牛卵泡微环境对颗粒细胞和卵母细胞及早期胚胎发育的影响

Effects of Bovine Follicles Microenvironment on Granulosa Cells,Oocytes,and Early Embryos Development

[目的]研究卵泡微环境对颗粒细胞与卵母细胞质量及体外受精胚胎发育的影响。[方法]从不同直径大小的卵泡中收集卵母细胞和颗粒细胞,Hoechst33342染色观察卵母细胞核形态;采用Annexin-V-FITC/PI法染色、流式细胞仪检测颗粒细胞的凋亡情况;进行受精卵体外培养,并计算受精率及受精卵的囊胚率。[结果]大卵泡(>10 mm)和小卵泡(<3 mm)内颗粒细胞凋亡率明显高于3～7和7～10 mm卵泡内的颗粒细胞凋亡率(P<0.05)大卵泡和小卵泡内的大量卵母细胞呈现出染色质固缩、核碎裂及胞浆浓缩等凋亡特征。3～7和7～10 mm卵泡内的卵母细胞成熟率明显高于大卵泡和小卵泡(P<0.05)。来源于不同直径卵泡的卵母细胞,其卵裂率和囊胚率差异均不显著。[结论]卵泡微环境是影响牛胚胎体外生产的主要环节,直径为3～10 mm卵泡内颗粒细胞质量及卵母细胞的体外成熟率较高,卵泡直径对卵母细胞受精及受精卵的早期发育没有影响。

[Objective] The aim of this study was to investigate the effects of bovine follicles microenvironment on the quality of granulosa cells and oocytes as well as subsequent embryos development after in vitro fertilization.[Method] The oocytes and granulosa cells were collected from follicles of different diameters.Hoechst33342 staining was used to observe nuclear morphology of oocytes.Annexin-V-FITC/PI staining and flow cytometry were used to detect apoptosis of granulosa cells.After the oosperm were cultured in vitro,fertilization rate and blastocyst rate of oosperm were determined.[Result] The granulosa cells in the large follicles with diameter 10 mm or small follicles with diameter 3 mm had significantly higher apoptosis rate than those in the follicles with diameter of 3-7 or 7-10 mm（P 0.05）.Many oocytes in the follicles with diameter 10 mm or 3 mm showed chromatin condensation,nuclear fragmentation and pyknosis.The maturation rate of oocytes with diameter of 3-7 or 7-10 mm was significantly higher than that in the large or small follicles（P 0.05）.The cleavage rate and blastocyst rate were not significantly different between the oocytes in the follicles with different diameters.[Conclusion] The follicles microenvironment is a crucial aspect affecting in-vitro embryos production.In the follicles with diameter of 3-10 mm,the quality of granulosa cells as well as the in-vitro maturation rate of oocytes was higher.And the diameter of follicles does not affect oocytes fertilization and early embryonic development of oosperm.