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目的探讨卵母细胞发育过程中同型半胱氨酸(HCY)对组蛋白表观遗传修饰的影响。方法首先使用10只2周ICR雌性小鼠建立完整卵泡的体外培养体系,在卵母细胞发育早期,利用免疫组织化学方法,观察HCY对甲基化组蛋白H3K4、H3K9以及乙酰化组蛋白H3K9分布的影响;其次使用10只4周ICR雌性小鼠,利用卵母细胞的体外成熟培养体系,观察HCY对卵母细胞成熟过程的影响,同时利用实时定量PCR方法,观察在此成熟过程中HCY对卵母细胞内组蛋白乙酰化水平的调控酶GCN5和HDAC表达的影响。结果 HCY明显抑制卵母细胞的体外成熟,在HCY作用下,甲基化组蛋白H3K4、H3K9以及乙酰化组蛋白H3K9的分布没有变化,但是表达强度降低,核呈现去浓缩的趋势。成熟过程中HCY并不改变基因GCN5的表达水平,却明显抑制卵母细胞内HDAC基因的表达。结论卵母细胞发育过程中高水平的同型半胱氨酸影响组蛋白的表观遗传修饰,HCY对卵母细胞核内组蛋白甲基化和乙酰化修饰的影响有可能是造成核染色体稳定性下降的重要原因。
Objective To investigate the effect of homocysteine (HCY) on the epigenetic modification of histones during oocyte development. Methods To establish an in vitro culture system of intact follicles using 10 2-week ICR female mice. At the early stage of oocyte development, the distribution of methylated histone H3K4, H3K9 and acetylated histone H3K9 by HCY was observed by immunohistochemistry Followed by the use of 10 4-week ICR female mice, the use of oocytes in vitro maturation culture system to observe the effect of HCY on the maturation of oocytes, while using real-time quantitative PCR method to observe the maturation of HCY in this process Effect of Ovarian Histone Acetylation Regulating Enzymes GCN5 and HDAC Expression in Oocytes. Results HCY significantly inhibited the in vitro maturation of oocytes. Under the action of HCY, the distribution of methylated histone H3K4, H3K9 and acetylated histone H3K9 did not change, but the expression intensity decreased and the nucleus showed a tendency of decondensation. HCY does not change the expression of gene GCN5 during maturation, but significantly inhibits the expression of HDAC gene in oocytes. Conclusion High levels of homocysteine affect the epigenetic modification of histone during oocyte development. The effect of HCY on the histone methylation and acetylation of oocyte may result in the decrease of nuclear chromosomal stability important reason.