Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

中文翻译：

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牛胚胎体外发育后胚盘的形成


受胎伸长过程中的失败是有蹄类动物妊娠失败的主要原因，对农业产生了相关的经济影响。对这一时期发生的发育事件知之甚少，主要是因为这一过程无法在体外重现。先前的研究已经建立了一种体外孵化后发育（PHD）系统，该系统基于琼脂糖凝胶隧道以及富含血清和葡萄糖的培养基，支持囊胚阶段之后的牛胚胎发育。不幸的是，在这个系统下，胚胎盘的形成没有实现，并且胚胎显示出臭名昭著的细胞凋亡和坏死的迹象。本研究的目的是开发一种能够支持胚胎盘形成的体外系统。我们首先比较了琼脂糖隧道内或自由漂浮在富含血清和葡萄糖的培养基（PHD 培养基）中的琼脂糖包被培养皿上的孵化后发育。琼脂糖隧道内的培养通过物理收缩塑造胚胎形态，但它限制了胚胎生长，并且在下胚层和外胚层谱系的发育方面没有提供任何显着的优势。与 PHD 培养基相反，化学成分确定且富集的培养基 (N2B27) 即使在没有琼脂糖涂层的情况下，也能在体外支持完整的下胚层迁移和外胚层存活。在约 56% 的胚胎中观察到表达多能性标记 SOX2 的细胞，约 25% 的胚胎发育出由 SOX2+ 细胞形成的胚盘样结构。总之，我们在这里提供了一种支持胚泡阶段后滋养外胚层增殖、下胚层迁移和外胚层存活的培养系统。