Zhang Tiantian, Isayeva Anna, Adams Serean L, Rawson David M
Luton Institute of Research in the Applied Natural Science, University of Luton, 2 Adelaide Street, Luton, Bedfordshire LU1 5DU, UK.
Cryobiology. 2005 Jun;50(3):285-93. doi: 10.1016/j.cryobiol.2005.02.007. Epub 2005 Apr 1.
Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.
研究鱼卵母细胞膜通透性对于制定成功的冷冻保存方案至关重要。本研究的目的是在设计冷冻保存方案之前，研究斑马鱼（Danio rerio）卵母细胞膜对水和冷冻保护剂的通透性。该研究以III期和V期斑马鱼卵母细胞为对象。在22℃下暴露20分钟后，测量不同蔗糖浓度下III期卵母细胞的体积变化，并使用玻意耳 - 范特霍夫关系确定卵母细胞的非渗透活性体积（Vb）。还测量了卵母细胞暴露于不同冷冻保护剂溶液期间的体积变化。卵母细胞在22℃下暴露于2 M二甲基亚砜（DMSO）、丙二醇（PG）和甲醇中40分钟。III期卵母细胞也在0℃下暴露于2 M DMSO。使用Linkham软件在Olympus BX51低温显微镜上捕获卵母细胞图像以进行图像记录。使用Scion Image进行图像分析和直径测量。使用Berkeley Madonna 8.0.1软件将实验数据拟合到双参数模型。使用该模型估计水力传导率（L(p)）和溶质（冷冻保护剂）渗透率（Ps）。发现III期斑马鱼卵母细胞的非渗透活性体积为69.5%。假设卵母细胞的内部等渗值为272 mOsm，在22℃下，在DMSO和PG存在下，Lp的平均值±SE分别为0.169±0.02和0.196±0.01μm/min/atm。DMSO和PG的Ps值分别为0.000948±0.00015和0.000933±0.00005 cm/min。还表明III期卵母细胞的膜通透性随温度显著降低。在甲醇处理期间未观察到细胞体积的显著变化。本文首次报道了鱼卵母细胞膜通透性参数。III期斑马鱼卵母细胞获得的Lp和Ps值通常低于成功冷冻保存的哺乳动物卵母细胞获得的值，高于鱼胚胎和海胆卵获得的值。由于V期卵母细胞在暴露于冷冻保护剂期间经历了外卵膜与内卵黄膜的分离，因此使用本研究中采用的方法无法估计V期卵母细胞的膜通透性参数。
