猪卵母细胞玻璃化冷冻后的超微结构变化  

Ultrastructural changes of vitrified porcine oocytes

作　　者：武彩红[1] 芮荣[1] 戴建军[1] 谢冰[1] 剧世强[1] 卢晓[1] 侯加法[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《南京农业大学学报》2007年第1期99-104,共6页Journal of Nanjing Agricultural University

基　　金：上海市科技兴农重点攻关项目(沪农科攻字[2003]第14-1号);国家自然科学基金资助项目(30270958)

摘　　要：为研究猪未成熟期(GV期)和体外成熟期(MⅡ期)卵母细胞玻璃化冷冻后的超微结构变化,随机取新鲜与冷冻-解冻后的GV期和MⅡ期卵母细胞制备电镜标本。GV期和MⅡ卵母细胞分3组进行处理:Ⅰ组为对照组,Ⅱ组为GV期卵母细胞冷冻组,Ⅲ组为MⅡ期卵母细胞冷冻组。结果表明,所用玻璃化冷冻程序更适用于猪GV期卵母细胞的冷冻保存,GV期冷冻组卵母细胞的二乙酸荧光素(FDA)染色存活率、卵裂率均明显高于MⅡ期卵母细胞冷冻组。透射电镜观察结果发现,猪GV期卵母细胞经玻璃化冷冻后,主要表现为部分卵丘-卵母细胞复合体(COC)发生卵丘细胞脱落,透明带破损,卵丘细胞碎裂,卵丘细胞与卵母细胞间的连接受到破坏,卵母细胞微绒毛断裂、消失、数量减少,卵母细胞内脂滴多呈均质状。而MⅡ期卵母细胞冷冻后,皮质颗粒仍呈单层排列于质膜下,仅可见形态不规则的不均质脂滴,其周围常严重空泡化。试验表明,采用适当的冷冻方案,可以获得少量源于冷冻保存的猪GV期卵母细胞的胚胎。而猪MⅡ期卵母细胞冷冻后,经体外受精未见卵裂,脂滴严重空泡化是其最为明显的变化。To investigate the ultrastruetural changes of vitrified porcine immature （GV stage） and mature （M Ⅱ stage） oocytes, oocytes were divided into 3 groups： Group I was the control, Group Ⅱ was the freezing group for GV stage ooeytes and Group Ⅲ for M Ⅱ stage ooeytes. The results showed that vitrification procedures used in the experiment were more suitable for the eryopreserration of porcine GV stage ooeytes, resulting in significantly higher survival rate and cleavage rate in comparison with M Ⅱ stage ooeytes. To investigate the effects of vitrification on the ultrastructures of ooeytes, fresh and vitrified-warmed GV and M Ⅱ stage ooeytes were randomly adopted for electron transmission microscopic samples, it was found that partial vitrified porcine GV stage ooeytes behaved the separation of cumulus cells （ CC ） from cumulus-ooeyte complexes （ COC ） , the zona pellucida （ ZP ） adjacent to CC fractured and gap junctions between CC bottom and cytoplasm ruptured. The mierovilli （MV） on the oolemma surface fractured and disappeared. Lipid droplets （LD） were homogeneous. In vitrified M Ⅱ stage ooeytes, their cortical granules （CG） lined under the oolemma. Inhomogeneous LD showed irregular with many vacuoles. It showed that porcine embryos could be obtained from vitrified GV stage oocytes using suitable freezing protocol. However, no cleavage and further development were obtained from vitrified M Ⅱ stage oocytes. One of the obvious changes was the vacuolized LD, indicating that it might be the possible reason affecting viability of oocytes. The exact mechanism of the impact of these changes on the development of vitrified oocytes remained to be further studied.

关 键 词：猪 卵母细胞 玻璃化冷冻 体外成熟 体外发育 超微结构 

分 类 号：S852.162[农业科学—基础兽医学]