title: Critical factors affecting the permeabilization of Drosophila embryos by alkanes.
abstract: Because of waxes in the vitelline membrane, the Drosophila egg is effectively impermeable to liquid water and to aqueous solutes, and consequently it cannot be cryopreserved unless it can be permeabilized. The more successful of the few published permeabilization procedures involve the removal of the chorion mechanically or by hypochlorite solution, the removal of all surrounding water by air drying or alcohol, the exposure of eggs to pure alkanes like octane or hexane for some 30 s, the removal of the alkane and the transfer of the eggs to aqueous culture medium without their desiccation, and lastly incubation of the permeabilized embryos under mineral oil. In following these procedures we opted for a somewhat different approach to applying hypochlorite, water, alcohol, and alkane; namely, eggs were placed between two Nucleopore filters, and the fluids drawn sequentially through the filters by vacuum. Extensive initial attempts were mystifying and discouraging in that although permeabilization was good, survivals were poor, and modifications that increased the latter reduced the former. The explanation turned out to be that permeabilization and survival depended critically on the amount of carry-over alcohol that contaminated the alkane. To determine the effects of alcohol concentration in the alkane, it was essential first to effectively eliminate carry-over contamination and then re-add precise amounts of alcohol (isopropanol) to the alkane (n-hexane, heptane, or octane). When the alcohol concentration is less than or equal to 0.2%, permeabilization is poor; when it is greater than or equal to 0.5%, permeabilization is good but survival (hatching) is poor. There are strong interactions between alcohol concentration and exposure time to alkane/alcohol mixtures with respect to the fraction of embryos that become permeabilized and the percentage that survive. There are also significant but less critical effects from the type of alcohol and alkane. The best results for 12-h embryos (greater than or equal to 90% permeabilization and 70-80% hatching) were achieved with eggs exposed to 0.3 or 0.4% 1-butanol in n-heptane for 90 s. High survivals of permeabilized 12-h embryos did not require incubation under mineral oil. Permeabilized embryos are permeable to water, ethylene glycol, glycerol, and the stain rhodamine B (which was used to assess permeabilization). They are effectively impermeable to sucrose. Embryo age is important. Between 14 and 16 h the above permeabilization procedures become dramatically less effective.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: University of Tennessee--Oak Ridge Graduate School of Biomedical Sciences 37831-8077.
authors.forename: P
authors.initials: P
authors.lastname: Mazur
authors.forename: K W
authors.initials: KW
authors.lastname: Cole
authors.forename: A P
authors.initials: AP
authors.lastname: Mahowald
mesh_terms: D000388:Air; D000438:Alcohols; D000473:Alkanes; D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004331:Drosophila melanogaster / Q000196:embryology; D005069:Evaluation Studies as Topic; D005260:Female; D010063:Ovum; D010539:Permeability; D013997:Time Factors
chemical_list: D000438:Alcohols; D000473:Alkanes; D003451:Cryoprotective Agents