【摘要】The objective of this study was to evaluate polyampholyte---Poly--L--lysine (PLL) as a new cryoprotectant for ovine oocyte vitrification, alternative to the traditionally used cryoprotectant dimethyl sulfoxide (DMSO). Recently, Matsumura et al. (2009) demonstrated that PLL effectively protects L929 mouse stem cells during cryopreservation. Oocytes were screened and collected from fresh ovine ovarian tissues, then randomly classed into two developmental stages, namely, germinal vesicle (GV) stage oocytes (immature) and metaphase 2 (M2) stage oocytes (mature). GV stage oocytes were vitrified immediately upon collection, while M2 stage oocytes were vitrified after 24 h in vitro culture for oocyte maturation. Each developmental group was subjected to two different vitrification solutions: (1) PLL based formula (5%, 10% PLL); (2) and DMSO based formula (10%, 20% DMSO, ethylene glycol, and holding medium), resulting in four groups: PLL: GV, PLL-M2, DMSO-GV, and DMSO-M2. In the first experiment, there are six replicated batches (block) for each group, and each batch was processed on the same day under the same condition. The normality of developing oocytes was examined under microscope. Normal oocytes showed the characteristics of homogeneous cytoplasm and intact zona pellucid, with abundant organelles uniformly dispersed within the plasma; while abnormal oocytes displayed a reduced amount of organelles and low density of granular cells. The data were analyzed with proc Glimmix procedure of SAS. The results showed that the oocyte survival rate (%) of PLL-M2 (80.13) was significantly (P<0.05) higher than PLL-GV (62.28); DMSO-M2 (63.99) was significantly (P<0.05) higher than DMSO-GV (40.50), whereas PLL-GV was significantly (P<0.05) higher than DMSO-GV, and PLL-M2 was in turn significantly (P<0.05) higher than DMSO-M2. There was no difference in survival rate after vitrification between group PLL-GV and DMSO-M2. For the trypan blue staining comparison, the number of PLL-M2 (25+/-2.89) was significantly (P<0.05) higher than PLL-GV (16+2.31). There was no significant difference between the density of the PLL based formula and DMSO based formula, at the same time, there were no different injuries found among layer 1, 2 and 3. However, more frequent injuries were found in vitrified groups than the control groups. In the process of vitrification, the removal of granular cell after 24 h in vitro maturation appeared to be important to the success of cryopreserved oocytes and more disrupted spindle with dispersion of chromosomes were found in GV stage vitrified groups than M2 stage ones. Furthermore, M2 stage oocytes have shown a high viability. In conclusion, PLL can be used as an alternative reagent for ovine oocytes vitrification, with a high efficiency in a lower concentration compared with the traditional DMSO protocol.
【摘要机译】这项研究的目的是评估聚两性电解质-聚-L-赖氨酸（PLL）作为绵羊卵母细胞玻璃化的一种新型防冻剂，替代传统的防冻剂二甲基亚砜（DMSO）。最近，松村等人。 （2009年）证明了PLL在冷冻保存过程中可以有效保护L929小鼠干细胞。从新鲜的绵羊卵巢组织中筛选并收集卵母细胞，然后随机分为两个发育阶段，即生泡（GV）阶段卵母细胞（未成熟）和中期2（M2）阶段卵母细胞（成熟）。 GV期卵母细胞在收集后立即玻璃化，而M2期卵母细胞在体外培养24小时后玻璃化以使卵母细胞成熟。每个发育组都要接受两种不同的玻璃化溶液：（1）基于PLL的公式（5％，10％PLL）； （2）和基于DMSO的公式（10％，20％DMSO，乙二醇和保持介质），得出四组：PLL：GV，PLL-M2，DMSO-GV和DMSO-M2。在第一个实验中，每个组有六个重复的批次（块），并且每个批次在相同的条件下于同一天进行处理。在显微镜下检查发育中的卵母细胞的正常性。正常的卵母细胞具有均匀的细胞质和完整的透明带的特征，丰富的细胞器均匀地分散在血浆中。而异常卵母细胞则显示细胞器数量减少和颗粒细胞密度低。用SAS的proc Glimmix程序分析数据。结果表明，PLL-M2（80.13）的卵母细胞存活率（％）显着（P <0.05）比PLL-GV（62.28）高； DMSO-M2（63.99）显着（P <0.05）高于DMSO-GV（40.50），而PLL-GV显着（P <0.05）高于DMSO-GV，而PLL-M2则显着（P <0.05 0.05）高于DMSO-M2。 PLL-GV组和DMSO-M2组之间玻璃化后的存活率没有差异。对于锥虫蓝染色比较，PLL-M2（25 +/- 2.89）的数量显着（P <0.05）比PLL-GV（16 + 2.31）高。基于PLL的公式和基于DMSO的公式的密度之间没有显着差异，同时，在第1层，第2层和第3层之间没有发现不同的伤害。但是，玻璃化组中的伤害比对照组更常见组。在玻璃化过程中，体外成熟24小时后去除粒状细胞似乎对冷冻保存的卵母细胞的成功至关重要，在GV期玻璃化组中发现比M2期玻璃化组更多的破坏了纺锤体的染色体分散。此外，M2期卵母细胞显示出高生存力。总之，与传统的DMSO协议相比，PLL可以在较低的浓度下以较高的效率用作羊卵母细胞玻璃化的替代试剂。
机译蔗糖胁迫下玻璃化绵羊卵母细胞的体外受精和胚胎发育午夜国产免费视频亚洲-在线欧美 精品 第1页