Animal Breeding and Husbandry Group, Institute of Animal Science, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany.
Reproduction. 2010 May;139(5):857-70. doi: 10.1530/REP-09-0312. Epub 2010 Feb 22.
This study was conducted to investigate the effect of suppressing transcription factor gene MSX1 on the development of in vitro produced bovine oocytes and embryos, and identify its potential target genes regulated by this gene. Injection of long double-stranded RNA (LdsRNA) and small interfering RNA (siRNA) at germinal vesicle stage oocyte reduced MSX1 mRNA expression by 73 and 37% respectively at metaphase II stage compared with non-injected controls. Similarly, injection of the same anti-sense oligomers at zygote stage reduced MSX1 mRNA expression by 52 and 33% at 8-cell stage compared with non-injected controls. Protein expression was also reduced in LdsRNA- and siRNA-injected groups compared with non-injected controls at both stages. Blastocysts rates were 33, 28, 20 and 18% in non-injected control, scrambled RNA (scRNA), LdsRNA- and siRNA-injected groups respectively. Cleavage rates were also significantly reduced in Smartpool siRNA (SpsiRNA)-injected group (53.76%) compared with scRNA-injected group (57.76%) and non-injected control group (61%). Large-scale gene expression analysis showed that 135 genes were differentially regulated in SpsiRNA-injected group compared with non-injected controls, of which 54 and 81 were down- and up-regulated respectively due to suppression of MSX1. Additionally, sequence homology mapping and gene enrichment analysis with known human pathway information identified several functional modules that were affected due to suppression of MSX1. In conclusion, suppression of MSX1 affects oocyte maturation, embryo cleavage rate and the expression of several genes, suggesting its potential role in the development of bovine preimplantation embryos.
本研究旨在探讨抑制转录因子基因 MSX1 对体外生产牛卵母细胞和胚胎发育的影响，并鉴定其潜在的受该基因调控的靶基因。在生发泡期注射长双链 RNA（LdsRNA）和小干扰 RNA（siRNA）可使中期 II 期卵母细胞中的 MSX1mRNA 表达分别降低 73%和 37%，与未注射对照组相比。同样，在合子期注射相同的反义寡核苷酸可使 8 细胞期的 MSX1mRNA 表达降低 52%和 33%，与未注射对照组相比。与未注射对照组相比，LdsRNA 和 siRNA 注射组的蛋白质表达在两个阶段均降低。非注射对照组、乱序 RNA(scRNA)、LdsRNA 和 siRNA 注射组的囊胚率分别为 33%、28%、20%和 18%。Smartpool siRNA（SpsiRNA）注射组的卵裂率（53.76%）也明显低于 scRNA 注射组（57.76%）和非注射对照组（61%）。大规模基因表达分析显示，与未注射对照组相比，SpsiRNA 注射组有 135 个基因差异表达，其中 54 个和 81 个基因分别因 MSX1 的抑制而下调和上调。此外，与已知的人类途径信息进行序列同源性映射和基因富集分析，确定了由于 MSX1 的抑制而受影响的几个功能模块。综上所述，MSX1 的抑制会影响卵母细胞成熟、胚胎卵裂率和几个基因的表达，提示其在牛胚胎植入前发育中的潜在作用。
