微流控法去除低温保护剂对卵母细胞发育的影响

为了减小低温保护剂去除过程对卵母细胞造成的渗透损伤和毒性损伤,本文利用微流控芯片对猪二次减数分裂中期(MⅡ-stage)卵母细胞低温保护剂的去除方案进行了优化研究。首先分析了微流控去除方法中去除时间、去除液成分及浓度对卵母细胞存活率及体外发育情况的影响,然后将微流控去除方法与传统的一步法、两步法进行了比较。研究结果表明,微流控法中去除总时间为8 min时,卵母细胞存活率(95.99%±4.64%)及桑椹胚率(74.17%±1.18%)与新鲜细胞(98.53%±2.94%;78.22%±1.34%)相比,差异无统计学意义;1 mol/L蔗糖去除液最有利于卵母细胞低温保护剂去除后的存活及体外发育;微流控法去除低温保护剂后,卵母细胞的存活率、体外发育情况等,均好于传统去除方法。本文研究结果提示,以微流控法去除低温保护剂可减小对卵母细胞的损伤,从而可能进一步提高卵母细胞的低温保存效果。     

微流控法低温保护剂添加及去除线型优化研究

在卵母细胞低温保存过程中,低温保护剂的添加与去除是必不可少的步骤。近年来微流控芯片被用于低温保护剂的添加及去除,而不同线型添加和去除低温保护剂会影响卵母细胞存活率及体外发育情况。利用以微流控芯片为中心的微混合系统,分别设计线性加载和去除保护剂的方案,并以分段线性衍生出凸型、凹型保护剂加载和去除方案,最终用上述方案共组合出9种添加-去除联用方案,研究不同线型添加-去除保护剂对卵母细胞存活率及体外发育情况的影响。结果表明:凹型添加-凸型去除联用方案下,猪MII期卵母细胞的存活率及桑椹胚率达到97.22%、46.03%,均显著优于其他添加-去除方法(P<0.05);低温保护剂添加及去除过程中,连续性添加及去除方案间具有一定的匹配关系,这将为优化微流控法连续性添加-去除低温保护剂方案提供新思路。     

用于低温保护剂混合的微流控芯片设计及优化

卵母细胞在低温保护剂的加载和去除过程中,会受到渗透损伤和毒性损伤,采用微流控技术可实现保护剂浓度连续变化,减小细胞损伤。本文设计了用于低温保护剂加载及去除的5种不同参数的Y型微流控芯片,测定了在不同入口流速、芯片入口角度、通道深宽比及转弯半径下,微通道内保护剂溶液和缓冲溶液的混合程度。实验结果表明:随着溶液入口流速的减小、通道深宽比的增大及转弯半径的减小,溶液在微通道内混合长度减小,混合速度加快,而微通道入口角度对流体混合影响很小。但实际芯片的操作条件及结构参数应根据低温保护剂加载和去除时需要达到的效果以及芯片加工工艺等因素确定。本文研究结果可为其他用于低温保护剂混合的微流控芯片的设计提供参考。     

纳米颗粒对猪GV 期卵母细胞低温保存效果的影响

纳米低温保存技术很可能是新一代低温保存技术的重要发展方向,但纳米颗粒应用于卵母细胞玻璃化保存的报道较少。本研究将羟基磷灰石（HA）、二氧化硅、三氧化二铝、二氧化钛等4种纳米颗粒添加到低温保护剂中,使用Cryotop法冷冻猪GV期卵母细胞,使用形态观察和荧光染色的方法, 研究其对细胞存活率和发育率的影响。结果显示在实验浓度范围内,HA较其它纳米颗粒对猪卵母细胞的毒性低,当浓度低于0.5%时,细胞发育率为100%;低温保护剂中添加0.1%HA纳米颗粒,发育率比其它组显著提高,可以达到22%, 且HA的粒径对结果影响不大;当低温保护剂中添加0.05%粒径为60 nm的HA颗粒时,卵母细胞冷冻复温后发育率会进一步从14.7%提高达到30.4%。在低温保护剂中添加适宜浓度的HA纳米颗粒,可以减少复温过程中的重结晶现象,促进细胞的冷冻存活率和发育率,保存效果与浓度相关,而与纳米颗粒的粒径关系不大。     

卵母细胞深低温保存的策略分析

背景：卵母细胞具有特殊而复杂的低温生物学性质,致使卵母细胞在深低温保存中易受到多因素的损伤而影响复温后的存活率、受精率和受精后发育潜能。 目的：综述卵母细胞深低温保存技术的研究进展,阐明存在的技术缺陷以及解决问题的思路与研究路线。 方法：以“卵母细胞,深低温保存/慢速冷冻,玻璃化”为中文捡索词,以“oocyte, cryopreservation/slow freezing, vitrification”为英文检索词,在中国知网(CNKI)期刊全文数据库和PubMed数据库检索2004 年1月至2014年10月有关卵母细胞深低温保存文献,排除陈旧性、重复性研究,最终纳入41篇文献进行综述。 结果与结论：慢速冷冻、玻璃化方法深低温保存卵母细胞得到较好的临床应用,但复温存活率、受精能力、发育能力损伤等仍困扰医生们。卵母细胞深低温保存技术的进一步完善或突破在于对深低温保存技术环节的细化、卵母细胞低温生物学特性的研究以及相关技术的创新,如深低温保护剂选择、冷冻载体的改进、卵母细胞时相选择、卵母细胞体外成熟技术、卵巢组织保存与移植等。     

生殖细胞的细胞膜渗透特性研究进展

生殖细胞的低温保存在多个领域具有重要应用价值。为提高保存后细胞的存活率与活性,研究细胞膜对水和低温保护剂的渗透特性十分必要。本文综述了近年来动物生殖细胞的细胞膜渗透特性研究概况,列出了典型的精子细胞和卵母细胞的细胞膜对水和常见低温保护剂的渗透系数,分析了这些细胞的渗透特性对制定其低温保存方案的影响。论文还介绍了细胞膜渗透特性的最新测量方法。     

玻璃化冷冻技术在卵母细胞冻存上的应用

玻璃化冷冻保存技术由于具有操作简便、冻融损伤小并且不需要仪器精密控温的特点，已经成为最具应用前景的冷冻保存技术。玻璃化冷冻受到冷冻保护剂成分、浓度、作用时间、作用温度以及降温速率的影响。卵母细胞由于其特殊的细胞学特性，冷冻保存相对困难。玻璃化冻存技术应用于卵母细胞的冷冻保存能获得较好的存活率、受精率以及卵裂率。但是，经过玻璃化冻融的卵母细胞受精后囊胚形成率还不理想，高浓度的冷冻保护剂是否具有细胞毒性，是否会影响胚胎发育等问题尚待解答。因此，还需要更深入的研究来改善卵母细胞的玻璃化冷冻保存技术。     

微流体技术在低温保存领域的研究进展

微流体技术可能是解决低温保存过程中所遇到问题的一种有效方法。本文详细综述了微流体技术在细胞膜渗透性、保护剂添加与去除、生物材料玻璃化保存三方面的研究进展,总结了在低温保存领域应用微流体技术仍可能存在的问题,并指出了未来的研究方向。     

水通道在冷冻保存中的应用研究进展

水通道(aquaporins,AQPs)是一类参与水快速转运的跨膜蛋白家族,广泛分布于动物、植物及微生物的生物膜表面,精细参与调节细胞跨膜的渗透平衡。近年来,AQPs在细胞转运水和冷冻保护剂中的作用倍受关注,AQPs能提高某些细胞和组织的冻融存活率,在低温保存中具有一定的应用前景。     

深低温冷冻保护剂研究进展

【摘要】作为再生医学的重要组成部分,组织工程在受损组织和器官的修复再生方面显示了良好的临床应用前景。组织工程相关细胞及组织的深低温保存是组织工程技术平台的重要部分。冷冻保护剂是深低温保存细胞和组织不可缺少的部分,具有很高的临床应用和研究价值。就传统低温冷冻保护剂的分类和组成、其他冷冻剂的研究进展、改善深低温冷冻保护剂效果的技术手段及冷冻保护剂在组织工程研究中的重要价值进行了综述;探讨最优的深低温保护剂,为组织工程产品的保存及临床应用提供理论依据,为冷冻保护剂的研究指出了新的发展方向;最后指出了冷冻保护剂在组织工程应用方面存在的相关问题。     

A new technique for evaluating volume sensitivity of ion channels

 A new technique is described for evaluating the volume sensitivity of both endogenous and expressed ion channels in Xenopus oocytes. The technique utilizes vesicles derived from inside-out, excised patches and permits evaluation of volume sensitivity at the single-channel level, which is not possible with whole oocytes since removal of the vitelline membrane renders oocytes too fragile to withstand osmotic stress. Using this method, the volume sensitivity of the normally occurring oocyte mechanosensitive (SA-cat) channel was assessed. In 12 experiments, osmotic swelling increased vesicle volume by an average of 60 ± 8% and increased the surface area of the inner side of the vesicle by 47 ± 10%. This was associated with an increase in mean number of open channels (NP _o) from zero (unswollen) to 0.1 ±0.02 (after 5 min). Hence, oocyte SA-cat channels are volume as well as stretch sensitive, where the volume sensitivity may involve cytoskeletal elements adhering to the membrane but probably does not involve freely diffusible components of the original oocyte. Received: 11 July 1996 / Received after revision: 28 August 1996 / Accepted: 9 September 1996     

Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration

BACKGROUND: This study compared the viability of human pronuclear oocytes subjected to vitrification followed by post-thaw step-wise removal of cryoprotectants versus direct rehydration, in terms of their subsequent in vitro survival and ultrastructural features. METHODS: A total of 115 three-pronuclei stage oocytes were cryopreserved in super-open-pulled straws by vitrification in 40% ethylene glycol + 0.75 mol/l sucrose for either 1 min or 10 s at 38 degrees C, followed by direct plunging into liquid nitrogen. After thawing, oocytes vitrified for 1 min (group 1) or 10 s (group 2) were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) for removal of the cryoprotectant in five 2.5 min steps. A second batch of oocytes vitrified for either 1 min (group 3) or 10 s (group 4) were directly expelled into culture medium at 38 degrees C after thawing. Finally, the ultrastructural changes occurring in oocytes in each of the treatment groups were evaluated. RESULTS: Oocyte development (division to two-blastomere stage) rates after in vitro culture were 82, 83, 0 and 0% for groups 1, 2, 3 and 4, respectively. The harsh osmotic process involved in direct rehydration provoked ultrastructural changes, including the disruption of cytoplasmic and pronuclear membranes as well as intracellular organelles. CONCLUSION: The direct post-thaw rehydration of human pronuclear oocytes has lethal osmotic effects, such that protocols for vitrifying human pronuclear oocytes should include the step-wise removal of the cryoprotectant.     

Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation

BACKGROUND: To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and P(EG) (cryoprotectant permeability) of mature human oocytes after exposure to ethylene glycol (EG) and tested the efficiency of a multi-step slow cooling protocol based on this cryoprotectant. METHODS: Oocytes were perfused with 1.5 mol/l EG for 10 min. Oocyte volume at each time point was calculated and normalized to the original volume. Slow cooling was conducted by exposing oocytes to increasing EG concentrations (0.5, 1.0 and 1.5 mol/l n = 155) or 1.5 mol/l of propane-1,2-diol (PrOH) n = 102. Oocytes which survived cryopreservation n = 80 and fresh oocytes n = 73 were prepared for confocal microscopy analysis of the meiotic spindle. RESULTS: During EG exposure, oocytes underwent an abrupt 50% volume reduction. Complete recovery of the initial volume was not observed. From the values of a best fit plot, the coefficients Lp = 0.82 +/- 0.29 microm min(-1) atm(-1) (mean +/- SD) and P(EG) 0.10 +/- 0.01 microm s(-1) were generated. Survival rates after freezing with EG were lower than with PrOH (51.6 versus 71.5%, respectively, P < 0.05). The frequencies of normal spindle configuration were lower in frozen EG and frozen PrOH oocytes compared with fresh oocytes (53.8, 50.9 and 66.7%, respectively, P < 0.05). CONCLUSIONS: The oocyte plasmalemma possesses limited permeability to EG and EG exposure causes considerable osmotic stress. However, post-thaw rates of survival and normal meiotic spindle organization may be preserved by protocols which are designed in order to minimize osmotic stress.     

Permeability characteristics of human oocytes in the presence of the cryoprotectant dimethylsulphoxide.

Equilibration of oocytes with cryoprotectants is a prerequisite of low temperature storage. However, cryoprotectant exposure may induce damage via osmotic stress. Knowledge of cell membrane permeability characteristics and their temperature dependence would facilitate the design of cryopreservation protocols in which osmotic stress is minimized and the incidence of intracellular freezing is reduced. To obtain such data, the volume change of donated human oocytes following exposure to cryoprotectant was measured at a variety of temperatures. After removal of cumulus cells, each oocyte was placed in a 5 microl droplet of phosphate-buffered medium. The oocyte was held in position by suction generated using a fine pipette and perfused with 1 ml 1.5 mol/l dimethylsulphoxide (DMSO) at 30, 24 or 10 degrees C. The volume of the oocyte before, during and after perfusion was recorded by videomicroscopy. Oocyte volume was calculated from radius measurements and the Kedem-Katchalsky (K-K) passive coupled transport coefficients, namely L(p) (hydraulic permeability), P(DMSO) (permeability to DMSO) and sigma (reflection coefficient) were derived. The resulting coefficients were L(p) = 1. 65 +/- 0.15, 0.70 +/- 0.06 and 0.28 +/- 0.04 microm/min.atm; P(DMSO) = 0.79 +/- 0.10, 0.25 +/- 0.04 and 0.06 +/- 0.01 microm/s and sigma = 0.97 +/- 0.01, 0.94 +/- 0.03 and 0.96 +/- 0.01 at 30, 24 and 10 degrees C respectively. The activation energy for L(p) was 14.70 and for P(DMSO) was 20.82 kcal/mol. The permeability parameters of human oocytes are higher than those of murine oocytes, suggesting that they require a shorter period of exposure to DMSO with concomitantly reduced toxic effects.     

High-throughput optofluidic system for the laser microsurgery of oocytes

This study combines microfluidics with optical microablation in a microscopy system that allows for high-throughput manipulation of oocytes, automated media exchange, and long-term oocyte observation. The microfluidic component of the system transports oocytes from an inlet port into multiple flow channels. Within each channel, oocytes are confined against a microfluidic barrier using a steady fluid flow provided by an external computer-controlled syringe pump. This allows for easy media replacement without disturbing the oocyte location. The microfluidic and optical-laser microbeam ablation capabilities of the system were validated using surf clam (Spisula solidissima) oocytes that were immobilized in order to permit ablation of the 5 μm diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection.     

Volume changes of mature human oocytes on exposure to cryoprotectant solutions used in slow cooling procedures

BACKGROUND: Despite the recent increase in pregnancies from cryopreserved human oocytes, success in terms of births per thawed oocyte is still poor. Modifications to cryopreservation protocols have not been based on measurement of the osmotic response of oocytes, and methodologies are often poorly described or protocols not strictly adhered to, inevitably resulting in variability. METHODS: Volume change of mature human oocytes was measured on exposure to cryoprotectant. Oocytes were exposed to either 0.75 mol/l propane-1,2-diol (PrOH) for 10 min; 1.5 mol/l PrOH for 10 min, having been exposed to 0.75 mol/l PrOH for 7.5 min; or 1.5 mol/l PrOH plus 0.2 or 0.3 mol/l sucrose for 10 min, having been exposed to 1.5 mol/l PrOH for 10 min. RESULTS: On exposure to PrOH alone, oocytes shrank and then re-expanded, having reached 75 and 84% of their starting volume in 0.75 and 1.5 mol/l, respectively. Oocytes shrank continuously in PrOH plus sucrose, reaching 67 or 55% of their initial volume in 0.2 or 0.3 mol/l sucrose, respectively. CONCLUSIONS: To improve consistency following cryopreservation, protocols must be strictly adhered to; small changes in duration of exposure to cryoprotectant can result in drastic changes in cellular hydration and thus the fate of the cell during freezing/thawing.     

Human oocyte cryopreservation: new perspectives regarding oocyte survival

The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Various attempts to cryopreserve human oocytes have been performed with contrasting results. Therefore the effect of some factors, such as the presence or absence of the cumulus oophorus, the sucrose concentration in the freezing solution and the exposure time to cryoprotectants, on human oocyte survival after thawing were investigated. The oocytes were cryopreserved in 1,2-propanediol added with sucrose, using a slow-freezing-rapid-thawing programme. After thawing, the oocytes were inseminated by intracytoplasmic sperm injection (ICSI) and the outcomes of insemination and subsequent embryo development were also recorded. The post-thaw cryosurvival rate was not different for the oocytes cryopreserved with their cumuli partially removed mechanically (56%) when compared with those cryopreserved with their cumuli totally removed enzymatically (53%). On the contrary, a significantly higher survival rate was obtained when the oocytes were cryopreserved in the presence of a doubled sucrose concentration (0.2 mol/l) in the freezing solution and the survival rate was even higher when the sucrose concentration was tripled (0.3 mol/l) (60 versus 82% P < 0.001). Furthermore, a longer exposure time (from 10.5 to 15 min) to cryoprotectants, before lowering the temperature, significantly increased the oocyte survival rate (P < 0.005). Intracytoplasmic sperm injection produced a good fertilization rate (57%) of thawed oocytes and a high embryo cleavage rate (91%) and a satisfactory embryo morphology was observed (14 and 34% for grade I and grade II embryos respectively).     

Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI

BACKGROUND: The value of oocyte cryopreservation remains controversial. Two major problems exist: poor survival and injury to the oocyte meiotic spindle after freezing and thawing. METHODS: For slow oocyte cryopreservation, we used 1.5 mol/l 1,2-propanediol and 0.3 mol/l sucrose. We waited 3 h after thawing for possible recovery of the meiotic spindles before performing ICSI. RESULTS: Forty-three women undergoing IVF or ICSI cycles cryopreserved some or all of their harvested oocytes; of these, 20 thawed their cryopreserved oocytes for personal use and one for donation. The survival rate of oocytes after thawing was 75%, with 67% of oocytes fertilizing normally after ICSI. All 21 cycles (100%) resulted in fertilization and embryo transfers. Seven pregnancies (33%) resulted. Four women delivered five babies with normal karyotypes. Three conceptions are ongoing. Compared to 38 cycles of frozen-thawed embryos at the pronuclear stage in the same period, the percentages of survival, pregnancy and implantation were similar. Additionally, four unmarried women with white blood cell diseases underwent oocyte freezing before preconditioning treatment for haematopoietic stem cell transplantation. CONCLUSIONS: This protocol achieved reproducible success of survival, fertilization and pregnancy for freezing and thawing of human oocytes. The 3 h post-thaw incubation could permit restoration of the meiotic spindles, thus facilitating normal fertilization.     

Cell cycle regulation: Egg activation induced by osmotic pressure change and the effects of amiloride on the cryopreservation of mouse oocytes

Activation of oocytes is caused by osmotic pressure change insome species. However, cryopreservation of oocytes occurs inthe presence of osmotic pressure change induced by cryoprotectants.We investigated the effect of 5-(N,N,-dimethyl)-amiloride (NNDMA),a selective inhibitor of Na⁺/H⁺ exchange, on the cryopreservationand osmotic activation of mouse oocytes. The percentage (23.2%)of degenerate oocytes after cryopreservation in the presenceof NNDMA was found to be lower than that (39.5%) of untreatedoocytes. After thawing, the percentage (23.6%) of oocytes whichcould be fertilized following cryopreservation in the presenceof NNDMA was significantly higher than that of untreated (18.0%)oocytes. These results suggest that amiloride increased thesurvival rate after thawing following cryopreservation. To investigatethe effect of NNDMA on oocyte activation caused by the cryoprotectant,dimethyl sulphoxide (DMSO) was used to induce osmotic pressurechange. NNDMA was found to inhibit cortical granule exocytosis,the second polar body emission and pronuclear formation whichoccurs upon activation due to osmotic pressure change. It alsoinhibited the increase in phosphorylation of many proteins including33 and 45 kDa proteins, which occurs during fertilization andchemical oocyte activation. In contrast, protein phosphorylationwas not inhibited by W7, a calmodulin inhibitor. The actionsof these inhibitors suggest that oocyte activation induced byosmotic pressure change involves a pathway mediated by Na⁺/H⁺exchange which may be distinct from the Ca-calmodulin pathway.Amiloride may be a useful drug for increasing the rate of survivalof cryopreserved oocytes. amiloride/cryopreservation/oocyte activation/osmotic pressure change