Department of Anatomy, Mahidol University, Bangkok, Thailand.
Reprod Biol Endocrinol. 2010 Jun 22;8:70. doi: 10.1186/1477-7827-8-70.
Cryopreservation of oocytes, which is an interesting procedure to conserve female gametes, is an essential part of reproductive biotechnology. The objective of the present study was to investigate the effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes.
Immature oocytes (germinal vesicles) isolated from ovaries of normal bitches (> 6 months of age) were either vitrified in open pulled straw (OPS) using 20% ethylene glycol (EG) and 20% dimethyl sulfoxide (DMSO) as vitrification solution or exposed to vitrification solution without subjected to liquid nitrogen. After warming, oocytes were investigated for nuclear maturation following in vitro maturation (IVM), ultrastructural changes using transmission electron microscopy (TEM) and gene expression using RT-PCR. Fresh immature oocytes were used as the control group.
The rate of resumption of meiosis in vitrified-warmed oocytes (53.4%) was significantly (P < 0.05) lower than those of control (93.8%) and exposure (91.4%) groups. However, there were no statistically significant differences among groups in the rates of GV oocytes reaching the maturation stage (metaphase II, MII). The ultrastructural alterations revealed by TEM showed that cortical granules, mitochondria, lipid droplets and smooth endoplasmic reticulum (SER) were affected by vitrification procedures. RT-PCR analysis for gene expression revealed no differences in HSP70, Dnmt1, SOD1 and BAX genes among groups, whereas Bcl2 was strongly expressed in vitrified-warmed group when compared to the control.
Immature canine oocytes were successfully cryopreserved, resumed meiosis and developed to the MII stage. The information obtained in this study is crucial for the development of an effective method to cryopreserve canine oocytes for establishment of genetic banks of endangered canid species.
卵母细胞的冷冻保存是一种有趣的保存女性配子的方法，是生殖生物技术的重要组成部分。本研究的目的是研究玻璃化对犬卵母细胞核成熟、超微结构变化和基因表达的影响。
从正常母犬（> 6 月龄）卵巢中分离出未成熟卵母细胞（生发泡），使用 20%乙二醇（EG）和 20%二甲基亚砜（DMSO）作为玻璃化溶液，在开放式拉制吸管（OPS）中进行玻璃化，或者不接触液氮暴露于玻璃化溶液中。解冻后，通过体外成熟（IVM）研究卵母细胞核成熟情况，使用透射电子显微镜（TEM）观察超微结构变化，并使用 RT-PCR 研究基因表达。新鲜未成熟卵母细胞用作对照组。
玻璃化-解冻卵母细胞中减数分裂恢复的比率（53.4%）显著（P < 0.05）低于对照组（93.8%）和暴露组（91.4%）。然而，各组GV 卵母细胞达到成熟阶段（中期 II，MII）的比率没有统计学上的显著差异。TEM 显示的超微结构改变表明，皮质颗粒、线粒体、脂滴和光滑内质网（SER）受到玻璃化程序的影响。基因表达的 RT-PCR 分析表明，各组 HSP70、Dnmt1、SOD1 和 BAX 基因无差异，而与对照组相比，玻璃化-解冻组中 Bcl2 强烈表达。
犬未成熟卵母细胞成功冷冻保存，恢复减数分裂并发育至 MII 阶段。本研究获得的信息对于开发有效方法冷冻保存犬卵母细胞以建立濒危犬种的遗传库至关重要。
