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，父亲的先选暴露于环境扰动，例如 压力和童年不利的经历（ACE）与生殖结果的变化有关， 后代妊娠发展，最终是后代健康和疾病。动物模型的研究具有 暗示了小型非编码RNA（SNCRNA）的生殖细胞转移，包括miRNA和tRNA片段， 编程这些效果。我们最近发表了我们的基础作品，使我们能够建造一个脚手架 最初评估sncRNA的组成和动态变化（包括miRNA，piRNA和tRNA） 来自年轻，健康且相对同质的学生队列的精子样本。重复测量 设计使我们能够在人类中定义最丰富的精子中的参赛和内部变化 随着时间的流逝，sncrna含量。另外，通过利用个人之间关系的复杂建模 sncRNA和感知的应力状态在每个精子捐赠之前，我们能够识别特定的sncrna 响应先前压力的动力。最终，我们的模型确定了高度表达的miRNA共同 所有受试者，包括miR-34c-5p和miR-16-5p，以及三个miRNA，包括miR-181a-5p和let-7f-5p， 符合严格的标准，用于在受试者之间和受试者之间进行动态表达，并与先前感知到 压力。为了检验我们的假设，提供了以下目的：1）在目标1中测试我们当前的精子sncrna 在较大，更具代表性的学生中，我们将检查所确定的结果 在我们的第一个研究中，包括在6个月的精子收集中感知压力并测试SncRNA种群 表达，方差和对先前感知压力的反应； 2）在AIM 2中测试 我们模型中的受试者ACE是对成人当前压力感知的主要影响之一，以对 精子sncrna在低和高ace暴露的男性中； 3）在AIM 3中证实了因果关系的重要性 先前在我们的模型中鉴定出的精子相关miRNA，这些miRNA始终在高水平上表达 跨受试者，或动态表达与先前感知的应力相关 主题。我们将利用小鼠合子对miRNA抑制剂的合子显微注射来特别降低正常水平 高表达的精子相关miRNA，miR-22-3p，miR-16-5p和miR-34c-5p，miRNA模拟 特别提升了通常表达的单个miRNA，但在环境上有动态响应， miR-181a-5p，miR-4454或let-7f-5p。结果将检查这些微分注射对体外的影响 植入前胚胎发育，体内胚胎和胎儿发育以及转录组的变化 通过E7.5胚胎的RNA测序。