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: 10442142
• 负责人: Tracy L Bale
• 金额: $ 74.35万
• 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/10442142
• 关键词: Accounting; Adult; Animal Model; Biological; Chinese; Collection; Complex; Data; Development; Disease; Disease model; Embryo; Embryonic Development; Embryonic and Fetal Development; Environment; Epidemiology; Exposure to; Famines; Foundations; 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; 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; novel; offspring; perceived stress; piRNA; predictive modeling; preimplantation; 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，父亲的孕前暴露于环境扰动，如 压力和不良童年经历（ACE）与生殖结果的变化有关， 后代妊娠期发育，最终影响后代的健康和疾病。动物模型的研究 暗示了小的非编码RNA（sncRNA），包括miRNA和tRNA片段的生殖细胞转移， 设计这些效果。我们最近发表了我们的基础工作，使我们能够构建一个支架， 为了初步评估细胞中sncRNA（包括miRNA、皮尔纳和tRNA）的组成和动态变化， 精子样本来自年轻、健康和相对同质的学生群体。这种重复测量 设计允许我们在人类中定义最丰富精子的参与者之间和参与者内部的差异 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和每次捐精前的感知压力状态，我们能够识别特定的sncRNA， 对先前压力的动态反应。最终，我们的模型鉴定出了高表达的miRNA， 所有受试者，包括miR-34 c-5 p和miR-16- 5 p，以及三种miRNA，包括miR-181 a-5 p和let-7 f-5 p， 符合严格的标准，动态表达内和之间的主题，并与先前的感知 应力为了验证我们的假设，提供了以下目的：1）在目的1中，测试我们目前的精子sncRNA 在一个更大，更有代表性的学生群体的框架内，我们将研究确定的结果 在我们的第一项研究中，包括在6个月的精子收集和测试sncRNA群体的感知压力， 表达，方差和对先前感知压力的反应; 2）在目标2中，测试以下因素的额外影响： 在我们的模型中，ACE作为成年人当前压力感知的主要影响之一，对 精子sncRNA在低与高ACE暴露的男性;和3）在目标3，以证实因果关系的重要性， 先前在我们的模型中鉴定出的精子相关miRNA一直以高水平表达 跨学科，或动态表达与先前感知的压力内-和之间- 科目我们将利用小鼠合子显微注射miRNA抑制剂来特异性地降低正常的 高表达的精子相关miRNA，miR-22- 3 p，miR-16- 5 p和miR-34 c-5 p，以及miRNA模拟物， 特异性地升高通常低表达但动态环境响应性个体miRNA， miR-181a-5p、miR-4454或let-7f-5p。结果将检查这些微量注射对体外细胞的影响。 植入前胚胎发育、体内胚胎和胎儿发育以及转录组学变化 E7.5胚胎的RNA测序。