Pan-omic characterization of the molecular determinants of uterine receptivity

本研究的总体目的是确定影响胚胎着床接受窗口的子宫分子靶点。提出这项研究的理由是，更好地了解影响着床接受窗口的子宫源性介质将为子宫接受性提供多个靶点，从而可用于确定体外受精临床中胚胎移植的合适时机。在这个提议中，我们将使用野生型小鼠来比较来自子宫对胚胎着床敏感的前感受期、接受期和非接受期（难接受期）的分离上皮和基质细胞的蛋白质、脂质和代谢物谱。虽然目前对与子宫容受性相关的脂质介质的了解相对有限，但脂质代谢产物内源性大麻素和前列腺素已被证明发挥关键作用。子宫接受性小鼠模型的深入脂质组学表征从未进行过。我们的假设是脂质代谢的调节网络可以作为信号中枢控制以前未表征的目标；结构复杂的脂质，脂质代谢酶和脂质代谢产物，在子宫接受性中有不同的作用。为了探索这一假设，该项目将利用太平洋西北国家实验室最近开发的两项独特技术，用于发现和验证影响胚胎植入接受性的分子靶点：1)采用先进的纳米级液相色谱（nanoLC）方法的超灵敏电喷雾电离离子迁移谱-飞行时间质谱（IMS-TOF MS）和2)纳米喷雾解吸电喷雾电离（nanoDESI）成像质谱法。对于纳米olc - ims - tof质谱测量，将从适当的小鼠子宫细胞类型制备肽，脂质和代谢物样品的复杂混合物，并且该平台将能够同时定量所有三类分析物。由于我们的目标是利用这些数据来识别可能与子宫接受性直接相关的新分子靶点，我们将使用我们的网络和基于通路的数据集成方法来关注脂质信号。这些靶点将通过Dey实验室的分子分析和子宫组织切片的纳米desi成像质谱进一步验证，以确认细胞类型特异性表达。纳米desi成像将为脂质和代谢物提供一个独特的视角，这是原位杂交或免疫染色无法看到的。本研究将揭示来自不同子宫细胞类型并由其产生的特定分子介质的身份，所有这些分子介质都会影响胚胎-子宫在接受性过程中的相互作用。

The overall objective of this research is to identify uterine molecular targets that influence the window of receptivity for embryo implantation. The rational for the proposed research is that a better understanding of uterine-derived mediators that influence the window of receptivity to implantation will offer multiple targets for uterine receptivity, which may in turn be used to determine appropriate times for embryo transfer in the in vitro fertilization clinic. In this proposal we will use wild-type mice to compare protein, lipid and metabolite profiles in isolated epithelial and stromal cells from prereceptive, receptive and nonreceptive (refractory) phases of uterine sensitivity to embryo implantation. Although current knowledge of lipid mediators correlated with uterine receptivity is relatively limited, lipid metabolites endocannabinoids and prostaglandins have been shown to play critical roles. An in-depth lipidomic characterization of a mouse model of uterine receptivity has never been undertaken. Our hypothesis is that regulatory networks in lipid metabolism could be serving as signaling hubs controlling previously uncharacterized targets; structurally complex lipids, lipid metabolic enzymes and lipid metabolites, with distinct roles in uterine receptivity. To explore this hypothesis, this project will utilize two unique technologies recently developed at Pacific Northwest National Laboratory for discovery and validation of molecular targets that influence receptivity for embryo implantation: 1) ultra-sensitive electrospray ionization ion mobility spectrometry-time of flight mass spectrometry (IMS-TOF MS) with advanced nano-scale liquid chromatography (nanoLC) methods and 2) nanospray desorption electrospray ionization (nanoDESI) imaging mass spectrometry. For nanoLC-IMS-TOF MS measurements, a complex mixture of peptides, lipids and metabolite samples will be prepared from appropriate mouse uteri cell types, and the platform will enable simultaneous quantification of all three classes of analytes. As our goal is to use these data to identify novel molecular targets with the potential to be directly associated with uteri receptivity, we will use our network and pathway-based data integration approach to focus on lipid signaling. These targets will be further validated with molecular assays in the Dey laboratory and by nanoDESI imaging mass spectrometry of uterine tissue sections to confirm cell type-specific expression. NanoDESI imaging will provide a unique look at lipids and metabolites, which cannot be visualized by in situ hybridization or immunostaining. This research will reveal the identity of specific molecular mediators, derived from and incurred by different cell types of the uterus, all of which influence embryo-uterine interactions during receptivity.