Abstract：
It has been documented that the inefficacy of round spermatid injection (ROSI) might be caused by abnormal epigenetic modifications. Therefore, this study aimed to evaluate the effect of trichostatin A (TSA) as an epigenetic modifier of preimplantation embryo development in activated ROSI oocytes. Matured oocytes were collected from superovulated female mice. Testes were placed in human tubal fluid medium and masses were then cut into small pieces to disperse spermatogenic cells. Round spermatids were treated with TSA and subsequently injected into oocytes. The expression level of the development-related genes including Oct4, Sox2, Nanog, Dnmt and Hdac transcripts were evaluated using qRT-PCR. Immunohistochemistry was performed to confirm the presence of Oct-4 protein at the blastocyst stage. There was no statistically significant difference in fertilization rate following ROSI/+TSA compared with the non-treated ROSI and intracytoplasmic sperm injection (ICSI) groups. Importantly, TSA treatment increased blastocyst formation from 38% in non-treated ROSI to 68%. The relative expression level of developmentally related genes increased and Dnmt transcripts decreased in ROSI/+TSA-derived embryos, similar to the expression levels observed in the ICSI-derived embryos. In conclusion, our results indicate that spermatid treatment with TSA prior to ROSI would increase the success rate of development to the blastocyst stage and proportion of pluripotent cells.
摘要：
已经证明，圆形精子细胞注射（ROSI）的无效可能是由异常的表观遗传修饰引起的。因此,这项研究旨在评估曲古抑菌素A（TSA）作为激活的ROSI卵母细胞植入前胚胎发育的表观遗传修饰因子的作用。从超排卵的雌性小鼠收集成熟的卵母细胞。将睾丸置于人输卵管液培养基中，然后将肿块切成小块以分散生精细胞。用TSA处理圆形精子细胞并随后注射到卵母细胞中。发育相关基因Oct4,Sox2,Nanog,使用qRT-PCR评估Dnmt和Hdac转录物。进行免疫组织化学以确认胚泡阶段Oct-4蛋白的存在。与未治疗的ROSI和胞浆内单精子注射（ICSI）组相比，ROSI/TSA后的受精率没有统计学上的显着差异。重要的是,TSA处理将胚泡形成从未处理的ROSI中的38%增加到68%。ROSI/+TSA来源胚胎发育相关基因相对表达水平升高,Dnmt转录本降低,与ICSI衍生胚胎中观察到的表达水平相似。总之,我们的结果表明,在ROSI之前用TSA处理精子细胞可以提高发育到囊胚期的成功率和多能细胞的比例.
