He Yimeng, Dong Qiaoxiang, Tiersch Terrence R, Devireddy Ram V
Bioengineering Laboratory, Department of Mechanical Engineering, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Biol Reprod. 2004 May;70(5):1428-37. doi: 10.1095/biolreprod.103.025296. Epub 2004 Jan 21.
In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment "ball-on-stick" model with a "ball" 1.66 microm in diameter and a "stick" 41 microm in length and 0.14 microm wide. Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 microm in diameter and a "stick" 53 microm in length and 0.17 microm wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 Vo, where Vo is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: Lpg = 0.30 x 10(-15) m(3)/Ns (0.0017 microm/min-atm) and ELp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.27 x 10(-15) m(3)/Ns (0.0015 microm/min-atm) and ELp[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: Lpg = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp[cpa] = 37.6 kJ/mole (9.0 kcal/mole). The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70 degrees C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C. gigas.
在本研究中，采用一种与形状无关的差示扫描量热法（DSC）技术，来测量二倍体和四倍体太平洋牡蛎（Crassostrea gigas）精子细胞冷冻过程中的脱水反应。这是DSC技术首次应用于非哺乳动物物种的精子细胞。在细胞外有冰且存在8%（v/v）浓度的二甲基亚砜（DMSO，一种常用的冷冻保护剂）的情况下，以5℃/min和20℃/min的冷却速率获得了牡蛎精子细胞悬液冷冻过程中的体积收缩情况。利用先前发表的数据，将二倍体牡蛎的精子细胞建模为一个双隔室“球-棒”模型，其中“球”的直径为1.66微米，“棒”的长度为41微米、宽度为0.14微米。同样，四倍体牡蛎的精子细胞建模为“球”的直径为2.14微米，“棒”的长度为53微米、宽度为0.17微米。假定两个倍性水平的精子细胞的渗透惰性细胞体积Vb均为0.6Vo，其中Vo为等渗（或初始）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（Lpg和ELp）。在不存在冷冻保护剂的情况下，单倍体精子细胞（或二倍体太平洋牡蛎的细胞）在5℃/min和20℃/min时的综合最佳拟合膜通透性参数为：Lpg = 0.30×10⁻¹⁵立方米/（牛顿·秒）（0.0017微米/（分钟·大气压）），ELp = 41.0千焦/摩尔（9.8千卡/摩尔）。在存在8% DMSO的情况下，相应参数为：Lpg[cpa] = 0.27×10⁻¹⁵立方米/（牛顿·秒）（0.0015微米/（分钟·大气压）），ELp[cpa] = 38.0千焦/摩尔（9.1千卡/摩尔）。同样，在不存在冷冻保护剂的情况下，二倍体精子细胞（或四倍体太平洋牡蛎的细胞）在5℃/min和20℃/min时的综合最佳拟合膜通透性参数为：Lpg = 0.34×10⁻¹⁵立方米/（牛顿·秒）（0.0019微米/（分钟·大气压）），ELp = 29.7千焦/摩尔（7.1千卡/摩尔）。在存在8% DMSO的情况下，相应参数为：Lpg[cpa] = 0.34×10⁻¹⁵立方米/（牛顿·秒）（0.0019微米/（分钟·大气压）），ELp[cpa] = 37.6千焦/摩尔（9.0千卡/摩尔）。本研究中获得的参数表明，太平洋牡蛎精子细胞的最佳冷却速率范围为40至70℃/min。这些理论冷却速率与通过实验确定的太平洋牡蛎（C. gigas）精子细胞的最佳冷却速率非常一致。
