A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2, required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells.

哺乳动物胚胎在胚泡期经历第一次细胞分离，其中将形成胚的细胞分为两个谱系。滋养外胚层（TE）和内细胞团（ICM）。此第一个细胞分离过程由细胞位置依赖性Hippo信号传导控制，该信号是磷酸化级联反应，确定是与Yippo相关的蛋白1（YAP1）（Hippo信号传导途径的关键组成部分之一）是否位于细胞核或细胞质内。YAP1定位确定关键基因Cdx2的转录开/关，是TE区分所必需的。但是，参与胚泡形成后YAP1核质穿梭的控制机制仍然未知。这项研究侧重于牛囊胚中囊胚腔收缩后，YAP1从TE核释放的机制。胚泡液抽吸收缩的胚泡表明，TE细胞中YAP1从核向细胞质的转运与肌动蛋白细胞骨架的突出有关。YAP1从收缩的胚泡中TE核的释放被肌动蛋白破坏和稳定所阻止。相反，发现Y27632是含有蛋白激酶1/2（ROCK）活性的Rho相关卷曲螺旋的有效抑制剂，可促进YAP1核在收缩胚泡TE细胞中的定位。与此同时，Lambda蛋白磷酸酶（LPP）处理诱导蛋白去磷酸化不能阻止YPS1从收缩胚泡中的TE核中释放，表明YAP1从TE核中的释放不依赖于Hippo信号通路。这些结果表明胚泡收缩导致YAP1通过肌动蛋白细胞骨架重塑以Hippo信号独立的方式从TE核释放。因此，本研究提高了胚泡形成后YAP1亚细胞定位受肌动蛋白细胞骨架组织控制的可能性。我们的结果证明了TE细胞中YAP1核质穿梭的多种调控机制。表明从TE核释放的YAP1不依赖于Hippo信号通路。这些结果表明胚泡收缩导致YAP1通过肌动蛋白细胞骨架重塑以Hippo信号独立的方式从TE核释放。因此，本研究提高了胚泡形成后YAP1亚细胞定位受肌动蛋白细胞骨架组织控制的可能性。我们的结果证明了TE细胞中YAP1核质穿梭的多种调控机制。表明从TE核释放的YAP1不依赖于Hippo信号通路。这些结果表明胚泡收缩导致YAP1通过肌动蛋白细胞骨架重塑以Hippo信号独立的方式从TE核释放。因此，本研究提高了胚泡形成后YAP1亚细胞定位受肌动蛋白细胞骨架组织控制的可能性。我们的结果证明了TE细胞中YAP1胞质穿梭的多种调控机制。本研究提出了胚泡形成后YAP1亚细胞定位受肌动蛋白细胞骨架组织控制的可能性。我们的结果证明了TE细胞中YAP1核质穿梭的多种调控机制。本研究提出了胚泡形成后YAP1亚细胞定位受肌动蛋白细胞骨架组织控制的可能性。我们的结果证明了TE细胞中YAP1核质穿梭的多种调控机制。