毛喉素对猪卵母细胞降脂及冷冻保护效果研究

Effects of Forskolin on Lipid Degradation and Cryopreservation of Porcine Oocytes

猪卵母细胞脂质含量高被认为是其冷冻效率低下的重要因素之一。本文通过在猪卵母细胞体外成熟过程中添加化学降脂剂毛喉素(forskolin),检测冷冻前后成熟卵母细胞的脂滴含量、脂滴超微结构、线粒体膜电位、活性氧水平、早期凋亡指标、冻后存活率及发育潜能变化等,研究其对猪卵母细胞降脂和冷冻保护的效果。结果显示,成熟过程中毛喉素处理可部分提高卵母细胞成熟率,但差异不显著(P>0.05)。尼罗红荧光染色显示,毛喉素处理后卵母细胞内脂滴数量及面积在冷冻前后均极显著低于未处理组(P<0.01);超微结构观察发现,经毛喉素处理的卵母细胞中非均质脂滴数量与均质脂滴数量的比例扩大,且比均质脂滴面积更大;冷冻后,卵母细胞中以非均质脂滴为主,脂滴变小变少,分布不均匀,毛喉素处理后非均质与均质间的脂滴数量比例进一步增加。毛喉素处理极显著上调冻后卵母细胞线粒体膜电位(1.04 vs. 0.51,P<0.01),减轻氧化应激,降低早期凋亡率(68.30%vs. 86.03%,P<0.01),从而有效提高了冷冻后卵母细胞的存活率(71.17%vs. 51.47%,P<0.01)和孤雌激活卵裂率(16.63%vs. 8.23%,P<0.05)。本研究表明,在猪卵母细胞体外成熟过程中添加毛喉素可有效降低细胞内脂质含量,并通过降低氧化损伤和改善线粒体功能,达到提高冻后存活率和发育能力的目的。

The high lipid content of porcine oocytes is considered one of the most important factors for its low freezing efficiency. The purpose of this study was to evaluate the effect of chemical lipid-lowering agent forskolin on the lipid degradation and anti-freezing ability of porcine oocytes during in vitro maturation. The content and ultrastructural changes of lipid droplets, mitochondrial membrane potential, reactive oxygen species level, early apoptosis rate, survival rate and developmental potential of matured porcine oocytes with or without forskolin treatment were detected before and after vitrification. The results showed that forskolin treatment partially improve the maturation rate of oocytes, but the difference was not significant(P>0.05). The results of nile red fluorescence staining showed that the number and area of lipid droplets before and after vitrification in forskolin-treated oocytes were significantly lower than those untreated(P<0.01). Ultrastructural observation showed that the ratio of the number of heterogeneous lipid droplets to homogeneous lipid droplets in the oocytes treated with forskolin increased, and the area of heterogeneous lipid droplets was larger than that of homogeneous lipid droplets. The heterogeneous lipid droplets were irregularly distributed in oocytes after vitrification as the main lipid droplets form, of which became smaller and less in number. After forskolin treatment, the proportion of lipid droplets between heterogeneous and homogeneous further increased. Forskolin treatment also significantly increased the mitochondrial membrane potential of vitrified oocytes(1.04 vs. 0.51, P<0.01), alleviated oxidative stress and reduced early apoptosis rate(68.30% vs. 86.03%, P<0.01), which effectively improved the survival rate of vitrified oocytes(71.17% vs. 51.47%, P<0.01) and its cleavage rate after parthenogenetic activation(16.63 vs. 8.23%, P<0.05). In conclusion, the addition of forskolin during the in vitro maturation of porcine oocytes effectively reduced the content of intracellular lipids, and improved the survival rate and development ability after vitrification through alleviating oxidative damage and improving mitochondrial function.