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: 10195913
• 负责人: KELLIE Breen Church
• 金额: $ 23.69万
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10195913
• 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.

项目总结/摘要 压力被认为是月经周期紊乱、闭经和不孕的主要因素， 影响了25%的育龄妇女。到目前为止，神经内分泌的原因，压力引起的不孕症是 不完全理解。糖皮质激素分泌增强（CORT）是一种常见的和关键的反应， 所有压力源我们证明应激水平的CORT通过以下途径破坏雌性小鼠的排卵周期： 抑制位于弓状（ARCKiss 1）和前腹侧脑室周围的kisspeptin（Kiss 1）神经元 （AVPVKiss 1）核，这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特别是，动情间期低水平的雌二醇使CORT损害ARCKiss 1 控制促黄体生成激素（LH）脉冲和高雌二醇水平使CORT能够预防AVPVKiss 1 LH分泌的神经元激活。雌激素受体和糖皮质激素受体（GR）都在 Kiss 1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 在ARCKiss 1或AVPVKiss 1神经元内抑制LH分泌的机制尚不清楚。该提案采用了一种切割- 边缘方法从异质ARC或AVPV细胞群中捕获和分离Kiss 1细胞。隔离 在特定细胞类型中标记的细胞核的表达（INTACT）方案采用标记的核膜的表达 蛋白（Sun 1）仅在Kiss 1细胞（即Cre依赖性）中用于快速分离标记的细胞核，用于下游 转录组和表观基因组测序。目的1将检验雌二醇使CORT改变的假设， Kiss 1神经元的转录组，促进ARCKiss 1和AVPVKiss 1神经元活性的抑制， 分别为脉冲式和激增式LH分泌。小鼠将进行OVX并接受间情期样（ARCKiss 1）或 雌二醇的峰水平替代（AVPVKiss 1），并用CORT或胆固醇（对照）治疗， 将通过RNA测序评价INTACT分离的Kiss细胞的转录组。目标2将检验假设 雌二醇驱动ARCKiss 1和AVPVKiss 1神经元染色质景观的变化，允许GR结合 并改变神经元的转录组INTACT将结合：（2.1）转座酶可降解性测定 染色质（ATAC-seq），以评估雌二醇和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）直接映射GR的全基因组结合谱， ARCKiss 1和AVPVKiss 1细胞，以检验GR结合位点可被雌二醇接近的假设 （目标1中描述的激素治疗）。成功完成这一提案将大大提高我们的 了解控制Kiss 1神经元生理反应的细胞内机制， 在随后的工作中进行测试，并可能确定影响生殖管理的药物靶点。 由于中枢调节受损而引起的紊乱。此应用程序福尔斯高风险-高 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，激素 治疗和动物模型（Kiss 1-CrexSun 1-GFP）在该研究小组内可用。