一种安全有效的卵母细胞封闭式玻璃化冷冻载体

目的探讨封闭式玻璃化冷冻载体冻存小鼠卵母细胞的可行性。方法以小鼠MII期卵母细胞为模型,以开放式玻璃微细管法（GMP）为对照组,比较两种玻璃化冷冻载体对小鼠卵母细胞冷冻后的存活率、受精率、卵裂率及囊胚率的影响。结果卵母细胞经冻融后,封闭式冷冻载体组和GMP组的存活率、受精率、卵裂率和囊胚率均没有明显差异（92.80%vs93.11%,49.80%vs51.67%,36.73%vs35.83%,12.65%vs14.17%%;P〉0.05）。结论封闭式冷冻载体能安全、有效的冷冻保存小鼠卵母细胞。     

猪卵母细胞玻璃化冷冻后细胞骨架的变化

为研究玻璃化冷冻后猪卵母细胞纺锤体、染色体和微丝的变化,从屠宰猪卵巢表面直径2—5mm卵泡中采集未成熟(GV)期卵母细胞,由GV期卵母细胞经成熟培养获得体外成熟(MⅡ)期卵母细胞。GV期和MⅡ期卵母细胞各分为3组对照组、冷冻保护剂处理组和玻璃化冷冻组。MⅡ期卵母细胞经分组处理后直接用于激光扫描共聚焦显微镜(LSCM)观察样本;而GV期卵母细胞处理后先经44h成熟培养,再用作LSCM观察样本。供试卵母细胞经固定、免疫荧光染色后,于LSCM下观察。结果表明,冷冻保护剂处理组GV期卵母细胞经成熟培养后,其纺锤体结构、染色体排列与微丝分布正常率分别为42.9%、89.6%和28.6%;玻璃化冷冻组此3项指标的正常率分别为10.1%、36.4%和16.9%,两组间差异显著(P<0.05);除冷冻保护剂处理组染色体正常率与对照组无较大差异外,两试验组的其他指标均明显低于对照组(分别为79.5%、93.1%和72.3%,P<0.05)。MⅡ期卵母细胞冷冻保护剂处理组的纺锤体结构、染色体排列与微丝分布正常率分别为34.4%、61.3%和47.9%,而冷冻组分别为12.9%、56.7%和37.2%,两组均显著低于对照组(分别为78.3%、90.1%和72.8%,P<0.05)。结果表明,猪GV期和MⅡ期卵母细胞经冷冻保护剂处理或玻璃化冷冻保存后,均造成了纺锤体、染色体和微丝不可逆的损伤,这可能是影响卵母细胞成熟、受精与发育的重要原因。     

玻璃化冷冻对猪体外成熟卵母细胞染色体与纺锤体的影响

以冷冻环为载体,探讨玻璃化冷冻对猪体外成熟卵母细胞染色体与纺锤体影响。单用40%乙二醇(ethyleneglycol,EG)或20%EG与20%二甲基亚砜(dimethylsulphoxide,DMSO)联合作冷冻保护剂,用直投液氮或使用玻璃化冷冻仪法制冷冷冻猪体外成熟卵母细胞;解冻2h后固定并免疫荧光法染色纺锤体及染色体;挑选各试验组形态正常卵母细胞进行体外受精实验。结果表明,与单用EG以及EG和DMSO联合直投液氮方案比较,EG和DMSO联合应用并采用玻璃化冷冻仪制冷方案卵母细胞染色体正常率为30.1%,纺锤体正常率为37.2%,可明显降低卵母细胞染色体及纺锤体结构损伤(P<0.05),并明显提高卵母细胞的激活效果(P<0.05)。采用联合冷冻保护剂及玻璃化冷冻仪高速冷冻可较好维持猪卵母细胞染色体与纺锤体形态,但玻璃化冷冻明显影响猪卵母细胞体外受精后的发育能力。     

牛卵母细胞玻璃化冷冻对基因mRNA表达量的影响

目的 将处于生发泡期(GV期)和体外成熟期(IVM)的牛卵母细胞进行玻璃化冷冻.解冻,对其卵裂率和囊胚率以及一些与发育相关基因的mRNA表达量进行评价.方法 玻璃化冷冻GV期(n=224)和IVM期(n=235)牛卵母细胞,解冻后对其进行体外培养并采用quantitative real time-PCR技术对冷冻.解冻...     

谷胱甘肽在小鼠卵母细胞玻璃化冷冻中的作用

目的：观察谷胱甘肽在小鼠玻璃化冷冻中的保护性作用。方法：通过卵母细胞是否玻璃化冷冻及是否添加GSH处理,将小鼠卵母细胞分为4组。检测卵母细胞内GSH浓度、ROS水平,以及通过彗星实验量化OTM值检测DNA碎片的生成。结果：在对照组、冷冻组、GSH处理组和GSH处理冷冻组细胞内GSH浓度分别为8．95±1．26、4．36±0．96、9．27±1．05和8．18±0．89;ROS水平分别为47．5±4．23、64．2±5．69、44．5±3．25and49．9±7．62。通过GSH处理,玻璃化冷冻卵母细胞出现彗尾百分比显著低于未处理组,差异具有统计学意义;通过GSH处理,玻璃化冷冻卵母细胞OTM值低于未处理组,差异无统计学意义。结论：玻璃化冷冻使小鼠卵母细胞产生一定的氧化应激损伤,表现为细胞内GSH浓度下降,ROS水平上升,DNA碎片增加,GSH处理可以在一定程度上改善。     

蔗糖和海藻糖对水牛卵母细胞玻璃化冷冻保护效果的比较

本实验比较蔗糖或海藻糖作为非渗透性保护剂对水牛成熟卵母细胞冷冻效果的影响。将体外成熟的水牛卵母细胞随机分成对照组、蔗糖组、海藻糖组,研究玻璃化冷冻后的存活率、细胞骨架和孤雌激活后发育潜能的变化。结果表明:玻璃化冷冻中蔗糖组(89.68%)与海藻糖组(91.81%)的存活率差异不显著;冻融后的卵母细胞在细胞骨架方面的表现为:蔗糖组和海藻糖组的纺锤体结构、染色体形态与微丝分布正常率分别为34.69%、42.83%、39.13%和39.51%、49.43%、42.61%,两组间差异不显著(p0.05),但均明显低于对照组(66.40%,71.82%,76.18%,p0.01);在进一步研究发育潜能中发现,冻融后卵母细胞的卵裂率、囊胚率和囊胚细胞数在实验组中均无显著性差异(p0.05),但两组的卵裂率、囊胚率均显著低于对照组(p0.01)。综上所述,玻璃化冷冻时添加蔗糖或海藻糖作为非渗透性保护剂对水牛成熟卵母细胞的保护作用差异不明显,表明两者均可在冷冻时添加使用。     

猪卵母细胞玻璃化冷冻后细胞骨架的变化

为研究玻璃化冷冻后猪卵母细胞纺锤体、染色体和微丝的变化，从屠宰猪卵巢表面直径2—5 mm卵泡中采集未成熟(GV)期卵母细胞，由GV期卵母细胞经成熟培养获得体外成熟(MⅡ)期卵母细胞。GV期和MⅡ期卵母细胞各分为3组：对照组、冷冻保护剂处理组和玻璃化冷冻组。MⅡ期卵母细胞经分组处理后直接用于激光扫描共聚焦显微镜(LSCM)观察样本；而GV期卵母细胞处理后先经44 h成熟培养，再用作LSCM观察样本。供试卵母细胞经固定、免疫荧光染色后，于LSCM下观察。结果表明，冷冻保护剂处理组GV期卵母细胞经成熟培养后，其纺锤体结构、染色体排列与微丝分布正常率分别为42.9%、89.6%和28.6%；玻璃化冷冻组此3项指标的正常率分别为10.1%、36.4%和16.9%，两组间差异显著(P<0.05)；除冷冻保护剂处理组染色体正常率与对照组无较大差异外，两试验组的其他指标均明显低于对照组(分别为79.5%、93.1%和72.3%，P<0.05)。MⅡ期卵母细胞冷冻保护剂处理组的纺锤体结构、染色体排列与微丝分布正常率分别为34.4%、61.3%和47.9%，而冷冻组分别为12.9%、56.7%和37.2%，两组均显著低于对照组(分别为78.3%、90.1%和72.8%，P<0.05)。结果表明，猪GV期和MⅡ期卵母细胞经冷冻保护剂处理或玻璃化冷冻保存后，均造成了纺锤体、染色体和微丝不可逆的损伤，这可能是影响卵母细胞成熟、受精与发育的重要原因。     

牛卵母细胞玻璃化冷冻保存影响因素的研究

采用毛细玻璃管法对牛卵母细胞进行玻璃化冷冻保存,解冻后再进行体外受精（IVF）和早期胚胎的体外培养（IVC）。在此技术的基础上,分别对冷冻前平衡时间、解冻处理、卵丘细胞层数以及卵母细胞所处的减数分裂阶段等影响卵母细胞冷冻保存的因素进行研究,以期筛选出适合牛卵母细胞冷冻保存的方法。结果发现,处于MⅡ期卵母细胞在10％二甲基亚砜（DMSO）＋10％乙二醇（EG）液（VSl）中平衡1～3min,然后进行玻璃化冷冻保存。解冻时将卵母细胞先移入VS1液中处理15s,然后移入蔗糖稀释液中。另外发现,冷冻保存时部分卵丘细胞对卵母细胞有保护作用。而减数分裂阶段不影响解冻后卵母细胞形态正常率,但对胚胎发育率有严重影响。     

活体肺癌组织玻璃化保存的研究

保存活体的肺癌组织将为肺癌发病基因筛查和靶向药物筛选等体外实验研究提供更完整的样本信息. 本文对活体肺癌组织的玻璃化保存方法进行研究，首先采用针浸法玻璃化保存单块肺癌组织，对所需低温保护剂的浓度和平衡时间进行了优化；其次采用冻存管对多块肺癌组织样本进行玻璃化保存，对低温保护剂溶液体积以及平衡时间进行了优化；最后对慢速冷冻、不加低温保护剂快速冷冻、玻璃化冷冻3种冷冻方法的冻存效果进行比较并通过低温显微分析其冰晶损伤机理.结果表明，20% EG+20% DMSO+0.5 mol/L海藻糖作为低温保护剂，在平衡溶液和玻璃化溶液分别加载3 min和1 min时，针浸法和0.25 ml冻存管内玻璃化冻存，复苏后组织活力最高，分别约为79.96%与80.44%. 免疫组化显示玻璃化保存肺癌组织经过复苏后，相比慢速冷冻和无保护剂快速冷冻，组织结构损伤较小，组织内细胞TUNEL阳性表达较少. 低温显微结果表明，玻璃化保存组织内部及周围只出现少量细小冰晶，而慢速冷冻、快速冷冻组织皆出现明显冰晶.     

精子冷冻保存技术及研究进展

精子冷冻是辅助生殖技术的基础,能够有效的保存有价值的基因资源。文章回顾了近些年来国内外精子冷冻保存的重要研究成果,分析了精子的来源、冷冻预处理、冷冻和解冻方法、防冻剂的选择及其加入去除方式的选择等因素对精子冷冻效果的影响。文章还总结了精子冷冻效果的评价方法,展望精子冷冻技术的发展方向和应用前景。     

牛精子和卵母细胞低温保存研究进展

牛的精子和卵母细胞的低温保存对于畜牧业的发展有重要价值。该文综述了近年来牛精子和卵母细胞低温保存的研究进展,包括保存方法、与低温保存有关的细胞特性、存在的问题和改进的方法等内容。     

Cryopreservation of Human Oocytes and Ovarian Tissue

Oocyte cryopreservation has the potential to be an important adjunct to assisted reproductive technologies and bypasses some ethical, moral, and religious dilemmas posed by human embryo cryopreservation. The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Among the morphological factors, the maturity, quality, size of the oocyte, the presence or the absence of the cumulus oophorus seems to play an important role in oocyte survival after thawing. The main biophysical factor of cellular disruption during cryopreservation process in the intracellular ice formation that can be avoided by an adequate cell dehydration; thus reducing the intracellular water by increasing the dehydration process we can limit the damages of the cryopreservation procedure. The dehydration process can be affected by the presence and concentration of the cryoprotectants in the freezing solutions (equilibration and loading solutions), and by the freezing and thawing rate. Two additional properties of cryoprotectants help to protect cells during slow cooling, when the cells are very dehydrated and are surrounded by concentrated salts. The cryoprotectants appear to reduce damage caused by high levels of salt, a property known as salt buffering. Some events occurring to the oocyte during cryopreservation procedure has been found to be a premature exocitosis of cortical granules, leading to an intempestive zona hardening and consequently to a reduction of fertilization rate, and the cryoinjury to the zona pellucida leading to a polispermic fertilization. ICSI is an efficient method to by pass these two events and to achieve a satisfactory outcome in terms of normal fertilization of cryopreserved oocytes. The application of the ICSI to cryopreserved oocytes did not seem to increase the degeneration rate after insemination with respect to fresh oocytes. The increased oocyte survival rate and the use of ICSI have facilitated the recent increase in the number of pregnancies and live birth.     

Cryopreservation of Plagiognathops microlepis sperm

Sperm was collected from cultured male fish and cryopreserved in 0.25 ml straws for the study of sperm cryopreservation. Different parameters were evaluated, including extender, dilution ratio, cryoprotectant type and concentration, equilibrium time, cooling height (in a two-step cooling protocol), and thawing temperature. The optimum result was obtained when the sperm was diluted at a 1:7 ratio in D-16 with 5% DMSO as a cryoprotectant, equilibrated for 20 min, held at 3 cm above liquid nitrogen for 10 min, and then stored in liquid nitrogen. After thawing in a water bath at 40 °C, the percentage of motile cells and fertilization rates of frozen-thawed sperm were 35.33 ± 2.52% and 39.00 ± 4.58%, respectively, while the corresponding rates for fresh sperm were 87.67 ± 3.06% and 88.67 ± 4.62%. We also used a programmed cooling protocol in which temperature was decreased from 4 °C to −80 °C by a rate of 30 °C/min, and then straws (0.25 ml) were placed above the surface of liquid nitrogen for 2 min before being stored in liquid nitrogen. This protocol provided a post-thaw activation rate of 36.67 ± 4.77%. Further parametric optimization is required to improve the quality of frozen-thawed sperm.     

两种冷冻保护剂和冷冻程序对兔精子冷冻效果的比较

目的比较不同冷冻保护剂和冷冻程序对兔精子冷冻保护的影响,以期提高兔精子冷冻保存的效果和效率。方法用三步降温法（程序Ⅰ）和两步降温法（程序Ⅱ）两种冷冻程序与终浓度分别为2%,3%,4%,5%的甘油和乙酰胺两种冷冻保护剂配合进行精液冷冻保存,统计精子复苏率。结果使用程序Ⅱ添加3%乙酰胺的冷冻保护剂实验组的精子复苏率较高,同其它组比较差异有显著性意义（P〈0.05）;程序Ⅱ比程序Ⅰ节省约70%的时间,同种浓度冷冻保护剂的不同冷冻程序组之间精子复苏率差异无显著性意义（P〉0.05）。结论程序Ⅱ与3%乙酰胺配合可以取得良好的冷冻保存效果;用程序Ⅱ进行兔精液冷冻保存可以大幅缩短操作时间。     

猪卵母细胞的体外受精及多精受精

对用于猪体外受精(IVF)的研究方法和技术,如传统的液滴IVF、透明带下注射精子受精(SUZI)、卵母细胞质内单精注射受精(ICSI)及细管IVF等进行了简述。与其它动物相比,进行猪卵的体外受精研究,多精受精现象特别明显。大量的研究表明,猪卵的多精受精不但与其品种特性有关,而且与卵母细胞成熟的程度、透明带的异常、受精时获能精子的浓度、输卵管分泌物、受精液蛋白添加成分、NaHCO3浓度、咖啡因、pH值以及温度等因素密切相关。     

Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants

In the present study, effects of concentration and pretreatment time of cytochalasin B (CB), and of two types of cryoprotectant solutions on the nuclear maturation of vitrified-warmed porcine oocytes were examined. Also, the developmental capacity of vitrified immature porcine oocytes following intracytoplasmic sperm injection (ICSI) was investigated. The nuclear maturation rate (46.8%) of the vitrified-warmed oocytes treated with 7.5 microg/mL CB for 30 min was significantly higher (P < 0.05) than those (13.9-39.2%) of the vitrified-warmed oocytes treated with 0, 2.5, or 5.0 microg/mL CB for 10 or 30 min. Additionally, the nuclear maturation rate of oocytes treated with CB and vitrified in ethylene glycol (EG) (37.1%) was significantly higher (P < 0.05) than that of EG + dimethyl sulfoxide (Me(2)SO) (23.9%). However, no significant differences were observed in the cleavage and blastocyst development rates among the control (45.2 and 20.0%, respectively), the EG group (37.8 and 13.5%, respectively) and the EG + Me(2)SO group (39.3 and 14.3%, respectively). These results demonstrated that: (1) pretreatment with 7.5 microg/mL CB was beneficial for the vitrification of immature porcine oocytes; (2) the combination of EG and Me(2)SO as a cryoprotectant was not advantageous for in vitro maturation (IVM) of vitrified immature porcine oocytes; and (3) vitrified-warmed porcine oocytes matured after IVM, developed to the blastocyst stage without distinct differences compared to fresh oocytes following ICSI.