Characterization of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopy and differential scanning calorimetry (DSC) were used to characterize the incidence of intracellular ice formation (IIF) in 12- to 13-hr-old embryos of Drosophila melanogaster (Oregon-R strain P2) as influenced by the state of the eggcase (untreated, dechorionated, or permeabilized), the composition of the suspending medium (with and without cryoprotectants), and the cooling rate. Untreated eggs underwent IIF over a very narrow temperature range when cooled at 4 or 16 °C/min with a median temperature of intracellular ice formation (TIIF 50) of −28 °C. The freezable water volume of untreated eggs was approximately 5.4 nl as determined by DSC. IIF in dechorionated eggs occurred over a much broader temperature range (−13 to −31 °C), but the incidence of IIF increased sharply below −24 °C, and the cumulative incidence of IIF at −24 °C decreased with cooling rate. In permeabilized eggs without cryoprotectants (CPAs), IIF occurred at much warmer temperatures and over a much wider temperature range than in untreated eggs, and the TIIF 50 was cooling rate dependent. At low cooling rates (1 to 2 °C/min), TIIF 50 increased with cooling rate; at intermediate cooling rates (2 to 16 °C/min), TIIF 50 decreased with cooling rate. The total incidence of IIP in permeabilized eggs was 54% at 1 °C/min, and volumetric contraction almost always occurred during cooling. Decreasing the cooling rate to 0.5 °C/min reduced the incidence of IIF to 43%. At a cooling rate of 4 °C/min, ethylene glycol reduced the TIIF 50 by about 12 °C for each unit increase in molarity of CPA (up to 2.0 M) in the suspending medium. The TIIF 50 was cooling rate dependent when embryos were preequilibrated with 1.0 M propylene glycol or ethylene glycol, but was not so in x1.0 M DMSO. For embryos equilibrated in 1.5 M ethylene glycol and then held at −5 °C for 1 min before further cooling at 1 °C/min, the incidence of IIF was decreased to 31%. Increasing the duration of the isothermal hold to 10 min reduced the incidence of IIF to 22% and reduced the volume of freezable water in embryos when intracellular ice formation occurred. If the isothermal hold temperature was −7.5 or −10 °C, a 10- to 30-min holding time was required to achieve a comparable reduction in the incidence of IIF.