褪黑素对绵羊卵母细胞体外成熟的影响

Effect of melatonin on the maturation of sheepoocytes in vitro

【目的】本试验旨在探索不同浓度褪黑素(melatonin,MLT)对绵羊体外成熟卵母细胞发育的影响,以期优化绵羊卵母细胞体外成熟体系。【方法】本试验分别添加10-3、10-5、10-7、10-9、10-11、0mol/L褪黑素,以检测褪黑素对绵羊体外成熟卵母细胞的极体率、孤雌激活后卵母细胞的卵裂率、囊胚率及bcl-2(抗凋亡基因)、bax(促凋亡基因)的表达水平的影响。【结果】10-7、10-9、10-11 mol/L MLT组的极体排出率(41.04%±3.37%、43.13%±3.82%、46.28%±0.36%)显著高于对照组(23.43±2.43%,P<0.05),10-11 mol/L MLT组极体率最高。10-9、10-11 mol/L MLT组(55.23%士3.19%、54.30%±0.85%)卵裂率显著高于对照组(40.9%1±4.67%,P<0.05),且10-11 mol/L MLT卵裂率最高。10-9、10-11 mol/L MLT组(19.23%±4.31%、18.84%±5.35%),囊胚率显著高于对照组(7.51%±1.40%),10-9 mol/L MLT组囊胚率最高。10-9 mol/L MLT极显著上调卵母细胞中bcl-2mRNA的表达量,10-7 mol/L MLT可极显著下调卵母细胞中bax mRNA的表达量。在8、16h,10-9 mol/L MLT组的bcl-2与bax比值较大。【结论】在绵羊卵母细胞体外成熟培养基中添加10-7、10-9、10-11 mol/L的褪黑素可促进卵母细胞体外成熟以及孤雌激活后卵母细胞的胚胎发育,添加10-9 mol/L褪黑素是促进绵羊卵母细胞体外成熟的最佳浓度。褪黑素通过上调bcl-2的表达,下调bax的表达,可促进绵羊卵母细胞体外成熟。

[Objective]This study was to explore effects of different concentrations of melatonin （melatonin, MLT） on the devel- opmental potential of ovine oocytes matured in vitro, in order to optimize ovine ooeytes in vitro maturation system. [Methods] The different concentration of MLT （10.3 ,10-5 ,10 7,10-9,10-11 and 0 mol/L） was added, respectively, to the maturation medi- um during ovine oocytes maturation in vitro . The polar body rate of oocytes after maturation , the cleavage rate and blastoeyst rate of oocytes after IVF were counted. Meanwhile, the expression level of bcl-2 （Anti-apoptotic genes） and bax （pro-apoptot- ic genes） were detected by RT-PCR. [Results]The polar body rates of 10.7 , 10 9 and 10-11 mol/L MLT group（41.04%±3.37%, 43.13%±3.82% ,46.28%±0.36%）were higher than that of the control group（23.43%±2.43% ,P〈0. 05）, and the polar body rate of 10-11 mol/L MLT group is the highest. The cleavage rates of 10-9 and 10-11 mol/L MLT group （55. 23%±3.19%, 54.30%±0.85 %） were higher than that of the control group（40.91%±4.67 %,P〈0.05）, and the eleavage rate of 10-11 mol/L MLT group is the highest. The blastocyst rates of 10-9 and 10-11 mol/L MLT group （19.23%±4.31% ,18.84%±5.35%）in- creased compared with the control group（7.51%±1.40%, P〈0.05）, and the blastocyst rate of 10 9mol/L MLT group is the highest. 10-gmol/L MLT was significantly upregulated oocytes bcl-2 mRNA expression levels, 107 mol/L MLT can signifi-cantly down-regulated expression of bax mRNA in oocytes . At 8h and 16h, the ratio of bcl-2 and baz 10,9 mol/L MLT is the maximum . [Conclusion] Concentrations of 10 7,10 s, 10-11 mol/L melatonin added in sheep oocyte maturation medium may pro- mote oocyte maturation and embryonic development after parthenogenetic activation , 10-9 mol/L MLT was the optimum con- centration to promote maturation of sheep oocytes. Melatonin promotes sheep oocytes maturation in vitro by ascending the ex- pression of bcl-2, descending of bax expression.