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.

项目摘要/摘要 压力被认为是月经周期紊乱、闭经和不孕的主要因素， 影响到25%的育龄妇女。到目前为止，应激性不孕的神经内分泌原因有 不能完全理解。糖皮质激素分泌增强(CORT)是一种常见和关键的反应 都是压力源。我们证明，应激水平的皮质醇通过以下途径扰乱雌性小鼠的排卵周期 弓状核(ArCKiss1)和前腹侧脑室周围区Kispeptin(Kiss1)神经元抑制 (AVPVKiss1)核，它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。具体地说，发情间期低水平的雌二醇会使皮质醇损害ARCKiss1 控制黄体生成素(LH)脉冲和高峰值雌二醇水平使CORT能够预防AVPVKiss1 神经元激活促黄体生成素的分泌。雌激素受体和糖皮质激素受体(GR)都在 Kiss1神经元；然而，GR和ER的相互作用调节转录和表观基因组格局 在ARCKiss1或AVPVKiss1内，抑制促黄体生成素分泌的神经元仍不清楚。这项提案采用了一种削减- EDGE方法从异种ARC或AVPV细胞群中捕获和分离Kiss1细胞。这种隔绝 在特定细胞类型中标记的核(完整)协议使用标记的核膜的表达 蛋白质(SUN1)仅在Kiss1细胞中(即依赖于Cre)用于快速分离下游标记的核 转录和表观基因组测序。目标1将检验雌激素使皮质醇能够改变 Kiss1神经元的转录组，促进ARCKiss1和AVPVKiss1神经元活性的抑制和降低 促黄体生成素的分泌分别为搏动性和波浪型。小鼠将成为OVX并接受间情期(ARCKiss1)或 雌激素的浪涌水平替代(AVPVKiss1)和皮质醇或胆固醇治疗(对照)，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将检验这一假设 雌激素驱动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。完整的将与：(2.1)转座酶可及的检测相结合 染色质(ATAC-SEQ)评价雌激素和皮质醇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)直接定位GR全基因组结合图谱 ARCKiss1和AVPVKiss1细胞，以检验雌激素使GR结合位点可达的假设 (目标1所述的激素治疗)。这项提案的成功完成将大大增加我们的 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，并可能确定影响生殖管理的可用药目标 中枢调节受损引起的紊乱。此应用属于高风险-高风险的范围 奖励机制，并将导致新的试剂，将影响生物医学研究。所有方法，荷尔蒙 治疗和动物模型(Kiss1-CrexSun1-GFP)可在该调查小组内获得。