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目的应用透射电子显微镜观察人未受精及受精未分裂卵母细胞的超微结构,分析卵母细胞受精失败、受精后不分裂的原因。方法分别将收集的体外受精(in vitrofertilization,IVF)及卵胞浆内精子注射(intracytoplasmic sperm injection,ICSI)时受精失败、受精未分裂的卵母细胞进行固定、脱水、包埋、超薄切片,透射电子显微镜下观察超微结构。结果常规IVF受精失败成熟卵母细胞主要呈现的超微结构:①透明带正常,但卵膜下未见皮质颗粒,精核呈浓缩状态,周围未见线粒体、滑面内质网(SER)泡等细胞器围绕;②透明带为异常紧密状结构,卵膜下有一层线形排列皮质颗粒,线粒体为其主要的细胞器,缺少SER集合管、SER泡等细胞器,有时可见透明带内存在顶体完整的精子。ICSI时未受精的第二次减数分裂中期(metaphaseⅡ,MⅡ)卵母细胞呈现的超微结构:①可见卵膜下一层线形排列皮质颗粒,SER集合管、SER泡常与线粒体相连;注射的精子已褪去核膜,部分染色质已去浓缩;②皮质颗粒极少或分散存在,未在卵黄膜下排列;缺乏线粒体、SER集合管等细胞器或者线粒体孤立存在。受精未分裂卵母细胞呈现的超微结构:卵周隙内可见皮质颗粒排放的皮质内容物,线粒体数量少,胞质内有许多小脂滴填满的脂褐体;环纹片层(annulate lamellae,AL)异常聚集且成丛分布于胞浆中。结论常规IVF卵胞质中细胞器异常分布导致卵母细胞胞质不成熟影响其受精能力;透明带结构异常及诱导顶体反应缺陷可导致精子穿透障碍;卵母细胞激活失败与ICSI受精失败有关;脂褐体的出现及AL异常聚集与受精卵不分裂有关。
Objective To observe the ultrastructure of human unfertilized and fertilized undifferentiated oocytes by transmission electron microscopy and analyze the reasons why oocytes failed fertilization and did not divide after fertilization. Methods Fetal non-dividing fertilized oocytes were harvested for fixation, dehydration, embedding, ultrathin sectioning, in vitro fertilization in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) The ultrastructure was observed under a transmission electron microscope. Results The main ultrastructures of mature oocytes failed to fertilize conventional IVF. ①The zona pellucida was normal, but no cortical granules were found in the subependymal layer. The seminiferous nucleus was concentrated and there were no mitochondria and SER (endoplasmic reticulum) bubbles And other organelles around; (2) zona pellucida is an abnormal tight structure, there is a layer of cortical particles arranged linearly under the egg membrane, mitochondria as its major organelles, the lack of SER collection tube, SER bubbles and other organelles, sometimes visible in the zona pellucida with acrosome intact sperm. The ultrastructures of metaphase II (MII) oocytes during ICSI unimpregnated showed the following: (1) cortical granules arranged linearly under the subependymal layer, SER collection tube, and SER vacuoles often connected with mitochondria; injection Of the sperm have faded the nuclear membrane, some chromatin has been to enrichment; ② little or scattered cortical particles exist, not arranged in the yolk membrane; lack of mitochondria, SER collection tube organelles or mitochondria exist in isolation. Ultrastructure of fertilized non-dividing oocytes: The cortical granules in the perivitelline space showed cortical contents with a small number of mitochondria and many lipid droplets filled with liposomes in the cytoplasm. The annulate lamellae, AL) abnormally clustered and distributed in the cytoplasm. Conclusion The abnormal distribution of organelles in the cytoplasm of IVF oocytes causes the immature cytoplasm of oocytes to affect its fertilization ability. The structural defects of zona pellucida and the defects of acrosome reaction can lead to sperm penetration failure. The failure of oocyte activation is related to the failure of ICSI fertilization. The appearance of lipofuscin and AL abnormal aggregation and fertilized eggs are not divided.