Stress modeling of the human sperm sncRNA transcriptome and causal importance of dynamic miRNA in reproductive and developmental outcomes

人类精子 sncRNA 转录组的压力模型以及动态 miRNA 在生殖和发育结果中的因果重要性

• 批准号: 10707015
• 负责人: Tracy L Bale
• 金额: $ 75.73万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707015
• 关键词: Accounting; Adult; Animal Model; Biological; Chinese; Collection; Complex; Conceptions; Data; Development; Disease; Disease model; Embryo; Embryonic and Fetal Development; Environment; Epidemiology; Exposure to; Famines; General Population; Genetic; Germ Cells; Health; Human; Immune; In Vitro; Individual; Linear Regressions; Measures; Messenger RNA; Metabolic; Methods; MicroRNAs; Microinjections; Modeling; Mus; Outcome; Participant; Plasma; Population; Population Heterogeneity; Pre-implantation Embryo Development; Pregnancy; Psychological Stress; Publishing; Role; Sampling; Seminal fluid; Signal Transduction; Small RNA; Stress; Structure; Students; Testing; Time; Transfer RNA; Untranslated RNA; Variant; Work; adverse childhood events; cohort; design; early childhood; experience; fetal; human subject; in vivo; inhibitor; insight; male; men; model building; novel; offspring; perceived stress; piRNA; predictive modeling; psychiatric symptom; psychologic; recruit; reproductive; reproductive outcome; response; scaffold; sperm cell; stress reactivity; stress state; transcriptome; transcriptome sequencing; transcriptomics; zygote

Epidemiological evidence shows that paternal preconception exposures to environmental perturbations, such as stress and adverse childhood experiences (ACEs), are associated with changes in reproductive outcomes, offspring gestational development, and ultimately, offspring health and disease. Studies in animal models have implicated the germ cell transfer of small non-coding RNA (sncRNA), including miRNA and tRNA fragments, in programming these effects. We recently published our foundational work which allowed us to construct a scaffold to initially assess the composition of, and dynamic changes in, sncRNA (including miRNA, piRNA, and tRNA) in sperm samples from a young, healthy and relatively homogenous student cohort. This repeated-measures design allowed us to define in humans the between- and within-participant variation in the most abundant sperm sncRNA content over time. In addition, by utilizing complex modeling of the relationships between individual sncRNA and perceived stress states preceding each sperm donation, we were able to identify specific sncRNA responsive to the dynamics of prior stress. Ultimately, our model identified highly expressed miRNA common to all subjects, including miR-34c-5p and miR-16-5p, and three miRNA, including miR-181a-5p and let-7f-5p, that fit strict criterion for dynamic expression within- and between-subjects, and were associated with prior perceived stress. To test our hypothesis, the following Aims are provided: 1) in Aim 1 to test our current sperm sncRNA framework within a larger and more representative cohort of students, we will examine the outcomes identified in our first study, including perceived stress across 6 months of sperm collection and test the sncRNA populations for expression, variance, and responses to prior perceived stress; 2) in Aim 2 to test the additional influence of subject ACEs in our model, as one of the major influences on adult current stress perception, for effects on sperm sncRNA in low vs high ACE-exposed males; and 3) in Aim 3 to substantiate a causal importance of the sperm-associated miRNA previously identified in our model that were consistently expressed at high levels across subjects, or dynamically expressed in association with prior perceived stress within- and between- subjects. We will utilize mouse zygotic microinjection of miRNA inhibitors to specifically reduce levels of normally highly expressed sperm-associated miRNA, miR-22-3p, miR-16-5p, and miR-34c-5p, and miRNA mimics to specifically elevate individual miRNA normally lowly expressed, but dynamically environmentally responsive, miR-181a-5p, miR-4454, or let-7f-5p. Outcomes will examine the impact of these microinjections on in vitro preimplantation embryonic development, in vivo embryonic and fetal development, and transcriptomic changes by RNA sequencing of E7.5 embryos.

流行病学证据表明，父亲的成见会暴露于环境扰动，例如 压力和不良童年经历（ACE）与生殖结果的变化有关， 后代妊娠发育，最终影响后代的健康和疾病。动物模型研究已 涉及小非编码 RNA (sncRNA)（包括 miRNA 和 tRNA 片段）的生殖细胞转移 对这些效果进行编程。我们最近发表了我们的基础工作，使我们能够建造一个脚手架 初步评估 sncRNA（包括 miRNA、piRNA 和 tRNA）的组成和动态变化 精子样本来自年轻、健康且相对同质的学生群体。这种重复测量 设计使我们能够定义人类中最丰富精子的参与者之间和参与者内部的变异 sncRNA 含量随时间的变化。此外，通过利用个体之间关系的复杂建模 sncRNA 和每次精子捐赠前感知的压力状态，我们能够识别特定的 sncRNA 对先前压力的动态做出反应。最终，我们的模型鉴定了常见的高表达 miRNA 所有受试者，包括 miR-34c-5p 和 miR-16-5p，以及三种 miRNA，包括 miR-181a-5p 和 let-7f-5p， 符合受试者内部和受试者之间动态表达的严格标准，并且与先前的感知相关 压力。为了检验我们的假设，提供了以下目标：1）在目标 1 中测试我们当前的精子 sncRNA 在更大、更具代表性的学生群体的框架内，我们将检查确定的结果 在我们的第一项研究中，包括在 6 个月的精子采集过程中感受到的压力并测试 sncRNA 群体 表达、差异和对先前感知压力的反应； 2）在目标2中测试额外的影响 我们模型中的受试者 ACE，作为成人当前压力感知的主要影响因素之一，对 低 ACE 暴露男性与高 ACE 暴露男性的精子 sncRNA； 3) 在目标 3 中证实以下因素的因果重要性 先前在我们的模型中发现的与精子相关的 miRNA 始终以高水平表达 跨学科，或动态表达与先前感知到的内部和之间的压力相关 科目。我们将利用小鼠合子显微注射 miRNA 抑制剂来特异性降低正常情况下的水平 高表达的精子相关 miRNA、miR-22-3p、miR-16-5p 和 miR-34c-5p，以及 miRNA 模拟物 特异性升高通常低表达但动态环境响应的单个 miRNA， miR-181a-5p、miR-4454 或 let-7f-5p。结果将检查这些显微注射对体外的影响 植入前胚胎发育、体内胚胎和胎儿发育以及转录组变化 通过 E7.5 胚胎的 RNA 测序。