Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program, and Genetics Institute, University of Florida, Gainesville, Florida, United States of America.
PLoS One. 2013 Jun 14;8(6):e66230. doi: 10.1371/journal.pone.0066230. Print 2013.
There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6-8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6-8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation.
在胚胎植入前发育过程中，DNA 甲基化的控制存在物种差异。牛胚胎中确切的甲基化模式尚未建立，其性别或调节发育的母体信号（如集落刺激因子 2（CSF2））的调节也尚未建立。通过使用抗 5-甲基胞嘧啶的免疫荧光标记和用 X 染色体分选的精子产生的胚胎，证明甲基化从 2 细胞期到 6-8 细胞期减少，然后在此后增加到囊胚期。在第二个实验中，通过用 X 或 Y 分选的精子受精产生特定性别的胚胎。发育模式与第一个实验相似，但存在阶段×性别相互作用。在 8 细胞期，雌性的甲基化程度更高，但在囊胚期，雄性的甲基化程度更高。CSF2 处理对囊胚中 DNA 甲基化的标记没有影响。内细胞团（即未用抗 CDX2 标记的细胞）的甲基化程度低于滋养外胚层（CDX2 阳性）。通过确定基因表达和甲基化程度来评估 DNMT3B 在甲基化发育变化中的可能作用。DNMT3B 的稳定态 mRNA 从 2 细胞期到 D5 胚胎>16 细胞的低谷下降，然后在囊胚期增加。高分辨率熔解分析用于评估 DNMT3B 内含子区域中富含 CpG 的区域的甲基化。6-8 细胞期和囊胚期之间的甲基化百分比下降，但 ICM 和 TE 之间的甲基化没有差异。结果表明，DNA 甲基化在胚胎植入前期间以依赖于性别和细胞谱系的方式发生动态变化。DNMT3B 表达的发育变化表明其在甲基化变化中可能发挥作用。此外，DNMT3B 本身似乎受到甲基化的表观遗传控制。
