猪卵母细胞在GV期与MⅡ期的冷冻保存效果比较研究

摘要

This study was designed to compare the efficiency of porcine oocytes vitrified at the GV and MⅡ stages.The vitrified GV oocytes were matured in vitro and then evaluated their cortical granule distribution with immunofluorescence, CD9 protein level with Western blotting and zona pellucida dissolution time with pronse digestion method, as compared to vitrified MⅡ oocytes.The results showed that the survival percentages of oocytes vitrified at the GV stage were significantly lower than those vitrified at the MⅡ stage after 2 h of warming (P<0.05);However, there was no difference in maturation rate between vitrified and fresh oocytes (P>0.05).The oocytes vitrified at MⅡ stage resulted in significantly decreased normal cortical granule distribution and CD9 protein level (P<0.05), but no such results were found in vitrified GV oocytes after maturation (P>0.05).In addition, vitrification did not affect the dissolution time of zona pellucida in GV and MⅡ oocytes (P>0.05).The data demonstrated that despite of lower survival rate compared with MⅡ oocytes, vitrified porcine GV oocytes after maturation showed normal polar body extrusion, cortical granule distribution and CD9 protein level.%为比较猪卵母细胞在GV期与MⅡ期的冷冻保存效果,试验在这两个成熟阶段对其进行玻璃化冷冻,GV期卵母细胞解冻后培养至成熟,MⅡ期卵母细胞解冻后恢复2 h,然后采用免疫荧光标记、Western blotting和链霉蛋白酶溶解方法分别检测它们的皮质颗粒分布、CD9蛋白表达水平和透明带消化时间上的差异.结果表明,GV期卵母细胞在解冻后2 h的存活率显著低于MⅡ期卵母细胞(P<0.05),但极体排出率与对照卵母细胞无明显差异(P>0.05);在冷冻MⅡ期卵母细胞中,皮质颗粒的皮质区分布比例和CD9的蛋白表达水平显著下降(P<0.05),但冷冻GV期卵母细胞经体外成熟后则无明显变化(P>0.05);冷冻GV期与MⅡ期卵母细胞均不会影响透明带的消化时间(P>0.05).由此可见,猪卵母细胞在GV期的冷冻存活率虽然较MⅡ期低,但其体外成熟后极体排出率、皮质颗粒分布和CD9蛋白表达水平均未受到冷冻的影响.