ASH1L和JMJD3在人卵子成熟和种植前胚胎发育中的作用及调控机制

A good developmental potential of embryos is critical for the patients undergone in vitro fertilization - embryo transfer (IVF-ET). Low embryonic development potential usually leads to a serious waste of embryos, and also increases the economic burden of patients. It has been documented that histone lysine methylation is involved in the mammalian oocyte maturation and early embryo development. However, the molecular mechanism remains poorly understood. Our previous studies found that the levels of H3K4me3 and H3K27me3 exhibited dynamic changes in the process of oocyte maturation and embryo development.Namely, the level of H3K4me3 decreased steadily from germinal vesicle to metaphase II stage, obviously increased from zygote stage to four-cell tage, and reached the lowest at eight-cell stage. A sharp increase was then observed at blastocyst stage. On the other hand, the level of H3K27me3 slightly changed from germinal vesicle stage to zygote stage, then decreased steadily and reached the lowest at eight-cell stage, followed by a significant increase at blastocyst stage. We also found that the expression levels of ASH1L,the H3K4me3's methylase,and JMJD3, the H3K27me3's demethylase, were higher than those of others,respectively. Interestingly, the excpresion levels of ASH1L and JMJD3 ,showed dynamic changes along with the oocyte maturation and preimplantation embryo development,were in accordance with the changs of H3K4me3 and H3K27me3 during this period.Based on these important findings, our project will keep to carry out the following work: Analysis of upstream regulator of ASH1L and JMJD3 genes using co-transfection and luciferase reporter assays; Analysis the function of ASH1L and JMJD3 genes in HeLa cells using gene over-expression and interference; Screen of ASH1L and JMJD3 target genes using RNA-seq and RT-PCR. Demonstration of these issues will provide new theoretical basis and intervention targets to improve embryo developmental potential in IVF-ET cycles.

胚胎具有良好发育潜能是体外受精-胚胎移植（IVF-ET）成功的关键因素，胚胎发育潜能低造成胚胎资源严重浪费。组蛋白赖氨酸甲基化参与了哺乳动物卵和胚胎发育的调控，但机制不详；我们前期发现在人卵成熟和胚胎发育中：H3K4me3水平从GV到MII期逐渐下降，4细胞期略有上升，8细胞期最低，囊胚期升高；H3K27me3水平变化与之类似；ASH1L 、MLL1、RBP2、PRC2、UTX和JMJD3分别是H3K4me3和H3K27me3的甲基化酶和去甲基化酶，其中ASH1L和JMJD3的基因表达在mRNA水平较高，且与H3K4me3和H3K27me3的变化基本一致。本项目将进一步研究：分析调控ASH1L和JMJD3基因表达的转录因子；探讨ASH1L和JMJD3基因的功能；筛选ASH1L和JMJD3调控的靶基因；通过这些问题的阐释，有望为提高IVF-ET周期中胚胎发育潜能提供新的理论依据和干预靶点。

胚胎具有良好发育潜能是体外受精-胚胎移植（IVF-ET）成功的决定因素，胚胎发育潜能低造成胚胎资源严重浪费。组蛋白赖氨酸甲基化参与了哺乳动物卵和胚胎发育的调控，但机制不详。我们研究发现甲基化酶JMJD3和去甲基化酶ASH1L的表达水平与H3K4me3 和 H3K27me3强度在人卵子成熟和种植前胚胎的发育中呈相似的动态变化趋势，可能共同参与调控卵子成熟和种植前胚胎的发育。利用现有的形态学评级标准无法准确预测卵子及胚胎的质量，而颗粒细胞在卵泡发育并排卵的过程中发挥着重要作用，因此通过检测卵丘颗粒细胞转录水平的变化，可能可以用于预测卵子及胚胎质量，以期发现预测卵子发育和胚胎质量的生物学marker。通过对原发性卵不熟患者/继发性卵不熟患者颗粒细胞进行RNA-seq分析，得到160显著差异表达基因，经GO和KEGG分析，聚焦研究LH受体相关信号通路。本课题成功对一例29岁原发性卵子不熟患者（2次IVF未获成熟卵），行颗粒细胞测序及质谱分析，发现其存在LHGCR关键位点突变，T694P，该突变为新发现位点。对患者家系进行了测序分析，其父亲和兄长均为杂合子T694P变异，蛋白质二维结构和三维结构模型的预测显示此位点恰好位于受体的膜内区域。后期在临床促排卵阶段使用孕激素等药物，促使该女性获得单胎妊娠。这为针对无成熟卵子患者的治疗提供了新的临床干预手段。