人卵巢黄素化颗粒细胞生长液对体外成熟小鼠卵母细胞皮质颗粒分布的影响

Effect of luteinized granulosa cell conditioned medium on cortical granule of mouse oocytes matured in vitro

目的 探讨人卵巢黄素化颗粒细胞生长液对体外成熟小鼠卵母细胞皮质颗粒分布的影响。方法 将 6 35个小鼠未成熟卵母细胞随机分为对照组 (35 1个未成熟卵母细胞 )和实验组 (2 84个未成熟卵母细胞 )。实验组在培养基中加入 5 0 %人卵巢黄素化颗粒细胞生长液、卵泡刺激素 (终浓度为 75U/L) ,和雌二醇 (终浓度为 1nmol/L) ;对照组不加人卵巢黄素化颗粒细胞生长液 ,其余同实验组。每组分别于体外培养 16h和 18h ,对细胞核成熟的卵母细胞进行体外受精 ,采用直接免疫荧光标记法 ,用异硫酸酯荧光素 扁豆凝集素标记卵母细胞皮质颗粒 ,在激光扫描共聚焦显微镜下观察皮质颗粒的分布。结果 两组细胞核成熟的卵母细胞共 12 6个。卵母细胞细胞核成熟率 ,对照组培养16h为 70 0 % ,培养 18h为 75 1% ;实验组培养 16h为 76 5 % ,培养 18h为 83 1% ,两组比较 ,差异无显著性 (P >0 0 5 )。两组培养 16h的成熟卵母细胞的体外受精均失败 ,培养 18h后成熟卵母细胞均出现受精及胚胎发育。两组卵母细胞皮质颗粒呈皮质分布型 (即卵母细胞成熟的特征 )的比例 ,对照组培养 16h为 10 0 % ,培养 18h为 5 7 1% ;实验组培养 16h为 5 0 0 % ,培养 18h为 91 6 % ;每组培养 18h的皮质颗粒呈皮质分布型的比例均高于培养 16h者 (P

Objective To study the effe ct o f luteinized granulosa cell conditioned medium on cortical granule(CG) of the mo use oocytes matured in vitro. Methods Oocytes in germina l vesicle (GV) stage of Kunming mice were randomly divided into 2 groups according to different in vitro maturation(IVM) culture media. The study group medium co ntained 50% granulosa cell condition medium, follicle stimulating hormone 75 U/L and estrodial 1 nmol/L. The control group medium contained follicle stimulatin g hormone 75 U/L and estrodial 1 nmol/L. Oocytes were cultured for 16 or 18 hou rs. CG was examined by fluorescein isothiocyanate labeled Lens culinaris agglut inin under a confocal scanning laser microscope. Results A fter cultured for 16 hours, the nuclear maturation rates of control and study gr oups were 70.0% and 76.5%. After cultured for 18 hours, the maturation rates were 75.1% and 83.1%, respectively. There was no significant difference betwe en the two groups. After cultured for 16 hours, there was no pronuclear formati on in both groups. When culture was extended to 18 hours, fertilization occurr ed. After cultured for 16 hours, the rates of CGs forming a line under membrane were 10.0% and 50.0% in control and study groups respectively. When c ulture was extended to 18 hours, the rates rose to 57.1% and 91.6% accordingly. The rate of 18 h of each group was significantly higher than that of 16 h(both P <0.001). The rate of study group of 18 h was significantly higher than that of control group(P<0.05). Conclusion Granulosa c el l conditioned medium could improve the mouse oocytes maturation competence in vi tro.