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

流行病学证据表明，父亲的先入为主暴露在环境扰动中，例如 压力和不良童年经历(ACE)与生殖结果的变化有关， 后代的妊娠发育，并最终影响后代的健康和疾病。在动物模型中的研究已经 涉及到小分子非编码RNA(SncRNA)的生殖细胞转移，包括miRNA和tRNA片段，在 对这些效果进行编程。我们最近发表了我们的基础性工作，使我们能够构建一个脚手架 初步评估SncRNA(包括miRNA、piRNA和tRNA)的组成和动态变化 精子样本来自一个年轻、健康和相对同质的学生队列。这是重复的措施 设计使我们能够定义人类中最丰富的精子在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，通过利用个体之间的关系的复杂建模 在每次精子捐献之前，我们能够识别出特定的SncRNA 对先前压力的动态反应。最终，我们的模型确定了共同的高表达miRNA 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， 符合对象内部和对象之间动态表达的严格标准，并与先前感知的内容相关联 压力。为了验证我们的假设，提供了以下目标：1)在目标1中，测试我们当前的精子SncRNA 在更大和更具代表性的学生队列中，我们将检查确定的结果 在我们的第一项研究中，包括在6个月的精子收集和测试SncRNA群体中感受到的压力 对先前感知到的压力的表达、变化和反应；2)在目标2中测试 本模型中的受试者A，作为成人当前压力知觉的主要影响因素之一，对 精子SNcRNA在低与高ACE暴露的男性中；以及3)在目标3中证实 先前在我们的模型中发现的精子相关miRNA持续高水平表达 跨受试者，或与先前感觉到的内部和之间的压力相关联的动态表达- 研究对象。我们将利用小鼠受精卵显微注射miRNA抑制剂来特异性地降低正常水平 高表达的精子相关miRNA、miR-22-3p、miR-16-5p和miR-34c-5p，以及miRNA模拟 具体提升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。结果将检验这些微量注射对体外试验的影响 植入前胚胎发育、体内胚胎和胎儿发育以及转录变化 通过对e7.5胚胎进行RNA测序。

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.