玻璃化冷冻对小鼠卵母细胞透明带的影响

本文旨在通过研究玻璃化冷冻小鼠卵母细胞透明带超微结构、透明带厚度(ZPT)、厚度变量(ZPTV)及双折射分值(ZPB)的影响,分析三者间的相关性,探求玻璃化冷冻液的最佳配方。选用4组应用最广泛的冷冻液配方对小鼠卵母细胞进行处理,与未处理的卵母细胞比较存活率、受精率、卵裂率和囊胚率,选出最适冷冻液配方。以新鲜卵母细胞为对照组,进行冷冻程序但并没有进行实际冷冻的卵母细胞为处理组,进行玻璃化冷冻复苏的卵母细胞为冷冻组,扫描电镜观察各组的透明带超微结构变化;检测3组细胞的ZPT和ZPB,并对ZPT和ZPB、ZPTV和ZPB之间相关性进行分析。结果发现HM+7.5%(DMSO+EG),HM+15%(DMSO+EG)+0.5 mol/L Su冷冻液组对卵母细胞发育潜力影响较小。玻璃化冷冻对ZPT(6.05±0.10μm vs 5.77±0.60μm)和ZPB(0.30±0.38 vs 1.22±0.21)有显著影响(P<0.05),且ZPT与ZPB呈负相关(r=-0.299)(P<0.05)。扫描电镜发现玻璃化冷冻造成卵母细胞透明带呈熔融状,表面凹凸不平,窗孔基本不可见,甚至出现透明带部分脱落的情况;冷冻组透明带超微结构的正常率较对照组和处理组下降,其中粗ZP(44.9%对92.9%和84.8%)(P<0.01)和光滑ZP(31.0%对7.4%和9.1%)(P<0.01)。结果表明,玻璃化冷冻影响卵母细胞的发育潜能,低温对透明带的超微结构有较大的负面影响。

Effect of vitrification on zona pellucida of mouse oocytes

This study was aimed to explore the effects of vitrification with different protocols on the ultrastructure,zona pellucida thickness(ZPT),zona pellucida thickness variance(ZPTV),and zona pellucida birefringence(ZPB)of mouse oocyte and the corresponding correlations.The rates of survival,fertilization,cleavage and blastocyst were analyzed using different preservation protocols(Test groups 1,2,3,and 4)and fresh oocytes.Consequently,the optimal protocol was selected for further study.The ultra-structures of the zona pellucida and the ZPT/ZPB correlations were analyzed.The results showed that HM+7.5%(DMSO+EG),HM+15%(DMSO+EG)+0.5 mol/L Su of the cryopreservation group had little effect on the development potential of the mouse oocyte.Vitrification exerted significant effects on ZPT(6.05±0.10μm vs 5.77±0.60μm)and ZPB(0.30±0.38 vs 1.22±0.21)(P<0.05),and a negative correlation was detected between ZPT and ZPB(r=-0.299)(P<0.05).Further exploration using a scanning electron microscope(SEM)revealed that the zona pellucida(ZP)of vitrified oocytes was fused,uneven with rough surface and invisible holes,and partially separated from the oocyte.The normality of ZP ultrastructure,including the rough ZP(44.9%vs 92.9%and 84.8%)(P<0.01)and the smooth ZP(31.0%vs 7.4%and 9.1%)(P<0.01)in the vitrification group,was decreased compared with that of the other groups.The above results indicated that vitrification protocols affected the development potential of mouse oocyte,and low temperature exerted a considerable negative effect on the ultrastructure of zona pellucida.