uPA对体外成熟牛卵母细胞生发泡破裂的影响

Effect of urokinase-type plasminogen activator on GVBD of maturebovine oocyte in vitro

为了探明尿激酶型纤溶酶原激活剂（uPA）对体外成熟牛卵母细胞核成熟的可能作用,试验根据uPA及其特异性抑制剂（amiloride）的有效浓度分成4组,即对照组、uPA（0.5 ng/mL）组、amiloride（1μg/mL）组及联合添加（uPA＋amiloride）组,然后采用地衣红染色方法分别研究体外成熟培养6 h与12 h后uPA对牛卵母细胞生发泡破裂（GVBD）的影响,采用ELISA方法研究体外成熟培养6 h后uPA对牛卵丘-卵母细胞复合体（COCs）中cAMP水平变化的可能调节作用。结果表明：减数分裂不同时期（GV期、GVBD期及MⅠ~MⅡ期）卵母细胞染色质（体）形态呈现不同特征。卵母细胞在体外成熟培养6 h后各组细胞均处于GV^GVBD期,其中uPA组GVBD期卵母细胞率（12.72%）显著低于对照组（24.83%）与联合添加组（30.42%,P〈0.05）;体外成熟培养12 h后,uPA组GVBD期卵母细胞率（54.81%）显著高于对照组（37.49%）与联合添加组（25.64%,P〈0.05）,uPA组MⅠ~MⅡ期卵母细胞率（44.85%）显著低于对照组（62.51%）与联合添加组（74.36%,P〈0.05）;体外成熟培养6 h后,uPA组cAMP水平显著高于对照组与联合添加组（P〈0.05）。说明uPA通过上调cAMP水平阻滞牛卵母细胞的减数分裂进程。

The aim of the present study was to investigate the effect of urokinase-type plasminogen activator （uPA） on nuclear maturation of bo- vine oocyte in vitro. According to the effective concentration of uPA and its specific inhibitor amiloride, the experiment was divided into four groups： control group, uPA （0.5 ng/mL） group, amiloride （ 1 Ixg/mL） group and combined group （uPA ＋ amiloride）.Then the effects of uPA on germinal vesicle break down （GVBD） of bovine oocytesafter in vitro maturation for 6 h and 12 hwere studied by orcein staining. And the ELISA method was used to study the possible regulatory effect of uPA on the changes of cAMP level in bovine cumulus oocyte complex （COCs） after in vitro maturation for 6 h.The results showed that the chromatin morphology of oocytes at different stages of meiosis （ GV stage, GVBD stage and M I-M tl stage） had different chm＇acteristics.The oocytes in each group were in GV-GVBD stage after in vitro maluration for 6 h. The ratio of oocytes in GVBD stage in the uPA grop （12. 72%） was significantly lower than that in the control group （24. 83%） and the combined group （30. 42%,P〈0. 05）. After in vitro maturation for 12 hours, the ratio of oocytes in GVBD stage in uP A group （54.81%） was significantly higher than that in the control group（37.49%） and the combined group（25.64%,P〈0.05）. The ratio of oocytes in M I-M II stage in uPA group （44. 85% ） was significantly lower than that in control group （62. 51% ） azld combined group （74. 36%, P〈0. 05 ）. The level of cAMP in uPA group was significantly higher than that in control group and combined group after maturation for 6 h in vitro （P〈0. 05） .The results indicated that uPA could block the meiosis process of bovine oocytes by up-regulating the level of cAMP.