Division of Biological Science, Graduate School of Biology-Oriented Science and Technology, Kinki University, Kinokawa, Wakayama 649-6493, Japan.
Biochem Biophys Res Commun. 2013 Apr 26;434(1):1-7. doi: 10.1016/j.bbrc.2013.03.083. Epub 2013 Apr 6.
Previous studies show that treatment of zygotes with trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), impacts the subsequent development to a blastocyst as well as full-term development. To reveal the dynamics of protein acetylation, with and without TSA treatment during one-cell stage, we examined oocytes and zygotes by immunofluorescence and Western Blot analyses using anti-acetylated lysine and acetylated α-tubulin antibodies. In unfertilized oocytes, lysine acetylation level was extremely low over all but faintly detected in the spindle. Once oocyte activation occurs, a dramatic increase of lysine acetylation signal was observed mostly in the pronuclei and a fiber-like structure, the so called midbody, suggesting activation coupled up-regulation of lysine acetylation presumably in histones and α-tubulin. TSA treatment resulted in significantly more hyperacetylation not only in the midbody structure and pronuclei but also in the whole cytoplasm. Consistently, Western Blot analysis revealed that acetylation of proteins about 53 kDa and 11 kDa in size, corresponding to α-tubulin and histone H4 sizes respectively, were increased mainly after oocyte activation and exclusively enhanced by TSA treatment in zygotes. To confirm this behavior of acetylated nonhistone proteins, acetylated α-tubulin was examined and found to be faintly detected in the spindle of MII oocytes but later in whole in the cell of zygotes including the midbody, which was enhanced by TSA treatment. To elucidate the mechanism underlying up-regulation of lysine acetylation following oocyte activation, we assayed the HDAC activity, and found significant reduction of HDAC activity from MII to zygotic stages. Taken together, our data indicate that HDACs play an important role in maintaining low acetylated status in a MII oocyte. However, once an oocyte has been activated, histone and nonhistone proteins including α-tubulin are hyperacetylated partly due to a reduction of HDAC activity. TSA treatment of zygotes enhances their acetylation, which could affect subsequent embryonic development.
先前的研究表明，组蛋白去乙酰化酶抑制剂（HDACi）曲古抑菌素 A（TSA）处理受精卵会影响囊胚和全期胚胎的发育。为了揭示单细胞期 TSA 处理和不处理时蛋白质乙酰化的动态变化，我们通过免疫荧光和 Western blot 分析，用抗乙酰化赖氨酸和乙酰化α-微管蛋白抗体检测了卵母细胞和受精卵。在未受精的卵母细胞中，除纺锤体外，赖氨酸乙酰化水平极低，但在纺锤体中可检测到微弱的信号。一旦卵母细胞激活，赖氨酸乙酰化信号显著增加，主要在原核和纤维状结构，即所谓的中体中，提示激活伴随着赖氨酸乙酰化的上调，可能在组蛋白和α-微管蛋白中。TSA 处理不仅导致中体结构和原核中乙酰化程度显著增加，而且导致整个细胞质中乙酰化程度显著增加。Western blot 分析结果一致表明，约 53 kDa 和 11 kDa 大小的蛋白质的乙酰化，分别对应于α-微管蛋白和组蛋白 H4 的大小，在卵母细胞激活后增加，仅在受精卵中被 TSA 处理增强。为了证实非组蛋白乙酰化蛋白的这种行为，我们检测了乙酰化的α-微管蛋白，发现它在 MII 卵母细胞的纺锤体中可检测到，但在受精卵中稍后在整个细胞中均可检测到，包括中体，且 TSA 处理可增强其乙酰化。为了阐明卵母细胞激活后赖氨酸乙酰化上调的机制，我们检测了 HDAC 活性，发现从 MII 到合子阶段 HDAC 活性显著降低。综上所述，我们的数据表明，HDAC 在维持 MII 卵母细胞低乙酰化状态中发挥重要作用。然而，一旦卵母细胞被激活，组蛋白和非组蛋白蛋白，包括α-微管蛋白，就会被高度乙酰化，部分原因是 HDAC 活性降低。TSA 处理受精卵会增强其乙酰化，这可能会影响随后的胚胎发育。
