R/F-84

Description:

The goals of this proposed research are to understand the permeability of teleost embryos to water and cryoprotectants in order to develop an uncomplicated, low-cost germ plasm cryopreservation system that may be applicable to many fish species. Traditional cryobiological techniques have been unsuccessful in developing successful approaches because they revealed little about the permeability of the embryos (a crucial element for successful cryopreservation). In order to increase our understanding of permeability, we will use magnetic resonance (MR) microscopy on zebrafish embryos. MR microscopy allows accurate quantification of cellular composition, such as cellular water and lipids, and their mobility, and permits direct visualization of dynamic processes, such as permeation of solutes across membranes. In the first year, we will examine the permeability of the various components of the embryo, such as the yolk and the blastoderm to various cryoprotectants at a variety of developmental time points. At 12 hr of development, the separate embryo components are permeable to one cryoprotectant, DMSO. However, the whole embryo is not permeable. Possibly the vitelline membrane which surrounds the yolk acts as a barrier to the cryoprotectant entry. We will use molecular poration techniques; that form transient aqueous pores; in membranes to try and increase permeability without causing developmental abnormalities. If the membrane can be permeabilized, then cryopreservation will become an attainable goal, and we will begin cryopreservation trials based on our results. Current cryopreservation methods do not work on teleost embryos. We believe we know why. ln examining the cryoprotectant permeability of teleost embryos, we have found that a layer develops between the yolk and the blastoderm - the yolk syncytial layer (YSL) that significantly hinders the movement of certain cryoprotectants into the yolk. This may help to explain why cryopreservation of fish embryos has failed in the past. This research will focus on methods to permeabilize the YSL so that cryoprotectants can enter the yolk. lf successful, these proposed experiments will set the stage for future cryopreservation trials. Specifically we will: (l) determine the type of cellular damage that occurs to zebrafish embryos during cryopreservation without cryoprotectants entering the YSL/yolk; (2) measure baseline cryoprotectant permeability of the YSL/yolk using sensitive magnetic resonance (MR) techniques which will establish the baseline concentrations for natural permeation and will be used to model biophysical membrane parameters, such as cryoprotectant permeability, Ps; and (3) examine whether pore-forming chemicals can increase cryoprotectant entry into the YSL/yolk and reduce cryodamage to cells.