Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two-round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient-rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this “on-gel” culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on-gel-cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA-seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic reduction of pluripotency-related genes, TE cells highly expressed Wnt, FGF, and VEGF signaling pathways-related genes to facilitate the functional maturation required for feto-maternal interaction. Quantitative PCR analysis of TE cells derived from on-gel culture further confirmed time-dependent increments in the expression of key TE markers. Altogether, the present study provides platforms to understand species-specific strategies for mammalian preimplantation development.

中文翻译：

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破译牛胚胎植入前发育过程中的两轮细胞谱系分离

囊胚形成产生内细胞团 (ICM) 和滋养外胚层 (TE)，然后在 ICM 内分化出外胚层 (Epi) 和原始内胚层 (PrE)。尽管这些两轮细胞谱系分化支持每个哺乳动物的适当胚胎发生，但它们的时空动态在物种之间非常不同。在这里，使用优化的体外培养方法剖析了牛胚泡阶段的分子细节。将囊胚置于充满营养丰富培养基的琼脂糖凝胶上，使胚胎暴露于气相和液相。在受精后第 10 天 (D) 将源自这种“凝胶上”培养物的胚胎转移给代孕母亲并成功植入。使用凝胶培养胚胎的免疫荧光研究表明，表达多能 ICM 标记物 OCT4 的 TE 细胞比例在第 8 天超过 80%，在第 9 天后迅速降低，并在第 9.5 天达到 0%。第一个谱系分离过程与第二个谱系分离过程在时间上平行，通过表达 SOX2 的 Epi 细胞和表达 SOX17 的 PrE 细胞的空间分离来确定。来自 D8 体外受精胚胎和 D14 体内胚胎的 TE 细胞的 RNA-seq 比较表明，除了多能性相关基因的急剧减少外，TE 细胞还高度表达 Wnt、FGF 和 VEGF 信号通路相关基因，以促进所需的功能成熟用于母婴互动。来自凝胶培养的 TE 细胞的定量 PCR 分析进一步证实了关键 TE 标志物表达的时间依赖性增量。总而言之，本研究为了解哺乳动物胚胎植入前发育的物种特异性策略提供了平台。