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.

摘要： 为了使人类子宫能够接受植入胚胎，子宫内膜间质细胞必须 对卵巢激素有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的植入阶段起着不可或缺的作用，尽管许多 由于子宫缺陷，昂贵的体外受精手术被证明是不成功的，分子 可悲的是，蜕膜化的基本途径仍未得到充分的研究。研究中的一个重大进展 蜕膜化是永生化的人类子宫内膜间质细胞(HESCs)的产生，它对 对卵巢荷尔蒙黄体酮和雌激素有很强的转录作用。作为初步数据提供给 建议的研究，我们从hESCs亚克隆了一个报告细胞系，该细胞在 控制催乳素启动子，因此在治疗时表现出一致和强大的YFP诱导 用一种卵巢激素的鸡尾酒。这些细胞被称为hESC-PRLY，是一种强大的筛查工具 评估在各种基因组和环境压力下蜕膜的形成。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，发现其中 正常蜕膜形成所需的有影响力的基因家族是同源结构域转录因子。这个 拟议的研究旨在首次定位hESCs中同源结构域蛋白的时间诱导谱以响应 用qPCR法进行激素合成。接下来，将确定蜕膜化过程中对同源结构域蛋白的需求。 通过使用PGR-cre作为首要候选因子来产生条件性基因敲除小鼠。基因敲除小鼠会 进行评估，包括以每窝幼崽为终点的生育研究、妊娠监测、囊胚 冲洗、植入试验和人工蜕膜试验，以确定是否 女性的生育能力受到每个基因缺失的影响，在这一过程中，生育能力会下降。候选人 被确定对蜕膜化影响最大的基因将接受下游评估，以绘制其 蜕膜中的目标。这将包括对小鼠基因敲除子宫中潜在靶点的定量聚合酶链式反应，如果时间 许可，突变的小鼠怀孕子宫间质的RNA测序，Crispr基因敲除(或siRNA敲除，如果 CRISPR失败)，hESCs和SpDAM检测在hESC中无偏见地识别下游受影响的基因并重叠 人类和小鼠的数据集。这些数据将共同定义同源结构域转录的强大作用 蜕膜化的因素，并确定一个或多个潜在的药理调节的主要靶点 女性着床级不孕症的治疗。接触到最新的技术，有洞察力的讲座， 定期专题讨论会和培养合作气氛将是培训的组成部分 进程。华盛顿大学为最重要的领导人提供了一个无与伦比的顾问环境 翻译应用的基础研究。