Filliers Muriel, Goossens Karen, Van Soom Ann, Merlo Barbara, Pope Charles Earle, de Rooster Hilde, Smits Katrien, Vandaele Leen, Peelman Luc J
Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.
Reprod Fertil Dev. 2012;24(5):691-703. doi: 10.1071/RD11068.
During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of early lineage markers can be species specific. The present study also revealed a pool of maternal NANOG mRNA transcripts, the role of which remains to be elucidated. Comparing transcription levels of these genes between in vivo and in vitro blastocysts revealed low levels of OCT4 mRNA in the latter, which may contribute to the reduced developmental competence of embryos under suboptimal conditions.
在哺乳动物植入前发育过程中，两个连续的分化事件导致建立了具有不同命运的三个定向谱系：滋养外胚层、原始内胚层和多能外胚层。在小鼠胚胎中，这两个细胞命运决定的分子机制已被广泛研究，从而鉴定出谱系特异性转录因子。已报道关键调控基因表达模式存在物种特异性差异，这引发了关于它们在不同物种中作用的问题。本研究的目的是使用逆转录定量聚合酶链反应来表征猫早期发育过程中多能性（OCT4、SOX2、NANOG）和分化（CDX2、GATA6）相关标志物的基因表达模式。此外,我们通过比较体外和体内囊胚中所研究基因的转录水平，评估了体外发育对基因表达的影响。为了使不同植入前胚胎阶段的定量数据标准化，我们首先验证了一组稳定的参考基因。所有研究基因的转录水平在植入前发育过程中均存在且发生变化；观察到基因表达存在高度显著的胚胎阶段效应。与体内囊胚相比，体外囊胚中OCT4的转录水平显著降低。所研究的其他基因在体外条件下均未显示表达改变。猫胚胎中OCT4、SOX2、CDX2和GATA6的不同基因表达模式与小鼠胚胎中描述的相似，表明这些基因在早期分离过程中具有保留的作用。然而，由于胚胎基因组激活后NANOG转录水平没有任何上调，NANOG不太可能是谱系分离的关键调节因子。这些结果支持了早期谱系标志物的行为可能具有物种特异性的假设。本研究还揭示了一批母体NANOG mRNA转录本，其作用仍有待阐明。比较体内和体外囊胚中这些基因的转录水平发现，后者中OCT4 mRNA水平较低，这可能导致在次优条件下胚胎发育能力降低。
