罗汉果甜苷Ⅲ延缓猪卵母细胞体外老化的作用机制

Mechanism effects of mogroside Ⅲ on the alleviation of porcine oocyte aging

  • 摘要: 【目的】验证罗汉果甜苷Ⅲ是否具有延缓猪卵母细胞体外老化进程的功效,为采用植物提取物延缓卵母细胞体外老化提供参考依据。【方法】以体外培养44 h获得的猪卵母细胞为研究对象,通过孤雌激活筛选出罗汉果甜苷Ⅲ对猪卵母细胞的有效作用浓度;然后设新鲜卵母细胞(Fresh)、老化卵母细胞(Aging)及老化卵母细胞添加罗汉果甜苷Ⅲ(MogⅢ)3个处理组,采用免疫荧光染色和实时荧光定量PCR等分别检测猪卵母细胞的线粒体数量和分布、腺嘌呤核苷三磷酸(ATP)含量、线粒体膜电位、活性氧(ROS)水平、DNA损伤、抗氧化相关基因表达及早期凋亡水平等指标。【结果】老化导致猪卵母细胞孤雌激活后的胚胎分裂率及囊胚发育率极显著下降(P<0.01,下同),而添加罗汉果甜苷Ⅲ能有效提高老化卵母细胞孤雌激活后的胚胎分裂率和囊胚发育率,尤其以添加25.0 µmol/L罗汉果甜苷Ⅲ的效果最优,故确定25.0 µmol/L为罗汉果甜苷Ⅲ的有效作用浓度。与Fresh组猪卵母细胞相比,Aging组猪卵母细胞线粒体荧光强度显著下降(P<0.05,下同)、ATP含量极显著下降、线粒体膜电位显著下降、抗氧化相关基因相对表达量极显著下降,而ROS水平、γ-H2AX荧光强度和Annexin-V阳性率均极显著上升;但添加25.0 µmol/L罗汉果甜苷Ⅲ后上述问题均得到有效改善,即罗汉果甜苷Ⅲ能有效缓解猪卵母细胞的老化进程。【结论】罗汉果甜苷Ⅲ通过改善老化猪卵母细胞线粒体数量和ATP含量的异常、恢复ROS平衡、上调抗氧化相关基因表达,以及缓解猪卵母细胞老化引起的DNA损伤和早期凋亡,而有效延缓猪卵母细胞的老化进程,进而提高猪卵母细胞的体外发育能力。

     

    Abstract: 【Objective】The study aimed to verify whether mogroside Ⅲ had the effects to delay the aging process of porcine oocytes in vitro and provide reference for utilizing plant extracts to alleviate oocyte aging in vitro.【Method】Porcine oocytes cultured in vitro for 44 h were used as research subjects. The effective concentration of mogroside Ⅲ for porcine oocytes was screened by parthenogenetic activation (PA). Three treatment groups were set up:fresh oocytes (Fresh),aging oocytes(Aging),and aging oocytes supplemented with mogroside Ⅲ. Immunofluorescence staining and real-time fluorescence quantitative PCR were employed to detect mitochondrial content and distribution,adenosine triphosphate(ATP)content,mitochondrial membrane potential,reactive oxygen species(ROS)levels,DNA damage, antioxidant-related gene expression,and early apoptosis levels in porcine oocytes.【Result】Aging extremely significantly decreased the embryonic cleavage rate and blastocyst development rate of parthenogenetic activated embryos derived from porcine oocytes(P<0.01,the same below). The addition of mogroside Ⅲ effectively improved the embryonic cleavage rate and blastocyst development rate of aging oocytes,especially at 25.0 µmol/L. which was therefore determined as the effective concentration for mogroside Ⅲ. Compared with the Fresh group,the Aging group exhibited significant decrease in mitochondrial fluorescence intensity(P<0.05,the same below),and extremely significant decrease in ATP content,significant decrease in mitochondrial membrane potential,and extremely significant decrease in the relative expression levels of antioxidant-related genes,while ROS levels,γ-H2AX fluorescence intensity,and Annexin-V positive rate were extremely significantly increased. However,the addition of 25.0 µmol/L mogroside Ⅲ effectively improved the above problems,indicating that mogroside Ⅲ could effectively alleviate the aging process of porcine oocytes.【Conclusion】Mogroside Ⅲ effectively delays the aging process of porcine oocytes by improving mitochondrial number and ATP content,restoring ROS balance,up-regulating the expression of antioxidant-related genes,and alleviating DNA damage and early apoptosis caused by the aging of porcine oocytes,thereby enhancing the in vitro developmental competence of porcine oocytes.

     

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