Department of Biology, University of Dayton, Dayton, Ohio.
Department of Biology, Mount St. Joseph University, Cincinnati, Ohio.
J Exp Zool A Ecol Integr Physiol. 2019 Jun;331(5):308-313. doi: 10.1002/jez.2262. Epub 2019 Apr 1.
Dryophytes chrysoscelis (formerly Hyla chrysoscelis, Cope's gray treefrog) is a freeze-tolerant anuran that accumulates glycerol and urea during cold acclimation and freezing. It is hypothesized that glycerol and urea function as cryoprotectants by minimizing osmotically induced cell damage during freezing and thawing, thereby improving the postfreeze viability of red blood cells (RBCs) when frozen in medium containing those solutes. To test this, erythrocytes were obtained from warm (22°C) and cold-acclimated (4°C) frogs and suspended in 280 mOsM phosphate-buffered saline (PBS). RBCs were frozen in 280 mOsM, isosmotic/isotonic, PBS, or in PBS made hyperosmotic by addition of 150 mM solutes. Postfreeze viability was determined with a hemolysis assay. Postfreeze viability of cells from warm-acclimated frogs improved from 18.9 ± 1.3% in PBS to 47.4 ± 5.2% in PBS with urea ( p < 0.01). The addition of other solutes (glycerol, glucose, NaCl, or sorbitol) had no effect. RBCs from cold-acclimated frogs had 45.8 ± 3.4% viability when frozen in 280 mOsM PBS, and this improved to 71.6 ± 8.9% or 71.9 ± 1.6%, respectively, when frozen with glycerol ( p < 0.01) or urea ( p < 0.001). The viability of RBCs from cold-acclimated frogs was not different between unfrozen cells 86.7-88.4%) and those frozen with glycerol (71.6 ± 8.9%, p > 0.05) or with urea (71.9 ± 1.6%, p > 0.05). These data suggest that (a) cold acclimation induces cellular changes in RBCs that result in improved postfreeze viability, and (b) glycerol and urea are part of a complex cryoprotectant system in D. chrysoscelis.
雨滨蛙（原称斑腿树蛙，Cope's 灰色树蛙）是一种耐寒的两栖动物，在寒冷适应和冷冻过程中积累甘油和尿素。据推测，甘油和尿素通过在冷冻和解冻过程中最小化渗透压引起的细胞损伤，作为冷冻保护剂发挥作用，从而提高在含有这些溶质的介质中冷冻时红细胞（RBC）的冷冻后存活率。为了验证这一点，从温暖（22°C）和寒冷适应（4°C）的青蛙中获得红细胞，并悬浮在 280 mOsM 磷酸盐缓冲盐水（PBS）中。将 RBC 在 280 mOsM、等渗/等张、PBS 或添加 150mM 溶质使其高渗的 PBS 中冷冻。用溶血试验测定冷冻后存活率。来自温暖适应的青蛙的细胞在 PBS 中的冷冻后存活率从 18.9±1.3%提高到 PBS 中添加尿素时的 47.4±5.2%（p<0.01）。添加其他溶质（甘油、葡萄糖、NaCl 或山梨糖醇）没有影响。在 280 mOsM PBS 中冷冻时，来自寒冷适应的青蛙的 RBC 具有 45.8±3.4%的存活率，当用甘油（p<0.01）或尿素（p<0.001）冷冻时，该存活率分别提高到 71.6±8.9%或 71.9±1.6%。来自寒冷适应的青蛙的 RBC 的存活率在未冷冻细胞（86.7-88.4%）和用甘油（71.6±8.9%，p>0.05）或尿素（71.9±1.6%，p>0.05）冷冻的 RBC 之间没有差异。这些数据表明：（a）寒冷适应会引起 RBC 中的细胞变化，从而提高冷冻后的存活率；（b）甘油和尿素是雨滨蛙复杂冷冻保护剂系统的一部分。
