NEW APPROACHES FOR STUDYING THE PERMEABILITY OF FISH EMBRYOS - TOWARDSUCCESSFUL CRYOPRESERVATION

This paper describes some new approaches for understanding the permeab ility of teleost embryos. The dechorionated zebrafish (Brachydanio rer io) was used as a model for basic studies of water and cryoprotectant permeability. These embryos are composed of two compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blas toderm cells. Cellular water was distributed unequally in each compart ment. Measurements indicated that the total water in the embryo was 74 %, while the total water in the yolk was 42%, and total water in the b lastoderm was 82%. The internal isosmotic value for the zebrafish embr yo is unknown. However, for one-compartment modeling studies of membra ne permeability, the mean Lp (+/-SEM) values were 0.022 +/- 0.002 to 0 .049 +/- 0.008 mu m x min(-1) atm(-1) at 40 mOsm (assuming this was on e possible internal isosmotic value for the entire embryo) and 0.040 /- 0.004 to 0.1 +/- 0.017 mu m x min(-1) atm(-1) at 300 mOsm (assuming this was another possible internal isosmotic value for the entire emb ryo). When three- and six-somite embryos were placed in 1.5 and 2.0 M cryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurements of volume changes indicated no cryoprotectant permeation. However, similar measurements with methanol revealed a small volume d ecrease (ca. 8%) and recovery (ca. 5%) for six-somite embryos in a 2.0 M solution. Magnetic resonance (MR) images of the spatial distributio n of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that only methanol permeated the entire embryo within 15 min. The other cryoprotectants exhibited little or no permea tion into the yolk over 2.5 h. The results from MR spectroscopy and cr yoprotectant microinjections into the yolk suggested that the yolk syn cytial layer plays the critical limiting role for cryoprotectant perme ation throughout the embryo. (C) 1997 Academic Press.