牛卵母细胞皮层颗粒荧光染色法质成熟鉴定

THE IDENTIFICATION OF CYTOPLASMIC MATURATION IN BOVINE OOCYTE WITH CORTICAL GRANULES FLUORESCENT STAINING

以皮层颗粒 (CG)单层分布于质膜下做为卵母细胞质成熟的指标 ,利用CG荧光染色法 ,研究了不同类型牛卵母细胞的质成熟情况。结果表明 :(1)采自直径大于 5mm、2～ 5mm和小于 2mm卵泡的卵母细胞 ,成熟培养前的质成熟率分别为 4 2 %、1 8%和 0 ,三者间无显著差异。但所有的大卵泡卵以及 95 2 %的中卵泡卵 ,CG已分布于皮层 ,而小卵泡卵中则有 51 7%的卵子 ,CG尚未迁移至皮质区 ,仍以簇的形式分布于细胞质的中间。经 2 4h成熟培养后 ,三类卵子的质成熟率分别达到 54 5%、2 8 6 %、和 7 1% ,三者间存在显著差异 (P <0 0 5)。小卵泡卵内的CG趋向于聚集成更大的簇 ,并向皮质区迁移。 (2 )根据卵丘细胞情况而分的四类卵母细胞 :A(带有 5层以上致密卵丘细胞 )、B(带有 3～ 5层致密卵丘细胞 )、C(裸卵或半裸卵 )和D(卵丘细胞已扩散成蜘蛛网状 ) ,成熟培养前的质成熟率分别为 4 3%、0、0和 2 8 0 % ,第四者显著高于前三者 (P <0 0 1)。成熟培养后 ,A、B类卵子的质成熟率 (2 3 1%和 10 8% )显著低于D类卵子 (88 2 % ) (P <0 0 1)。裸卵表现为完全缺乏CG或形态异常。 (3)采卵结果表明 ,大、中、小卵泡卵中 ,A类卵子所占的比例依次减少 (97 7%、56 3%和 18 4 % ) ,B类卵子依次增多 (2 3%、36 6 %和 52 0 % )。

Cortical granules (CG) fluorescent staining was used to investigate the cytoplasmic maturation (CM) in bovine oocytes.The monolayer distribution of CG beneath the oolemma was considered as the criterion of CM.The results showed that (1)The CM rates of oocytes recovered from >5 mm,2 5 mm and <2 mm follicles in diameter were 4.2%,1.8% and 0,respectively.CG in all oocytes from >5 mm follicles and 95.2% oocytes from 2 5 mm follicles were located at the cortex of oocytes.CG in 51.7% oocytes from <2 mm follicles were randomly distributed in plasma in small cluster and had not migrated into the cortex.After 24h IVM (in vitro maturation) culture,the CM rates in three groups were 54.5%,28.6% and 7.1%,respectively,there were significant differences among them (P<0.05).The CG in oocytes from <2 mm follicles tended to aggregate into bigger cluster and migrate towards the cortex.(2)Based on the layer and morphology of cumulus cells,the oocytes were divided into 4 groups as follows,A:compact and thick,B:compact but thin,C:naked and D:expanded.The CM rates of A～D were 4.3%,0,0 and 28 0%,respectively.D was significant higher than the former three groups (P<0 01).After IVM,the CM rates of A and B (23.1% and 10.8%) were significantly lower than that of D (88 2%,P<0 01).But the naked oocytes showed disappearation of CG.(3)Oocyte collection results indicated that the percentages of A in >5 mm,2 5 mm,and <2 mm follicle oocytes were 97 7%, 56 3% and 18.4%,that of B were 2.3%,36.6% and 52.0%.It was concluded that the decreasing of CM rates of oocytes from big follicles to small follicles was coincident with the decreasing of CM rates of oocytes with cumulus cells from thick to thin.