title: Intracellular ice formation in mouse zygotes and early morulae vs. cooling rate and temperature-experimental vs. theory.
abstract: In this study, mature female mice of the ICR strain were induced to superovultate, mated, and collected at either zygote or early morula stages. Embryos suspended in 1 M ethylene glycol in PBS containing 10 mg/L Snomax for 15 min, then transferred in sample holder to Linkam cryostage, cooled to and seeded at 7 °C, and then observed and photographed while being cooled to -70 °C at 0.5-20 °C/min. Intracellular ice formation (IIF) was observed as abrupt ''flashing''. Two types of flashing or IIF were observed in this study. Extracellular freezing occurred at a mean of -7.7 °C. In morulae, about 25% turned dark within ±1 °C of extracellular ice formation (EIF). These we refer to as "high temperature'' flashers. In zygotes, there were no high temperature flashers. All the zygotes flashed at temperatures well below the temperature for EIF. Presumably high temperature flashers were a consequence of membrane damage prior to EIF or damage from EIF. We shall not discuss them further. In the majority of cases, IIF occurred well below -7.7 °C; these we call ''low temperature'' flashers. None flashed with cooling rate (CR) of 0.5 °C/min in either zygotes or morulae. Nearly all flashed with CR of 4 °C/min or higher, but the distribution of temperatures is much broader with morulae than with zygotes. Also, the mean flashing temperature is much higher with morulae (-20.9 °C) than with zygotes (-40.3 °C). We computed the kinetics of water loss with respect to CR and temperature in both mouse zygotes and in morulae based on published estimates of Lp and it is Ea. The resulting dehydration curves combined with knowledge of the embryo nucleation temperature permits an estimate of the likelihood of IIF as a function of CR and subzero temperature. The agreement between these computed probabilities and the observed values are good.
authors.affiliation: Fundamental and Applied Cryobiology Group, Reproductive and Genetic Medicine Center, Dalian Municipal Women and Children's Medical Center, Dalian, Liaoning 116031, China. Electronic address: jinboknox@sina.com.
authors.forename: Bo
authors.initials: B
authors.lastname: Jin
authors.affiliation: Division of Animal Sciences, Bioscience Education and Research Support Center, Akita University, Akita 010-8543, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Seki
authors.affiliation: Fundamental and Applied Cryobiology Group, Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, USA.
authors.forename: Estefania
authors.initials: E
authors.lastname: Paredes
authors.affiliation: Fundamental and Applied Cryobiology Group, Reproductive and Genetic Medicine Center, Dalian Municipal Women and Children's Medical Center, Dalian, Liaoning 116031, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Qiu
authors.affiliation: Fundamental and Applied Cryobiology Group, Reproductive and Genetic Medicine Center, Dalian Municipal Women and Children's Medical Center, Dalian, Liaoning 116031, China.
authors.forename: Yanbin
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Fundamental and Applied Cryobiology Group, Reproductive and Genetic Medicine Center, Dalian Municipal Women and Children's Medical Center, Dalian, Liaoning 116031, China.
authors.forename: Zhenqiang
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Fundamental and Applied Cryobiology Group, Reproductive and Genetic Medicine Center, Dalian Municipal Women and Children's Medical Center, Dalian, Liaoning 116031, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Ma
authors.affiliation: Fundamental and Applied Cryobiology Group, Reproductive and Genetic Medicine Center, Dalian Municipal Women and Children's Medical Center, Dalian, Liaoning 116031, China.
authors.forename: Shuyan
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Fundamental and Applied Cryobiology Group, Reproductive and Genetic Medicine Center, Dalian Municipal Women and Children's Medical Center, Dalian, Liaoning 116031, China.
authors.forename: Jiaqi
authors.initials: J
authors.lastname: Li
authors.affiliation: Fundamental and Applied Cryobiology Group, Reproductive and Genetic Medicine Center, Dalian Municipal Women and Children's Medical Center, Dalian, Liaoning 116031, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Yuan
authors.affiliation: Fundamental and Applied Cryobiology Group, Reproductive and Genetic Medicine Center, Dalian Municipal Women and Children's Medical Center, Dalian, Liaoning 116031, China.
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
authors.affiliation: Fundamental and Applied Cryobiology Group, Reproductive and Genetic Medicine Center, Dalian Municipal Women and Children's Medical Center, Dalian, Liaoning 116031, China. Electronic address: xiaoguangshao03@sina.com.
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Shao
authors.affiliation: Fundamental and Applied Cryobiology Group, Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Mazur
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D005615:Freezing; D007053:Ice; D007700:Kinetics; D051379:Mice; D008813:Mice, Inbred ICR; D009028:Morula; D013696:Temperature; D015053:Zygote
chemical_list: D003451:Cryoprotective Agents; D007053:Ice; D019855:Ethylene Glycol
keywords: Cooling rate; Extracellular ice formation; High temperature flasher; Intracellular ice formation; Low temperature flasher