular permeability to water must be understood. In this paper, the zebrafish (Brachydanio rerio) was used as a model for basic studies of the dist ribution to permeability to water. These embryos are a complex multi-c ompartmental system composed of two membrane-limited compartments, a l arge yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells (each surrounded by a plasma membrane). Due to the c omplexity of this system, a variety of techniques, including magnetic resonance microscopy and electron spin resonance, was used to measure the water in these compartments. Cellular water was distributed unequa lly in each compartment. At the 6-somite stage, the percent water (V/V ) was distributed as follows: total in embryo = 74%, total in yolk = 4 2%, and total in blastoderm = 82%. A one-compartment model was used to analyze kinetic, osmotic shrinkage data and determine a phenomenologi cal water permeability parameter, L-p, assuming intracellular isosmoti c compartments of either 40 or 300 mosm. This analysis revealed that t he membrane permeability changed (P < 0.05) during development. During the 75% epiboly to 3-somite stage, the mean membrane permeability rem ained constant (L-p = 0.022 +/- 0.002 mu m x min(-1)atm(-1) [mean +/- S.E.M.] assuming isosmotic is 40 mosm or L-p = 0.049 +/- 0.008 mu m x min(-1)atm(-1) assuming isosmotic is 300 mosm). However, at the 6-somi te stage, Lp increased twofold (L-p = 0.040 +/- 0.004 mu m x min(-1)at m(-1) assuming isosmotic is 40 mosm or L-p = 0.100 +/- 0.017 mu m x mi n(-1)atm(-1) assuming isosmotic is 300 mosm). Therefore, the low perme ability of the zebrafish embryo coupled with its large size (and conse quent low area to volume ratio) led to a very slow osmotic response th at should be considered before formulating cryopreservation protocols. (C) 1997 Wiley-Liss, Inc.