卵丘细胞凋亡对体外培养卵母细胞发育潜能的影响

THE EFFECTS OF CUMULUS CELL APOPTOSIS ON DEVELOPMENTAL POTENTIAL OF HUMAN OOCYTE IN VITRO

目的研究体外培养中卵丘细胞凋亡对卵母细胞结构的影响,探讨体外受精中可用卵丘细胞凋亡率预测卵母细胞发育潜能的原因。方法对GV期人卵丘-卵母细胞复合体进行体外成熟培养,用HE染色、DAPI染色和原位末端标记(TUNEL)法标记3种方法对单卵卵丘细胞凋亡率进行检测。分为凋亡率高和低的2组,用光镜和透射电镜观察卵母细胞的结构。对MⅡ期卵母细胞进行体外受精,培养2d后在光镜下评价胚胎质量。结果卵丘细胞凋亡率低的卵母细胞结构和发育潜能良好;卵丘细胞凋亡率高的卵母细胞发育潜能差,存在各种结构异常,包括围卵周隙不均,对应不同部位的细胞质发育不同步;细胞质中出现堆积不均的细胞器团、次级溶酶体、及可能由溶酶体降解造成的大量空隙;线粒体外膜和嵴模糊、膨大,呈现凋亡迹象;第一极体碎裂;透明带异常增厚或变薄。相应的卵丘细胞微绒毛和细胞连接减少。结论揭示了卵丘细胞凋亡对卵母细胞结构的影响,揭示了卵丘细胞凋亡率影响卵母细胞发育潜能的原因。

Objective To study the effect of cumulus cell apoptosis on the structure of human oocyte cultured in vitro and to probe into the reason why cumulus cell apoptosis rate can be used clinically to predict the developmental potential of oocyte. Methods Cumulus-enclosed oocyte complexes （COCs） at the GV stage were cultured in vitro. The apoptosis rate of cumulus cells for every COC was evaluated with HE staining, DAPI staining and TUNEL labeling methods. Oocytes were divided into two groups according to their cumulus cell apoptosis rate. Structure of oocytes was observed under light microscope and transmission electron microscope. Mature oocytes at M Ⅱ stage were fertilized and cultured in vitro for two days. Then the embryos were evaluated according to their morphology under light microscope. Results The oocytes with low cumulus cell apoptosis rate had a normal structure and a promising developmental potential, while the oocytes with high cumulus cell apoptosis rate had deformed structures and a poor developmental potential. Some deformed oocytes had uneven perivitelline space. For oocytes with a small pefivitelline space, the organelle development was slow compared with those with a large pefivitelline space. Some had unevenly piled organelles. Secondary lysosomes and large space probably caused by the decopmounding of lysosomes were found in the ooplasm of these oocytes. The deformed oocytes appeared to have lots of swollen mitochondria with blurred cfistae and membrane. Some of these oocytes had fractured first polar bodies and abnormally thick or thin zona pellucida. The cell junctions and microvilli of cumulus cells reduced as well. Conclusion The effect of cumulus cell apoptosis on the structure of oocyte cultured in vitro was revealed. The reason why cumulus cell apoptosis rate prognosticated the developmental potential of oocyte was demonstrated in the aspect of oocyte structure.