Table3_Understanding bovine embryo elongation: a transcriptomic study of trophoblastic vesicles.XLSX

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：在胚胎伸长过程中，胚胎于子宫内体积增大，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，卵球形胚胎先转变为管状形态，随后形成丝状结构。该过程受众多基因与通路调控，当出现发育异常时，例如孕体（conceptus）长度低于预期，或是胚胎经分裂后发育，相关基因的表达模式可能发生改变。在牛科动物中，为解析伸长过程的分子机制，研究者采用了滋养层囊泡（trophoblastic vesicles, TVs）——即不含胚胎的短管状胚外组织片段，其在体内同样可发生伸长。然而截至目前，尚无针对卵球形或丝状阶段滋养层囊泡的分子分析，而这类分析或可揭示相关的表达变化规律。 研究方法：基于体内发育进程，我们从卵球形阶段（妊娠第12天，D12）至丝状阶段（妊娠第18天，D18）收集牛科孕体，将其切割为带有或不带有胚盘（embryonic disc, ED）的小块，随后移植到受体牛子宫中以评估其伸长能力。我们还在体外将球形囊胚培养至第8天（D8），并对其开展相同处理。此外，我们利用10K牛基因芯片以及覆盖24条通路、共224个基因的扩展型定量聚合酶链式反应（quantitative PCR, qPCR）芯片，检测不同样本与不同伸长阶段完整对照样本之间的基因表达差异。 研究结果：在体内环境中，滋养层囊泡的伸长程度主要取决于其形成时的发育阶段以及在子宫内的停留时长。其每日伸长速率与完整胚外组织存在差异，部分滋养层囊泡的伸长速率与早期胚外组织相近。整体而言，在D12至D18阶段，滋养层囊泡的分子特征与完整胚外组织遵循相似的发育轨迹。但在同一发育阶段，滋养层囊泡与完整胚外组织展现出不同的表达动态，其中部分动态特征与其他短上皮模型存在共通性。 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受限、子宫信号不足引发的伸长延迟，以及孕体与子宫之间的串扰模式改变。本研究证实，子宫、胚胎与胚外组织之间的紧密协调是实现正常伸长过程的必要条件；同时基于观察到的部分分化现象，本研究也提出了若干疑问，例如某些发育信号是否存在，或是发育进程存在异步性。