R/A-02

Description:

Our proposed research is a basic study aimed at the cryopreservation of fish species. In the past, one of the main reasons fish embryos were thought difficult to cryopreserve was their large yolk. Our studies over the past 4 years, examining the yolk and the blastoderm using MR microscopy, suggest that the yolk interior is readily permeable. Rather, the yolk syncytial layer, which surrounds the yolk, may be the main barrier to allowing cryoprotectant permeation into the embryo (a critical element for successful cryopreservation). However, the impermeability of the zebrafish embryo's membranes may prove an insurmountable problem unless we can alter the embryo's membrane permeability. In contrast, the permeability of fish oocyte may help to solve this issue. This proposal will examine the permeability of fish oocytes to determine whether they might provide optimal candidates for cryopreservation. Fish oocytes gradually mature within the ovarian cavity and are permeable to the ovarian fluid. When extruded into a hyper- or hypotonic environment, mature oocytes face two challenges, becoming fertilized and avoiding osmotic shock. During fertilization, a calcium wave passing through the egg produces an impermeable oocyte. One goal in this project is to study approaches, such as calcium channel blockers, to prevent oocyte activation, so that oocytes remain permeable to water and cryoprotectants. A second goal is to determine if mature zebrafish oocytes can be fertilized in the presence of calcium blockers to prevent activation. These mature, fertilized permeable oocytes would be ideal candidates for cyropreservation. A third goal is to measure the water and cryoprotectant permeability and activation energy of the fertilized, permeable oocytes. These studies will form the theoretical foundation needed for formulating future cryopreservation protocols.