Isaji Yuuki, Murata Moeko, Takaguchi Naoya, Mukai Toshita, Tajima Yosuke, Imai Hiroshi, Yamada Masayasu
Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
J Reprod Dev. 2013;59(2):196-204. doi: 10.1262/jrd.2012-156. Epub 2013 Jan 22.
We examined effects of treatment with valproic acid (0, 0.2, 1 or 2 mM, VPA), an inhibitor of class I and IIa histone deacetylases (HDACs), of mouse somatic cell nuclear transfer (SCNT) embryos for 24 h from 48 h (4-cell stage), 24 h (2-cell stage) or immediately after oocyte activation on blastocyst formation rates and qualities of the resultant blastocysts. Blastocyst formation rates (33.4-37.0%) were not improved by VPA treatments compared with the untreated control (35.1-36.4%). However, immunofluorescence staining revealed that Oct4 expression levels, evaluated from percentages of embryos expressing Oct4 strongly and having more than 10 Oct4-positive cells, in blastocysts from SCNT embryos treated with 1 mM VPA for 24 h from the 4-cell stage (VPA-4C) were highest among all the groups and that the proportion of cells with a normal nuclear distribution of histone H3 trimethylated at lysine 27 (H3K27me3), a marker of the state of X-chromosome inactivation, significantly increased in the VPA-4C group (36.6%) compared with the control group (12.4%, P<0.05). Treatments with scriptaid and sodium butyrate, inhibitors of class I and IIa/b HDACs, for 24 h from the 4-cell stage also had beneficial effects on SCNT blastocysts. These findings indicate that treatment with 1 mM VPA from the 4-cell stage improves the Oct4 expression and nuclear distribution of H3K27me3 in mouse SCNT blastocysts and suggest that the inhibition of class I and IIa HDACs from the 4-cell stage plays an important role in these effects.
我们研究了组蛋白去乙酰化酶I类和IIa类（HDACs）抑制剂丙戊酸（0、0.2、1或2 mM，VPA）对小鼠体细胞核移植（SCNT）胚胎的处理效果。从48小时（4细胞期）、24小时（2细胞期）或卵母细胞激活后立即开始，用VPA处理SCNT胚胎24小时，观察其对囊胚形成率及所得囊胚质量的影响。与未处理的对照组（35.1 - 36.4%）相比，VPA处理并未提高囊胚形成率（33.4 - 37.0%）。然而，免疫荧光染色显示，从4细胞期开始用1 mM VPA处理24小时的SCNT胚胎（VPA - 4C）所形成的囊胚中，从Oct4强表达且Oct4阳性细胞超过10个的胚胎百分比评估的Oct4表达水平，在所有组中最高，并且赖氨酸27三甲基化组蛋白H3（H3K27me3）（X染色体失活状态的标志物）核分布正常的细胞比例在VPA - 4C组（36.6%）中相比对照组（12.4%，P<0.05）显著增加。从4细胞期开始用I类和IIa/b类HDACs抑制剂司立西肽和丁酸钠处理24小时，对SCNT囊胚也有有益影响。这些发现表明，从4细胞期开始用1 mM VPA处理可改善小鼠SCNT囊胚中Oct4表达和H3K27me3的核分布，并表明从4细胞期开始抑制I类和IIa类HDACs在这些作用中起重要作用。
