DataSheet1_Understanding bovine embryo elongation: a transcriptomic study of trophoblastic vesicles.PDF

Background: During the process of elongation, the embryo increases in size within the uterus, while the extra-embryonic tissues (EETs) develop and differentiate in preparation for implantation. As it grows, the ovoid embryo transforms into a tubular form first and then a filamentous form. This process is directed by numerous genes and pathways, the expression of which may be altered in the case of developmental irregularities such as when the conceptus is shorter than expected or when the embryo develops after splitting. In bovines, efforts to understand the molecular basis of elongation have employed trophoblastic vesicles (TVs)—short tubular EET pieces that lack an embryo—which also elongate in vivo. To date, however, we lack molecular analyses of TVs at the ovoid or filamentous stages that might shed light on the expression changes involved.
Methods: Following in vivo development, we collected bovine conceptuses from the ovoid (D12) to filamentous stages (D18), sectioned them into small pieces with or without their embryonic disc (ED), and then, transferred them to a receptive bovine uterus to assess their elongation abilities. We also grew spherical blastocysts in vitro up to D8 and subjected them to the same treatment. Then, we assessed the differences in gene expression between different samples and fully elongating controls at different stages of elongation using a bovine array (10 K) and an extended qPCR array comprising 224 genes across 24 pathways.
Results:In vivo, TVs elongated more or less depending on the stage at which they had been created and the time spent in utero. Their daily elongation rates differed from control EET, with the rates of TVs sometimes resembling those of earlier-stage EET. Overall, the molecular signatures of TVs followed a similar developmental trajectory as intact EET from D12–D18. However, within each stage, TVs and intact EET displayed distinct expression dynamics, some of which were shared with other short epithelial models.
Conclusion: Differences between TVs and EET likely result from multiple factors, including a reduction in the length and signaling capabilities of TVs, delayed elongation from inadequate uterine signals, and modified crosstalk between the conceptus and the uterus. These findings confirm that close coordination between uterine, embryonic, and extra-embryonic tissues is required to orchestrate proper elongation and, based on the partial differentiation observed, raise questions about the presence/absence of certain developmental cues or even their asynchronies.

研究背景：在胚胎延展（elongation）过程中，胚胎于子宫内体积持续增大，同时胚外组织（extra-embryonic tissues, EETs）发育分化，为着床做好准备。随着生长进程推进，卵圆形胚胎（ovoid embryo）会先转变为管状形态，后续进一步形成丝状形态。该过程受众多基因及通路调控，当出现发育异常（如孕体短于预期、胚胎分裂后发育等情况）时，相关基因及通路的表达可能发生改变。在牛科动物中，为解析胚胎延展的分子基础，研究者已采用滋养层囊泡（trophoblastic vesicles, TVs）作为研究模型——此类结构为缺乏胚胎的短管状胚外组织片段，同样可在体内发生延展。但截至目前，尚缺乏对处于卵圆形或丝状阶段的滋养层囊泡的分子分析，而此类分析或可阐明相关表达变化的机制。

研究方法：基于体内发育过程，我们收集了从第12天（D12）卵圆形阶段至第18天（D18）丝状阶段的牛孕体（bovine conceptuses），将其切割为带有或不带有胚盘（embryonic disc, ED）的小块，随后将这些小块移植至受纳态牛子宫中，以评估其延展能力。我们还将体外培养的球形囊胚（spherical blastocysts）培养至第8天（D8），并对其开展相同处理。后续，我们利用牛基因芯片（10K）以及覆盖24条通路、共计224个基因的扩展实时定量聚合酶链反应（quantitative real-time PCR, qPCR）芯片，对不同样本与不同延展阶段的完整对照样本之间的基因表达差异进行了分析。

研究结果：在体内环境中，滋养层囊泡的延展程度取决于其构建时所处的发育阶段以及在子宫内停留的时长。其每日延展速率与对照胚外组织存在差异，部分滋养层囊泡的速率与早期胚外组织相似。整体而言，在D12至D18阶段，滋养层囊泡的分子特征谱与完整胚外组织的发育轨迹基本一致。但在每个具体发育阶段，滋养层囊泡与完整胚外组织的基因表达动态存在显著差异，其中部分差异与其他短上皮模型的特征相符。

研究结论：滋养层囊泡与完整胚外组织之间的差异可能由多种因素导致，包括滋养层囊泡的长度与信号传导能力降低、子宫信号不足引发的延展延迟，以及孕体与子宫间串扰模式的改变。本研究结果证实，子宫、胚胎及胚外组织之间的紧密协调是完成正常延展过程的必要条件；同时基于观察到的部分分化现象，本研究也对部分发育信号的存在与否乃至其不同步现象提出了新的科学疑问。