Transcriptomic and epigenomic basis for reproductive dysfunction during stress

应激期间生殖功能障碍的转录组和表观基因组基础

• 批准号: 10394958
• 负责人: KELLIE Breen Church
• 金额: $ 19.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10394958
• 关键词: Address; Affect; Age; Amenorrhea; Animal Model; Binding; Binding Sites; Biological Assay; Biomedical Research; Breast Cancer Cell; Cardiac health; Cell Nucleus; Cells; ChIP-seq; Chromatin; Coupled; Cues; Data; Development; Diestrus; Employment; Epigenetic Process; Estradiol; Estrogen Receptors; Estrogens; Exposure to; Female; Fertility; Functional disorder; Future; Gene Expression; Generations; Genetic Transcription; Glucocorticoid Receptor; Glucocorticoids; Gonadal Steroid Hormones; Gonadotropin Hormone Releasing Hormone; Gonadotropins; Homeostasis; Hormone secretion; Hormones; Hypothalamic structure; Impairment; Implant; Infertility; KISS1 gene; Knowledge; Laboratories; Luteinizing Hormone; Maps; Mediating; Menstruation Disturbances; Methods; Molecular; Mus; Neurons; Neurosecretory Systems; Nuclear Envelope; Ovarian; Ovarian Cycles; Pattern; Physiologic pulse; Physiological; Population; Production; Protocols documentation; Reagent; Regulation; Reproduction; Research; Research Personnel; Resources; Signal Transduction; Steroids; Stress; Talents; Technology; Testing; Transgenic Organisms; Transposase; Woman; Women' s Health; Work; base; bone health; brain health; cell type; cholesterol control; chromatin immunoprecipitation; druggable target; env Gene Products; epigenome; epigenomics; falls; genome-wide; glucocorticoid-induced orphan receptor; high reward; high risk; innovation; interest; male; neuromechanism; neuroregulation; next generation sequencing; novel; prevent; receptor; receptor binding; reproductive; reproductive system disorder; response; stressor; tool; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

Project Summary/Abstract Stress is considered a major factor in the development of menstrual cycle disorders, amenorrhea, and infertility, affecting 25% of reproductive age women. To date, the neuroendocrine causes of stress-induced infertility are not completely understood. Enhanced secretion of glucocorticoids (CORT) is a common and critical response to all stressors. We demonstrated that a stress level of CORT disrupts the ovulatory cycle of female mice via suppression of kisspeptin (Kiss1) neurons located in both arcuate (ARCKiss1) and anteroventral periventricular (AVPVKiss1) nuclei, which are essential components of the neural control of reproduction. The effect of CORT depends on ovarian steroid milieu. Specifically, low diestrus levels of estradiol enable CORT to impair ARCKiss1 control of luteinizing hormone (LH) pulses and high surge estradiol levels enable CORT to prevent AVPVKiss1 neuronal activation of the LH secretion. Receptors for estrogen and glucocorticoid receptors (GR) are both in Kiss1 neurons; however, the interaction of GR and ER to modulate the transcriptomic and epigenomic landscape within ARCKiss1 or AVPVKiss1 neurons to suppress LH secretion remains unclear. This proposal employs a cutting- edge method to capture and isolate Kiss1 cells from heterogeneous ARC or AVPV cell populations. The Isolation of Nuclei Tagged in specific Cell-Types (INTACT) protocol employs expression of a tagged nuclear envelope protein (Sun1) only in Kiss1 cells (i.e. Cre-dependent) for rapid isolation of tagged nuclei for downstream transcriptomic and epigenomic sequencing. Aim 1 will test the hypothesis that estradiol enables CORT to alter the transcriptome of Kiss1 neurons, facilitating suppressed ARCKiss1 and AVPVKiss1 neuronal activity and lowered pulsatile and surge-type LH secretion, respectively. Mice will be OVX and receive diestrus-like (ARCKiss1) or surge-level replacement (AVPVKiss1) of estradiol and treated with CORT or cholesterol (control), and the transcriptome of INTACT-isolated Kiss cells will be evaluated by RNA-sequencing. Aim 2 will test the hypothesis that estradiol drives changes of the chromatin landscape of ARCKiss1 and AVPVKiss1 neurons allowing GR to bind and alter the neuronal transcriptome. INTACT will be coupled with: (2.1) Assay for Transposase-Accessible Chromatin (ATAC-seq) to evaluate the epigenome for open chromatin in mediated by estradiol and CORT and (2.2) chromatin immunoprecipitation (ChIP-seq) to directly map the genome-wide binding profile of GR in ARCKiss1 and AVPVKiss1 cells to test the hypothesis that GR binding sites are made accessible by estradiol (hormone treatments described in Aim 1). Successful completion of this proposal will significantly increase our knowledge of intracellular mechanisms that govern the physiologic responsiveness of Kiss1 neurons, which will be tested in subsequent work and may identify druggable targets to influence the management of reproductive disorders resulting from impaired central regulation. This application falls within the scope of the high risk-high reward mechanism and will result in novel reagents that will impact biomedical research. All methods, hormone treatments and the animal model (Kiss1-CrexSun1-GFP) are available within this investigative team.

项目概要/摘要 压力被认为是导致月经周期紊乱、闭经和不孕的主​​要因素， 影响25%的育龄妇女。迄今为止，应激性不孕症的神经内分泌原因是 没有完全理解。糖皮质激素 (CORT) 分泌增强是一种常见且关键的反应 所有压力源。我们证明，CORT 的应激水平会通过以下方式扰乱雌性小鼠的排卵周期： 抑制位于弓状 (ARCKiss1) 和前腹侧脑室周围的 Kisspeptin (Kiss1) 神经元 (AVPVKiss1) 细胞核，是生殖神经控制的重要组成部分。 CORT的作用 取决于卵巢类固醇环境。具体来说，低发情间期水平的雌二醇使 CORT 能够损害 ARCKiss1 控制黄体生成素 (LH) 脉冲和高雌二醇水平使 CORT 能够预防 AVPVKiss1 LH 分泌的神经元激活。雌激素受体和糖皮质激素受体 (GR) 均位于 Kiss1神经元；然而，GR 和 ER 的相互作用调节转录组和表观基因组景观 ARCKiss1 或 AVPVKiss1 神经元内抑制 LH 分泌的作用尚不清楚。该提案采用了切割- 边缘方法从异质 ARC 或 AVPV 细胞群中捕获和分离 Kiss1 细胞。隔离 特定细胞类型中标记的细胞核 (INTACT) 方案采用标记核膜的表达 蛋白质 (Sun1) 仅存在于 Kiss1 细胞中（即 Cre 依赖性），用于快速分离下游标记的细胞核 转录组和表观基因组测序。目标 1 将检验雌二醇使 CORT 改变的假设 Kiss1 神经元的转录组，促进 ARCKiss1 和 AVPVKiss1 神经元活动受到抑制并降低 分别为脉冲型和激增型 LH 分泌。小鼠将是 OVX 并接受发情间期样 (ARCKiss1) 或 雌二醇的激增水平替代（AVPVKiss1）并用 CORT 或胆固醇（对照）治疗，以及 完整分离的 Kiss 细胞的转录组将通过 RNA 测序进行评估。目标 2 将检验假设 雌二醇驱动 ARCKiss1 和 AVPVKiss1 神经元染色质景观的变化，从而允许 GR 结合 并改变神经元转录组。 INTACT 将与：(2.1) 转座酶可及性检测相结​​合 染色质 (ATAC-seq) 用于评估雌二醇和 CORT 介导的开放染色质的表观基因组 (2.2) 染色质免疫沉淀 (ChIP-seq) 直接绘制 GR 的全基因组结合谱 ARCKiss1 和 AVPVKiss1 细胞用于测试雌二醇可接近 GR 结合位点的假设 （目标 1 中描述的激素治疗）。该提案的成功完成将显着提高我们的 了解控制 Kiss1 神经元生理反应的细胞内机制，这将 在后续工作中进行测试，并可能确定可影响生殖管理的药物靶标 中枢调节功能受损导致的疾病。该应用属于高风险高风险范围 奖励机制并将产生影响生物医学研究的新型试剂。所有方法，激素 该研究团队提供了治疗方法和动物模型（Kiss1-CrexSun1-GFP）。