Cryobehavior of Drosophila embryos

ABSTRACTS, 24th ANNUAL

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able freeze-induced dehydration. When dechorionated embryos were cooled in the DSC, discrete exothermic events were observed over the range of - 15 to - 35°C Ultrarapid cooling of 1 mm3 samples of freshly dis- with median temperatures of -28°C when cooled at sected rat liver, kidney, skeletal muscle and a cultured 2”Clmin and -31°C when cooled at i6”Clmin. cell line was achieved by the technique of metal mirror Warming of frozen embryos in the absence of external cooling utilizing liquid helium and resulting in partially water and measurement of the enthalpy of transition vitrified samples. Power compensated differential allowed us to determine the average volume of freezscanning calorimetry (DSC) was used to follow able water in a single egg to be -5.4 nl, with no signifithermal changes in the samples on warming from cant difference between native and dechorionated em- 196°C. Three parameters were compared: the onset bryos. For embryos that were dechorionated and pertemperature (Ti) of the first thermal event, the glass meabilized by solvent extraction of the eggcase, the transition temperature (Tg) and the temperature of the TIIF,, was cooling-rate-dependent over portions of melt (Tm). All samples yielded Ti between - 150°C the cooling range of 0.5 to 16’Cimin. When the perand - 160°C while Tg and Tm varied in a tissue/cell meabilized embryos were suspended in BD.20 and specific manner. Parallel mass spectrometric analysis cooled at O.S”C/min, freeze-induced dehydration was of deuterium oxide-treated tissue samples showed the observed by cryomicroscopy in the majority of the evolution of D,O in the Tiregion. DSC and mass spec- embryos. However, intracellular ice formation octrometry analysis provide confirmatory data sug- curred in -50% of the embryos with a TIIFsO of - 7°C. gesting that thermal changes indicative of either or The incidence of intracellular ice formation reached both glass transitions and/or devitrification are taking 100% at 2”C/min, although the TIFF,, remained unplace in the samples at temperatures nearly 30°C changed. A pair-wise comparison of TIIF, indicated a lower than the published glass transition temperature significant difference between cooling rates @X/min for water. and 2 16”C/min. At the slower cooling rates the range over which intracellular ice formation occurred was 22. Cryobehavior of Drosophila Embryos. STANLEY rather narrow (e.g., -4 to - 10°Cat l”C/min), while at P. MYERS, DANIEL V. LYNCH, RONALD E. faster cooling rates the range increased considerably Prrr, AND PETERL. STEPONKUS(Departments (e.g., -6” to -29°C at 64”Cimin). Treating the permeabilized embryos with cryoprotectants (i.e., DMSO of Agronomy and Agricultural Engineering, or ethylene glycol) decreased the TIFF,. When emCornell University Ithaca, New York). bryos were suspended in ethylene glycol and cooled at Cryomicroscopy and differential scanning calorimetry 4”Cimin the TIIFS, was -21.5”C in 1.0 M, -28°C in (DSC) were used to determine the incidence of intra1.5 M, and -33.5”C in 2.0 M. For embryos equilicellular ice formation in embryos of Drosophila me- brated in BD.20 + 1.0 M DMSO (1.42 total osm) the lanogaster (Oregon R strain P2) as influenced by de- TIFF,, was - - 24°C regardless of the cooling rate chorionation and solvent permeabilization of the egg- over the range of 1 to 16”Cimin. However, at all case and, in the case of permeabilized embryos, as a cooling rates the range over which IIF occurred was function of cooling rate and cryoprotectants. When very broad (e.g., -4 to -45°C at l”C/min). For em12- to 13-hr embryos with the chorion intact were sus- bryos suspended in Ringer’s solution + 1.0 M ethpended in Drosophila basic cell culture medium ylene glycol the TIIF, was cooling rate dependent. BD.20 (0.26 osm, pH 6.8) and cooled at 4”C/min, in- The TIIF,, of embryos cooled at 4”C/min was - 22°C tracellular ice formation occurred over a very narrow while those cooled at 16”Cimin had a TIIF, of - 29°C. temperature range (-26 to - 30°C) with a median Although both DMSO and ethylene glycol decreased temperature for intracellular ice formation (TIIF,,) of the TIFF,, they did not lower the incidence of IIF -28°C. When cooled at 16”C/min, the TIFF,, was (-50%) at the slowest cooling rate investigated (i.e., -29°C. At both cooling rates, there was no observ- O.S”C/min). (Supported by a grant from the United able freeze-induced dehydration of the embryos. Exo- States Department of Health and Human Services, thermic transitions distinguishable from the freezing National Institute of General Medical Sciences Grant of external water and thought to reflect intracellular 1 ROl GM37575-01.) ice formation in embryos were observed by DSC. Discrete exothermic events corresponding to the freezing 23. The Kinetics of Heterogeneous Nucleation of Ice of individual eggs occurred over the range of -27 to by Silver Iodide-a Calorimetric Study. S. - 32°C for embryos cooled at either 2”Cimin or 16°C CHAROENREINAND D. S. REID (Department of min. Following dechorionation of the embryos by Food Science and Technology, University of treatment with 50% clorox, intracellular ice formation California, Davis, California 95616). occurred over a broader range (- 13 to - 30°C) with a The kinetics of both homogeneous and heterogeTIIF,, of -24°C when cooled at 4”C/min and -28°C when cooled at 16”C/min. Again, there was no observ- neous nucleation of ice in an emulsion of water The University of Texas Health Science Center at Houston, The Woodlands, Texas, 77380).