外源褪黑素对羔羊卵母细胞成熟及卵丘扩展相关基因表达的影响

Effects of Exogenous Melatonin on Maturation and Cumulus Expansion Related Gene Expression of Lamb Oocyte

目的本研究旨在探究在羔羊卵母细胞体外成熟培养过程中,添加外源褪黑素对羔羊体外受精胚胎发育质量以及卵丘扩展相关基因表达的影响。方法在成熟培养液中添加不同浓度(0, 10, 25和50μg/L)的褪黑激素,研究外源褪黑素对羔羊体外受精胚胎卵裂率、囊胚率以及囊胚DNA核碎片化的影响,通过DCFH-DA染色检测卵母细胞中活性氧含量,通过RT-qPCR技术检测卵丘扩展相关基因(PTX3、HAS2和PTGS2)表达的变化。结果结果表明成熟液中添加25μg/L褪黑素组的囊胚形成率(19.8%±1.9%)显著高于不含褪黑素的对照组(10.1%±4.1%)(P<0.05)。成熟液中添加50μg/L褪黑素产生的囊胚DNA碎片化的比例(2.2%±1.1%)显著低于不使用褪黑素的对照组(10.9%±2.9%)(P <0.05)。与对照组相比,添加外源褪黑素组细胞内的活性氧显著降低(P <0.05)。添加褪黑素组卵丘细胞扩展相关基因PTX3、HAS2表达量显著高于(P <0.05)对照组,而PTGS2的表达量在各组之间没有显著差异(P> 0.05)。结论综上表明,在成熟培养基中添加褪黑素(25μg/L)有益于羔羊早期胚胎的发育和质量。

Objective The purpose of this study was to investigate the effects of exogenous melatonin on the developmental quality of in vitro fertilized embryos and the expression of cumulus expansion-related genes during the in vitro maturation of lamb oocytes. Methods Different concentrations of melatonin(0, 10, 25, and 50 μg/L) were added to maturation medium, the cleavage rate, blastocyst rate and blastocyst DNA nuclear fragments of lamb IVF embryos were studied, the content of reactive oxygen species in oocytes was detected by DCFH-DA staining, the expression changes of cumulus expansion related genes(PTX3, HAS2 and PTGS2) were detected by RT-qPCR technology. Results The results showed that the blastocyst formation rate of the group with 25 μg/L melatonin in the maturation medium(19.8% ± 1.9%) was significantly higher than that of the control group without melatonin(10.1% ± 4.1%)(P<0.05). The proportion of blastocyst DNA fragment nuclei(10.9% ± 2.9%) after adding 50 μg/L melatonin to the maturation medium was significantly lower than that without melatonin(2.2% ± 1.1%)(P<0.05). Compared with the control group, the addition of exogenous melatonin significantly reduced the intracellular reactive oxygen species(P<0.05). The expression levels of cumulus expansion related genes PTX3 and HAS2 in the melatonin-added group were significantly higher than those in the control group(P<0.05), while the expression levels of PTGS2 were not significantly different between the groups(P>0.05).Conclusion In summary, the results show that the addition of melatonin(25 μg/L) to the maturation medium is beneficial to the development and quality of lamb early embryos.