雌激素受体α抑制剂MPP对小鼠合子型基因组激活的影响可能与S期启动相关

Effects of Estrogen Receptor α Antagonist MPP on Mouse Zygotic Gene Activation Possibly Correlate with S-phase Initiation

目的观察雌激素受体α(ERα)特异性抑制剂甲基哌啶吡唑(Methyl-Piperidino-Pyrazole,MPP)对小鼠早期胚胎发育的影响与合子型基因组激活(ZGA)发生的时间是否相关,并检测与小鼠ZGA密切相关的2-细胞胚DNA复制是否受MPP处理的影响,为进一步探讨ERα在小鼠ZGA过程中的作用机制提供实验基础。方法 1收集昆明小鼠1-细胞胚、2-细胞胚和4-细胞胚,以空白KSOM培养液(单纯型优化的胚胎培养基)为对照组,以KSOM中添加MPP为实验组;将各时间点收集的早胚培养3h后再移入新KSOM培养液中继续培养至囊胚阶段,并记录囊胚数;2收集昆明小鼠末期1-细胞胚,分别置于上述两组培养液中培养15h至2-细胞胚S期后期,用荧光显微镜技术检测2-细胞胚核像变化;Brd U标记法分析DNA复制情况。结果1于不同时间段经MPP 3h处理后,在1-细胞胚末期、2-细胞早期和2-细胞胚中后期,MPP处理对小鼠早胚发育具有显著的抑制作用,其阶段特异性影响与小鼠ZGA发生的时间一致。另外,MPP对2-细胞胚G1/S过渡期(36~39h)抑制明显,但是对S期中后期(39~42h)进程无显著影响。2与KSOM组相比,经MPP处理15h后,2-细胞胚核变圆;Brd U标记水平明显下降。结论MPP在小鼠早胚中的阶段性作用与ZGA有关;ERα可能通过促进S期启动来调控ZGA。

Objective To observe whether the effects of the estrogen receptor alpha( ERα) specific antagonist Methyl-Piperidino-Pyrazole( MPP) on early mouse embryo development correlate with the occurrence of zygotic gene activation( ZGA), and to investigate the effects of MPP treatment on 2-cell DNA replication, which is closely related with mouse ZGA, in order to set up the foundation for further researches regarding the functional mechanisms of ERα during mouse ZGA. Methods 1 Kunming( KM) mouse 1-cell, 2-cell and 4-cell embryos at different developmental stages were collected and cultured respectively in KSOM medium, a simplex optimized medium used for embryo cultivation( control group), and KSOM medium supplemented with MPP( experimental group). After3 h treatment, the embryos were transferred to fresh KSOM medium and cultured until the blastocyst stage when the number of blastocysts was recorded. 2 Late 1-cell embryos were collected and cultured in the above-mentioned two groups of media for 15 h until 2-cell late S phase. Immunofluorescence microscopy was performed to observe the nuclear shape change; Brd U incorporation assay was conducted to examine the DNA replication levels in 2-cell embryos. Results 1 After 3h MPP treatment, significant inhibitory effects were observed on the embryos collected at late 1-cell, early 2-cell and mid-to-late 2-cell stages; the stage-specific pattern correlated with the occurrence of mouse ZGA. Besides, MPP significantly inhibited the development of embryos treated at G1 / S transition of 2-cell stage( 36 ~ 39h), whereas MPP treatment in the mid-to-late S phase 2-cells( 39 ~ 42h) showed no significant effect on embryo development.2 After 15 h culture, the MPP treated 2-cell embryos showed round-shaped nuclei and reduced Brd U incorporation when compared with the control group. Conclusion The stage-specific effects of MPP on mouse preimplantation embryo development correlate with ZGA;ERα might possibly regulate mouse ZGA by promoting the initiation of S-phase in mouse 2-cell embryos.