Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation.
Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation.
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Podocalyxin 是人类子宫内膜上皮对胚胎植入容受性的关键负调节因子。
DOI：
10.1093/humrep/deab032
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Nie,Guiying
中科院分区：
文献类型：
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作者：
Paule,SarahG;Heng,Sophea;Samarajeewa,Nirukshi;Li,Ying;Mansilla,Mary;Webb,AndrewI;Nebl,Thomas;Young,StevenL;Lessey,BruceA;Hull,MLouise;Scelwyn,Maxine;Lim,Rebecca;Vollenhoven,Beverley;Rombauts,LukJ;Nie,Guiying
研究问题：人类胚胎着床时，子宫内膜上皮接受性，特别是黏附性是如何在腔上皮表面调节的？podocalyxin （PCX）是一种跨膜蛋白，被认为是子宫内膜上皮接受性的关键负调控因子；分泌中期腔上皮中PCX的特异性下调，可能是由孕激素介导的，可能是子宫内膜表面从非接受状态转变为允许植入状态的关键步骤。人类子宫内膜必须经历重大的分子和细胞变化，才能从非接受状态转变为接受状态，以适应胚胎着床。然而，控制接受性的基本机制，特别是在胚胎最初与之相互作用的腔面，还没有得到很好的理解。一个广泛持有的观点是，上调粘合促进分子是重要的，但细节没有很好地表征。研究设计、大小、持续时间本研究首先旨在鉴定在原发性人子宫内膜上皮细胞（HEECs）接受性中具有潜在作用的新型粘附相关膜蛋白。随后，研究人员进行了进一步的实验，以确定候选基因在月经周期内在人子宫内膜中的体内表达模式、细胞培养中黄体酮的调节作用，以及在体外胚胎附着和侵袭模型中对接受性的功能重要性。从增殖期子宫内膜组织中分离初生HEECs (n = 9)，合并成三个池，通过超离心进行质膜蛋白富集，然后进行蛋白质组学分析，从而发现PCX作为新的候选物。免疫组织化学分析确定了它们在人子宫内膜中的体内表达模式和细胞定位（n = 23）。为了探讨PCX是否受子宫内膜分化的主要驱动因素黄体酮的调控，我们采用雌二醇和黄体酮对原代HEECs进行培养，并采用RT-PCR （n = 5）和western blot （n = 4）进行分析。为了证明PCX作为可接受性的负调节因子，PCX在石川细胞（一种接受系）中过表达，并通过体外附着（n = 3-5）和侵袭模型（n = 4-6）确定对可接受性的影响，其中石川单层细胞模拟子宫内膜表面，原代人滋养层球细胞模拟胚胎。Mann-Whitney u检验和ANOVA分析在*P≤0.05和**P≤0.01上均具有统计学意义。主要结果：在非接受性子宫内膜中，所有上皮细胞和内皮细胞的顶端表面都表达了CHANCEPCX，但在腔上皮中，从分泌中期开始选择性下调，与接受性的建立一致。孕酮被证实能够抑制原发性HEECs中的PCX，这表明这种激素可能介导了腔内PCXin体内接受性的下调。石川单层细胞中PCX的过表达不仅能抑制人胚胎代体细胞的附着，还能抑制其植入，表明PCX是上皮细胞植入接受性的重要负调控因子。局限性和注意的原因从…
STUDY QUESTIONHow is endometrial epithelial receptivity, particularly adhesiveness, regulated at the luminal epithelial surface for embryo implantation in the human?SUMMARY ANSWERPodocalyxin (PCX), a transmembrane protein, was identified as a key negative regulator of endometrial epithelial receptivity; specific downregulation of PCX in the luminal epithelium in the mid-secretory phase, likely mediated by progesterone, may act as a critical step in converting endometrial surface from a non-receptive to an implantation-permitting state.WHAT IS KNOWN ALREADYThe human endometrium must undergo major molecular and cellular changes to transform from a non-receptive to a receptive state to accommodate embryo implantation. However, the fundamental mechanisms governing receptivity, particularly at the luminal surface where the embryo first interacts with, are not well understood. A widely held view is that upregulation of adhesion-promoting molecules is important, but the details are not well characterized.STUDY DESIGN, SIZE, DURATIONThis study first aimed to identify novel adhesion-related membrane proteins with potential roles in receptivity in primary human endometrial epithelial cells (HEECs). Further experiments were then conducted to determine candidates’in vivoexpression pattern in the human endometrium across the menstrual cycle, regulation by progesterone using cell culture, and functional importance in receptivity usingin vitrohuman embryo attachment and invasion models.PARTICIPANTS/MATERIALS, SETTING, METHODSPrimary HEECs (n = 9) were isolated from the proliferative phase endometrial tissue, combined into three pools, subjected to plasma membrane protein enrichment by ultracentrifugation followed by proteomics analysis, which led to the discovery of PCX as a novel candidate of interest. Immunohistochemical analysis determined thein vivoexpression pattern and cellular localization of PCX in the human endometrium across the menstrual cycle (n = 23). To investigate whether PCX is regulated by progesterone, the master driver of endometrial differentiation, primary HEECs were treated in culture with estradiol and progesterone and analyzed by RT-PCR (n = 5) and western blot (n = 4). To demonstrate that PCX acts as a negative regulator of receptivity, PCX was overexpressed in Ishikawa cells (a receptive line) and the impact on receptivity was determined usingin vitroattachment (n = 3–5) and invasion models (n = 4–6), in which an Ishikawa monolayer mimicked the endometrial surface and primary human trophoblast spheroids mimicked embryos. Mann–Whitney U-test and ANOVA analyses established statistical significance at *P≤ 0.05 and **P≤ 0.01.MAIN RESULTS AND THE ROLE OF CHANCEPCX was expressed on the apical surface of all epithelial and endothelial cells in the non-receptive endometrium, but selectively downregulated in the luminal epithelium from the mid-secretory phase coinciding with the establishment of receptivity. Progesterone was confirmed to be able to suppress PCX in primary HEECs, suggesting this hormone likely mediates the downregulation of luminal PCXin vivofor receptivity. Overexpression of PCX in Ishikawa monolayer inhibited not only the attachment but also the penetration of human embryo surrogates, demonstrating that PCX acts as an important negative regulator of epithelial receptivity for implantation.LIMITATIONS, REASONS FOR CAUTIONPrimary HEECs isolated from the …