A two-step method for permeabilization of Drosophila eggs

Abstract

As a first step in developing a procedure for the cryopreservation of Drosophila melanogaster embryos, we have established a method for permeabilization of the eggcase and have initiated studies of the hydraulic conductivity of permeabilized embryos and the permeation of selected cryoprotective agents. The eggcase of D. melanogaster embryos has a wax layer that precludes any flux of water. A two-step procedure employing organic solvents was developed to effect removal of the wax layer with minimal deleterious effects on the embryos. Dechorionated embryos (Oregon-R strain P2, 12 to 13 hr old) were rinsed sequentially in isopropanol and hexane. After removal of solvent, embryos were held in a modified cell culture medium for further manipulation. This procedure routinely yielded 80 to 95% of the eggs permeabilized (as determined by osmotic contraction in 1 M sucrose) and 75 to 90% survival (incidence of hatching). Hydraulic conductivity of permeabilized embryos and permeation of cryoprotectants were determined using a microdiffusion chamber and computerized video microscopy. Regression analysis of the volumetric data from individual embryos yielded the Boyle-van't Hoff function FV eq = 0.124 (osm −1) + 0.541 with the standard deviations of slope and intercept ( V b) being 0.010 and 0.040, respectively. Permeabilized embryos exhibited ideal osmotic behavior over the range of 0.265 to 2.00 osm. The mean hydraulic conductivity coefficient ( L p) was 0.722 ± 0.366 (μm/(min · atm) at 20 °C, based on observations of contraction following a step change in concentration of Ringer's solution. The time course for the transient volumetric change of permeabilized embryos subjected to a step change to isotonic Drosophila Ringer's solution containing 1 M DMSO, ethylene glycol, propylene glycol, or glycerol demonstrated that all four cryoprotectants permeated the embryos. DMSO permeated embryos at the highest rate, followed closely by ethylene glycol and propylene glycol, while glycerol permeated very slowly.