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.

项目摘要/摘要 压力被认为是月经周期疾病，闭经和不育的发展的主要因素， 影响25％的生殖年龄妇女。迄今为止，压力引起的不育症的神经内分泌原因是 不完全理解。增强糖皮质激素（Cort）的分泌是对 所有压力源。我们证明了Cort的应力水平通过 抑制位于弧形（Arckiss1）和前腹膜周围的Kisspeptin（Kiss1）神经元（Kiss1）神经元 （AVPVKISS1）核，这是繁殖神经控制的重要组成部分。科特的效果 取决于卵巢类固醇环境。具体而言，雌二醇水平低的雌二醇水平使cort能够损害Arckiss1 控制黄体生成激素（LH）脉冲和高振兴雌二醇水平使Cort可以防止AVPVKISS1 LH分泌的神经元激活。雌激素和糖皮质激素受体（GR）的受体都在 Kiss1神经元；但是，GR和ER调节转录组和表观基因组景观的相互作用 在Arckiss1或AvpvKiss1神经元中抑制LH分泌的神经元尚不清楚。该提案采用了一项援助 - 从异质弧或AVPV细胞种群中捕获和分离Kiss1细胞的边缘方法。隔离 在特定细胞类型（完整）方案中标记的原子核采用标记的核包膜的表达 仅在Kiss1细胞（即CRE依赖性）中蛋白质（SUN1）快速分离标记的核的下游 转录组和表观基因组测序。 AIM 1将检验雌二醇使Cort改变的假设 Kiss1神经元的转录组，促进抑制Arckiss1和AvpvKiss1神经元活动 分别脉冲和潮流型LH分泌。小鼠将是OVX并接受Diestrus样（Arckiss1）或 雌二醇的激发级替代（AVPVKISS1），并用Cort或胆固醇（对照）处理 完整分离的亲吻细胞的转录组将通过RNA测序评估。 AIM 2将检验假设 雌二醇驱动Arckiss1和Avpvkiss1神经元的染色质景观的变化，使GR结合 并改变神经元转录组。完整将与：（2.1）转座酶可访问的测定 染色质（atac-seq）评估雌二醇和cort介导的开放染色质的表观基因组，以及 （2.2）染色质免疫沉淀（CHIP-SEQ）直接映射GR的全基因组结合曲线 arckiss1和avpvkiss1细胞测试以下假设：雌二醇可以使GR结合位点可访问 （AIM 1中描述的激素治疗）。成功完成此提案将大大增加我们的 对控制KISS1神经元生理反应性的细胞内机制的了解，这将 在随后的工作中进行测试，并可能识别出可药的目标以影响生殖的管理 中央法规受损引起的疾病。该应用程序属于高风险高的范围 奖励机制，并会导致新的试剂，从而影响生物医学研究。所有方法，激素 该调查团队可用治疗和动物模型（KISS1-CREXSUN1-GFP）。