紫草素对小鼠卵母细胞发育成熟的影响

Effects of shikonin on the maturation of mouse oocytes

目的观察紫草素对小鼠卵母细胞发育成熟的影响,并探讨其作用机制。方法采用6~8周龄ICR雌性小鼠的GV期未成熟卵母细胞,分别放入含有5、10、20、50μg/mL浓度紫草素的4组培养液,另外再配置4组含有3-异丁基-1-甲基黄嘌呤(IBMX)抑制剂的紫草素培养液,并以不含紫草素的培养液为对照组。单纯含紫草素的各组培养液及对照组直接进行卵母细胞培养,含IBMX抑制剂的紫草素培养液先静置12 h后再洗脱IBMX进行卵母细胞培养。各组均培养2 h后统计生发泡破裂(GVBD)率,12 h后统计第一极体排出率,并显微镜下观察紫草素对卵母细胞纺锤体及第一极体排出的情况。结果 20、50μg/mL紫草素对卵母细胞的发育成熟具有绝对抑制作用,GVBD率及第一极体排出率均为0;5μg/mL紫草素对卵母细胞无明显抑制作用(P>0.05);10μg/mL紫草素对卵母细胞的GVBD率无明显影响(P>0.05),但对第一极体排出率有明显抑制作用(P<0.05);显微镜下观察显示,对照组第一极体排出正常,仅有少量卵母细胞出现异常纺锤体形态,10μg/mL紫草素组则第一极体排出受到抑制,出现大量卵母细胞异常纺锤体形态,纺锤体呈现无极、散极或者多极等异常现象。结论高浓度的紫草素对卵母细胞发育成熟具有明显抑制作用,可影响纺锤体的形成和第一极体的排出,其作用机制可能与紫草素抑制卵母细胞能量代谢有关。

Objective To observe the effect of shikonin on the maturation of mouse ooeytes, and investigate its mechanism. Methods The immature oocytes of GV stage from 6 to 8 weeks old ICR female mice were put in four groups of shikonin nutrient solution that concentration was 5μg/mL, 10μg/mL, 20μg/mL and 50μg/mL respectively. In addition, there were other four groups that shikonin nutrient solution contained 3 - isobutyl 1 - methyl xanthine （IBMX） inhibitor, and the nutrient solution with no shikonin was treated as control group. The oocytes were directly cultured in four groups that merely contained shikonin and the control group. The shikonin nutrient solution containing IBMX inhibitor was set aside 12 h and then eluted with IBMX, finally the oocytes were cultured. The rate of germinal vesicle breaking down （GVBD） after culturing 2 h was evaluated, and the first polar body discharge rate after culturing 12 h were evaluated. The effects of shikonin on oocytes spindle and the discharge of first polar body were observed. Results The concentration of 20μg/mL and 50 μg/mL of shikonin had an absolute inhibitory effect on the maturation of ooeytes, and the rates of GVBD and the first polar body discharge were all zero. The concentration of 51μg/mL of shikonin had no obvious inhibitory effect on oocytes. The first polar body discharge in control group was normal, and only a few oocytes appeared abnormal spindle. The first polar body discharge in 10μg/mL shikonin group was restrained, and a large number of oocytes appeared abnormal spindle, and the spindle presents the anoma-lies such as the non -polar, sporadic polar, or muhi -polar. Conclusion The high concentration of shikonin has an obviously inhibitory effect on the maturation of oocytes, can affect the formation of spindle and the first polar body discharge, its mechanism can be linked to the inhibition of shikonin on the energy metabolism of oocytes.