目的:通过卵子老化过程中皮质颗粒的变化和自发孤雌激活比例,探讨老化卵子受精异常的原因.方法:对8～10周龄雌鼠注射孕马血清促性腺激素和绒毛膜促性腺激素14h后,于不同时间段取出MⅡ期成熟卵子分作体内不同时间老化组(MⅡ+ 0h、MⅡ+ 6h、MⅡ+ 12h、MⅡ+ 18h)；成熟MⅡ+0h卵子取出后体外分别培养6h、12h、18h作为体外老化组.对各组卵子免疫荧光染色,共聚焦显微镜成像显示染色体形态和皮质颗粒分布.体内各组卵子取出后继续体外培养8h统计自发孤雌激活的比例和碎裂卵子数.结果:体内和体外MⅡ+ 12h、MⅡ+ 18h组自发皮质颗粒排放比例和皮质颗粒内迁数明显高于MⅡ+ 0h、MⅡ+ 6h组.体外MⅡ+ 18h组自发皮质颗粒排放比例和皮质颗粒内迁数低于体内MⅡ+ 18h组.体内MⅡ+ 12h、MⅡ+ 18h组卵子取出继续体外培养8h后孤雌激活比例和死亡比例明显高于体内MⅡ+ 0h和MⅡ+ 6h组.结论:卵子在老化过程中存在皮质颗粒自发排放和皮质颗粒内迁,以及自发孤雌激活和碎裂化,这些因素均可能导致老化卵受精能力的降低.卵子老化过程中皮质颗粒排放和内迁可能受到体内环境的影响.%Objective; To explore the cortical granule (CG) change and parthenogenetic activation during mouse oocyte aging. Methods; Female mice, 8-10 weeks of age, were induced to superovulation with pregnant mare serum gonadotropin (PMSG; 10U, i. p. ) followed 48 h later by hCG (10 U, i. p. ). Oocytes were retrieved at different time intervals (MII +0h, +6h, + 12h, +18h) (in vivo aging) or MII + Oh oocytes cultured for different time periods ( +6h, +12h, + 18h) (in vitro aging) . CG distribution and release were assessed by fluorescene isothiocyanate - labelled Lens culinaris agglutinin ( FITC ?LCA) staining and laser confoeal microscopy. Spontaneous parthenogenetic activation and fragmentation of oocytes were observed after anther 8h of culture of different in vivo aging groups. Results; Spontaneous cortical granule exocytosis (CEG) and CG inward movement rates in MII + 12h, MII + 18h groups were statistically higher than those in MII +0h, MII +6h groups in both in vivo and in vitro aging oocytes. Spontaneous CGE and CG inward movement in M + 18h group was lower in in - vitro aging group than those of in vivo aging counterpart. Spontaneous parthenogenetic activation and oocyte fragmentation ratios after further 8h of culture were higher in MII + 12h,MII + 18h groups than those in MII +0h, MII +6h groups. Conclusion; Spontaneous CGE and CG inward movement increased during oocyte aging both in vivo and in vitro. There were also increased parthenogenetic activation and oocyte fragmentation ratios as oocyte aged. All these may cause the low fertility of aged oocyte. Spontaneous CGE and CG inward movement may be accelerated by maternal environment changes.
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