Institute of Biotechnology, National Taiwan University, Taipei, Taiwan.
Theriogenology. 2013 Jun;79(9):1284-93. doi: 10.1016/j.theriogenology.2013.03.003. Epub 2013 Apr 6.
The first successful rabbit SCNT was achieved more than one decade ago, yet rabbits remain one of the most difficult species to clone. The present study was designed to evaluate the effects of two histone deacetylase inhibitors (HDACis), namely trichostatin A (TSA) and scriptaid (SCP), on cloning efficiency in rabbits. The in vitro development, acetylation levels of histone H4 lysine 5 (H4K5), and octamer-binding transcription factor 4 (Oct-4) expression patterns of cloned embryos were systemically examined after various HDACi treatments. Supplementation of TSA (50 nM) or SCP (250 nM) in the culture medium for 6 hours improved blastocyst development rates of cloned embryos compared with the treatment without HDACi. The combined treatment with TSA (50 nM) and SCP (250 nM) further enhanced morula (58.6%) and blastocyst (49.4%) rates in vitro. More importantly, compared with single HDACi treatments, embryos with the combined treatment had a higher level of H4K5 and an increased total cell number (203.7 ± 14.4 vs. 158.9 ± 9.0 or 162.1 ± 8.2; P < 0.05) with a better Oct-4 expression pattern in hatching blastocysts, indicating substantially improved embryo quality. This was apparently the first report regarding Oct-4 expression in cloned rabbit embryos. We inferred that most cloned rabbit embryos had an aberrant inner cell mass (ICM) structure accompanied with abnormal spatial distribution of Oct-4 signals. This study demonstrated a synergistic effect of TSA and SCP treatments on cloned rabbit embryos, which might be useful to improve cloning efficiency in rabbits.
首例成功的兔体细胞核移植（SCNT）是在十多年前实现的，但兔仍然是最难克隆的物种之一。本研究旨在评估两种组蛋白去乙酰化酶抑制剂（HDACis），即曲古抑菌素 A（TSA）和 scriptaid（SCP），对兔克隆效率的影响。系统研究了不同 HDACi 处理后克隆胚胎的体外发育、组蛋白 H4 赖氨酸 5（H4K5）的乙酰化水平和八聚体结合转录因子 4（Oct-4）的表达模式。在培养基中添加 TSA（50 nM）或 SCP（250 nM）6 小时可提高克隆胚胎的囊胚发育率，与未添加 HDACi 的处理相比。TSA（50 nM）和 SCP（250 nM）联合处理进一步提高了体外桑葚胚（58.6%）和囊胚（49.4%）的形成率。更重要的是，与单一 HDACi 处理相比，联合处理的胚胎具有更高的 H4K5 水平和更多的总细胞数（203.7 ± 14.4 比 158.9 ± 9.0 或 162.1 ± 8.2；P < 0.05），孵化囊胚中 Oct-4 的表达模式更好，表明胚胎质量有了显著提高。这显然是首例关于克隆兔胚胎中 Oct-4 表达的报道。我们推断大多数克隆兔胚胎的内细胞团（ICM）结构异常，Oct-4 信号的空间分布也异常。本研究表明 TSA 和 SCP 处理对克隆兔胚胎具有协同作用，这可能有助于提高兔的克隆效率。
