The Role of Homeodomain Proteins in Human Decidualization

同源域蛋白在人类蜕膜化中的作用

• 批准号: 10619084
• 负责人: Meade Elspeth Haller
• 金额: $ 2.39万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10619084
• 关键词: Affect; Basic Science; Biological Assay; Biological Models; Cell Line; Cells; Clinical; Complex; Contraceptive Agents; Data; Data Set; Decidua; Decidual Cell Reactions; Defect; Development; Developmental Process; Differentiation and Growth; Embryo; Endometrial Stromal Cell; Endometrium; Environment; Estrogens; Exhibits; Family; Female; Female infertility; Fertility; Fertility Study; Fertilization; Fertilization in Vitro; Flushing; Fostering; Gene Family; Generations; Genes; Genetic; Genetic Transcription; Genetic study; Genome; Genomics; Homeodomain Proteins; Hormone use; Hormones; Human; Human Cloning; Implant; Infertility; Influentials; Knock-out; Knockout Mice; Maps; Mediating; Modernization; Molecular; Monitor; Mus; Ovarian hormone; Ovulation; Pathway interactions; Pattern; Pharmacology; Phase; Placentation; Pregnancy; Pregnant Uterus; Procedures; Process; Progesterone; Prolactin; Proteins; Reaction; Reporter; Role; Screening procedure; Signal Pathway; Signal Transduction; Small Interfering RNA; Technology; Testing; Time; Training; Transcript; Universities; Uterus; Washington; base; blastocyst; collaborative environment; conditional knockout; design; differential expression; early pregnancy loss; endometrial stroma; environmental stressor; experimental study; female fertility; genome wide screen; healthy pregnancy; homeodomain; hormone sensitivity; implantation; knock-down; lectures; mutant; natural Blastocyst Implantation; novel; promoter; pup; response; symposium; transcription factor; transcriptome sequencing; translational applications; uterine receptivity; whole genome

ABSTRACT: In order for the human uterus to become receptive to an implanting embryo, the endometrial stromal cells must undergo a complex proliferation and differentiation response to ovarian hormones, termed decidualization. Despite the integral role of uterine receptivity in the implantation phase of pregnancy, and despite many expensive in vitro fertilization procedures proving unsuccessful due to uterine deficiency, the molecular pathways underlying decidualization remain woefully understudied. One major development in the study of decidualization was the generation of immortalized human endometrial stromal cells (hESCs), which respond transcriptionally robustly to the ovarian hormones progesterone and estrogen. As preliminary data to the proposed study, we generated a reporter cell line sub-cloned from hESCs that expresses YFP under the control of the prolactin promoter, and therefore exhibits a consistent and robust induction of YFP when treated with a cocktail of ovarian hormones. These cells, termed hESC-PRLYs, represent a powerful screening tool to assess decidualization under various genomic and environmental stresses. Using these cells we performed a full genome siRNA screen to map the human decidualization genetic network, and found that among the most influential gene families required for normal decidualization are homeodomain transcription factors. The proposed study aims to first map the temporal induction profile of homeodomain proteins in hESCs in response to hormone using qPCR. Next, the requirement of homeodomain proteins in decidualization will be determined by generating conditional knockout mice using Pgr-cre for the top candidate factors. Knockout mice will undergo assessment including fertility study with endpoint of pups per litter, gestational monitoring, blastocyst flush, implantation assay, and artificial decidualization assay wherever appropriate to determine whether female fertility is affected by the loss of each gene, and at which point in the process fertility fails. The candiate gene determined to have the most impact on decidualization will undergo downstream assessment to map its targets in the decidua. This will include qPCR of potential targets in murine knockout uterus, and if time permits, RNA sequencing of mutant murine pregnant uterine stroma, Crispr knockout (or siRNA knockdown if Crispr fails) hESCs, and SpDAM assay in hESC unbiasedly identify downstream affected genes and overlap human and murine datasets. Together these data will define the robust role of homeodomain transcription factors in decidualization, and identify one or more major targets for potential pharmacological modulation in the treatment of female implantation-level infertility. Access to the newest technologies, insightful lectures, regular symposia and the fostering of a collaborative atmosphere will be an integral part of the training process. Washington University provides an unparalleled environment of advisement by the foremost leaders of basic research for translational applications.

抽象的： 为了使人类子宫能够接受植入的胚胎，子宫内膜基质细胞必须 对卵巢激素进行复杂的增殖和分化反应，称为蜕膜化。 尽管子宫容受性在妊娠着床阶段发挥着不可或缺的作用，并且尽管有许多因素 由于子宫缺陷，昂贵的体外受精程序被证明不成功，分子 令人遗憾的是，蜕膜化的潜在途径仍然没有得到充分的研究。研究的一大进展 蜕膜化是永生化人类子宫内膜基质细胞（hESC）的产生，它对 与卵巢激素黄体酮和雌激素的转录密切相关。作为初步数据 在拟议的研究中，我们生成了从 hESC 亚克隆的报告细胞系，该细胞系在以下条件下表达 YFP： 催乳素启动子的控制，因此在治疗时表现出一致且强大的 YFP 诱导 与卵巢激素的混合物。这些细胞被称为 hESC-PRLY，是一种强大的筛选工具 评估各种基因组和环境压力下的蜕膜化。使用这些细胞我们进行了 全基因组 siRNA 筛选绘制了人类蜕膜化遗传网络图谱，并发现其中最 正常蜕膜化所需的有影响力的基因家族是同源域转录因子。这 拟议的研究旨在首先绘制 hESC 中同源域蛋白响应的时间诱导图谱 使用 qPCR 检测激素。接下来，将确定蜕膜化中同源结构域蛋白的需求 通过使用 Pgr-cre 作为最佳候选因子生成条件敲除小鼠。淘汰小鼠将 接受评估，包括生育力研究，每窝幼仔的终点、妊娠监测、囊胚 在适当的情况下进行冲洗、着床试验和人工蜕膜化试验，以确定是否 女性的生育能力会受到每个基因丢失的影响，在这个过程中，生育能力会失败。候选人 确定对蜕膜化影响最大的基因将接受下游评估，以绘制其图谱 目标在蜕膜中。这将包括小鼠敲除子宫中潜在目标的 qPCR，如果时间 允许，突变小鼠怀孕子宫基质的 RNA 测序，Crispr 敲除（或 siRNA 敲除，如果 Crispr 失败）hESC 和 hESC 中的 SpDAM 检测无偏性地识别下游受影响的基因和重叠 人类和小鼠数据集。这些数据将共同定义同源域转录的强大作用 蜕膜化的因素，并确定潜在药理调节的一个或多个主要靶标 女性着床期不孕症的治疗。获得最新的技术、富有洞察力的讲座、 定期研讨会和营造协作氛围将是培训的一个组成部分 过程。华盛顿大学提供无与伦比的环境，由最重要的领导人提供建议 转化应用基础研究。