The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing
The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing
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DOI：
10.1093/humrep/15.5.1125
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发表时间：
2000-05-01
影响因子：
6.1
通讯作者：
Bischof, JC
中科院分区：
文献类型：
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作者：
Devireddy, RV;Swanlund, DJ;Bischof, JC
人类精子冷冻保存的坚实生物物理基础因缺乏在细胞外冰和冷冻保护剂（CPA）存在下冷冻过程中水分渗透特性的相关知识而受限。由于人类精子的形状极不规则且尺寸微小，处于光学显微镜分辨率的极限，冷冻显微镜无法用于测量人类精子冷冻过程中的脱水情况。利用一种新的与形状无关的差示扫描量热仪（DSC）技术，在细胞外冰和CPA存在的情况下，以5℃/min和10℃/min的冷却速率获得了人类精子细胞悬浮液在冷冻过程中的体积收缩情况。利用先前发表的数据，将人类精子细胞建模为长度为40.2μm、半径为0.42μm的圆柱体，其渗透不活跃细胞体积V - b为0.23Vₒ，其中Vₒ是等渗细胞体积。通过将水运输模型与实验获得的体积收缩数据进行拟合，确定了最佳拟合的膜通透性参数（Lₚ₉和E - Lₚ）。在改良培养基中，人类精子细胞在5℃/min和10℃/min时的“综合最佳拟合”膜通透性参数为：Lₚ₉ = 2.4×10⁻¹⁴ m³/Ns（0.14μm/min - atm），E - Lₚ = 357.7 kJ/mol（85.5 kcal/mol）（R² = 0.98）；在CPA培养基（含6%甘油和10%蛋黄）中为Lₚ₉[CPA] = 0.67×10⁻¹⁴ m³/Ns（0.04μm/min - atm），E - Lₚ[CPA] = 138.9 kJ/mol（33.2 kcal/mol）（R² = 0.98）。这些参数与先前在零上温度以及在无细胞外冰的零下温度下获得的人类精子参数显著不同。本研究中获得的参数还表明，根据CPA的浓度不同，在低至25 - 45℃/min的冷却速率下，人类精子细胞中可能会发生有害的细胞内冰晶形成（IIF）。这可能有助于解释使用先前发表的未考虑细胞外冰存在的零上人类精子通透性参数所确定的最佳冷冻保存冷却速率（7000℃/min）之间的差异。
A firm biophysical basis for the cryopreservation of human spermatozoa is limited by a lack of knowledge regarding the water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPA). Cryomicroscopy cannot be used to measure dehydration during freezing in human spermatozoa because of their highly non-spherical shape and their small dimensions which are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of human sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPA. Using previously published data, the human sperm cell was modelled as a cylinder of length 40.2 mu m and a radius of 0.42 mu m with an osmotically inactive cell volume, V-b, of 0.23V(o), where V-o is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (L-pg and E-Lp) were determined. The 'combined best fit' membrane permeability parameters at 5 and 10 degrees C/min for human sperm cells in modified media are: L-pg = 2.4 x 10(-14) m(3)/Ns (0.14 mu m/min-atm) and E-Lp = 357.7 kJ/mol (85.5 kcal/mol) (R-2 = 0.98), and in CPA media (with 6% glycerol and 10% egg yolk) are L-pg[cpa] = 0.67x10(-14) m(3)/Ns (0.04 mu m/min-atm) and E-Lp[cpa] = 138.9 kJ/mol (33.2 kcal/mol) (R-2 = 0.98). These parameters are significantly different from previously published parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in human sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPA. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates (7000 degrees C/min) obtained using previously published suprazero human sperm permeability parameters which do not account for the presence of extracellular ice.