Intracellular ice formation in mouse zygotes and early morulae vs. cooling rate and temperature-experimental vs. theory.
Intracellular ice formation in mouse zygotes and early morulae vs. cooling rate and temperature-experimental vs. theory.
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小鼠受精卵和早期桑葚中的细胞内冰形成与冷却速率以及温度实验与理论。
DOI：
10.1016/j.cryobiol.2016.07.014
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发表时间：
2016-10
期刊：
影响因子：
2.7
通讯作者：
Mazur P
中科院分区：
文献类型：
--
作者：
Jin B;Seki S;Paredes E;Qiu J;Shi Y;Zhang Z;Ma C;Jiang S;Li J;Yuan F;Wang S;Shao X;Mazur P
在这项研究中，诱导ICR品系的成熟雌性小鼠超数排卵，交配，并在受精卵或早期桑椹胚阶段收集。将胚胎悬浮于含10 mg/L Snomax的PBS中的1 M乙二醇中15 min，然后将其转移至样品保持器中的Linkam冷冻台，冷却至7 °C并在7 °C下接种，然后以0.5-20 °C/min的速度冷却至−70 °C进行观察和拍照。观察到细胞内冰形成（IIF）为突然的“闪烁”。在这项研究中观察到两种类型的闪烁或IIF。细胞外冷冻发生在平均-7.7 °C。在桑椹胚中，约25%在细胞外冰形成（EIF）的±1 °C内变暗。我们称之为“高温”闪光灯。受精卵中无高温闪光细胞。所有受精卵在远低于EIF温度的温度下闪光。据推测，高温闪蒸器是EIF之前膜损坏或EIF损坏的结果。我们将不再进一步讨论它们。在大多数情况下，IIF发生在-7.7 °C以下;我们称之为“低温”闪光器。在受精卵或桑椹胚中，冷却速率（CR）为0.5 °C/min时，均未出现闪光。几乎所有的闪光与CR 4 °C/min或更高，但温度的分布是更广泛的桑椹胚比受精卵。此外，桑椹胚的平均闪光温度（−20.9 °C）比受精卵（−40.3 °C）高得多。我们计算了水分流失的动力学相对于CR和温度在小鼠受精卵和桑椹胚的基础上发表的估计Lp和它是Ea。由此产生的脱水曲线结合胚胎成核温度的知识允许IIF的可能性作为CR和零度以下温度的函数的估计。这些计算的概率和观测值之间的协议是好的。
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.