Capturing stable embryonic stem cell (ESC) lines from domesticated animals still remains one of the challenges of non-rodent embryology. The stake is high, as stable ESCs derived from species such as cattle present high economic and scientific value. Understanding of the processes leading to the embryonic lineage segregation is crucial to provide species-orientated molecular environment capable of supporting self-renewal and pluripotency. Therefore, the aim of this study was to validate the action of the two core regulatory pathways (WNT and MEK/ERK) during bovine embryo development. In vitro produced bovine embryos were obtained in the presence of inhibitors (i), which enable activation of the WNT pathway (via GSK3i, CHIR99021) and suppression of MEK signalling by PD0325901 in the 2i system and PD184325 and SU5402 in the 3i system. We have followed the changes in the distribution of the key lineage specific markers both at the transcript and protein level. Our results showed that WNT signalling promotes the expression of key inner cell mass (ICM) specific markers in bovine embryos, regardless of the MEK/ERK inhibitor cocktail used. MEK/ERK downregulation is crucial to maintain OCT4 and NANOG expression within the ICM and to prevent their exclusion from the trophectoderm (TE). At the same time, the classical TE marker (CDX2) was downregulated at the mRNA and protein level. As a follow up for the observed pluripotency stimulating effect of the inhibitors, we have tested the potential of the 2i and the 3i culture conditions (supported by LIF) to derive primary bovine ESC lines. As a result, we propose a model in which all of the primary signalling pathways determining embryonic cell fate are active in bovine embryos, yet the requirement for pluripotency maintenance in cattle may differ from the described standards. WNT activation leads to the formation (and stabilisation of the ICM) and MEK/ERK signalling is maintained at low levels. Unlike in the mouse, GATA6 is expressed in both ICM and TE. MEK/ERK signalling affects HP formation in cattle, but this process is activated at the post-blastocyst stage. With regard to self-renewal, 2i is preferable, as 3i also blocks the FGF receptor, what may prevent PI3K signalling, important for pluripotency and self-renewal.

中文翻译：

---

MEK/ERK 抑制支持的 WNT 信号传导对于维持牛植入前胚胎的多能性至关重要。

从家养动物中捕获稳定的胚胎干细胞 (ESC) 系仍然是非啮齿动物胚胎学的挑战之一。风险很高，因为源自牛等物种的稳定 ESC 具有很高的经济和科学价值。了解导致胚胎谱系分离的过程对于提供能够支持自我更新和多能性的面向物种的分子环境至关重要。因此，本研究的目的是验证两个核心调控途径（WNT 和 MEK/ERK）在牛胚胎发育过程中的作用。在抑制剂 (i) 存在下获得体外生产的牛胚胎，其能够激活 WNT 途径（通过 GSK3i、CHIR99021）并通过 2i 系统中的 PD0325901 和 3i 系统中的 PD184325 和 SU5402 抑制 MEK 信号传导。我们在转录本和蛋白质水平上跟踪了关键谱系特异性标记的分布变化。我们的结果表明，无论使用何种 MEK/ERK 抑制剂混合物，WNT 信号都会促进牛胚胎中关键内细胞团 (ICM) 特异性标志物的表达。MEK/ERK 下调对于维持 ICM 内的 OCT4 和 NANOG 表达并防止它们被排除在滋养外胚层 (TE) 之外至关重要。同时，经典的 TE 标记（CDX2）在 mRNA 和蛋白质水平上被下调。作为观察到的抑制剂的多能性刺激作用的后续行动，我们测试了 2i 和 3i 培养条件（由 LIF 支持）衍生原代牛 ESC 系的潜力。其结果，我们提出了一个模型，其中所有决定胚胎细胞命运的主要信号通路都在牛胚胎中活跃，但牛的多能性维持要求可能与所描述的标准不同。WNT 激活导致形成（和稳定 ICM）并且 MEK/ERK 信号保持在低水平。与小鼠不同，GATA6 在 ICM 和 TE 中均有表达。MEK/ERK 信号会影响牛的 HP 形成，但该过程在胚泡后阶段被激活。关于自我更新，2i 更可取，因为 3i 还阻断 FGF 受体，这可能会阻止 PI3K 信号传导，这对多能性和自我更新很重要。WNT 激活导致形成（和稳定 ICM）并且 MEK/ERK 信号保持在低水平。与小鼠不同，GATA6 在 ICM 和 TE 中均有表达。MEK/ERK 信号会影响牛的 HP 形成，但该过程在胚泡后阶段被激活。关于自我更新，2i 更可取，因为 3i 还阻断 FGF 受体，这可能会阻止 PI3K 信号传导，这对多能性和自我更新很重要。WNT 激活导致形成（和稳定 ICM）并且 MEK/ERK 信号保持在低水平。与小鼠不同，GATA6 在 ICM 和 TE 中均有表达。MEK/ERK 信号会影响牛的 HP 形成，但该过程在胚泡后阶段被激活。关于自我更新，2i 更可取，因为 3i 还阻断 FGF 受体，这可能会阻止 PI3K 信号传导，这对多能性和自我更新很重要。