Mechanisms of EDC Effects via Small-RNA Cargo in Sperm Epididymosomes

精子附睾中小 RNA 货物的 EDC 作用机制

• 批准号: 10592593
• 负责人: ANDREA C GORE
• 金额: $ 19.81万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10592593
• 关键词: Adult; Agriculture; Alcohol consumption; Bioinformatics; Biology; Cell Differentiation process; Chemical Exposure; Chemical Industry; Chemical-Induced Change; Chronic stress; Competence; CpG Islands; DNA; DNA Methylation; Data; Development; Diet; Disease; Dose; Embryo; Endocrine Disruptors; Environment; Epididymis; Epigenetic Process; Epithelium; Exposure to; Fertilization; Fetal Development; Fetus; Functional disorder; Future Generations; Gene Expression; Germ Cells; Health; Heritability; Hormonal; Human; Industrialization; Inherited; Intervention; Lactation; Life; Link; Mammals; Mediating; Methylation; Modeling; Molecular; Oocytes; Outcome; Phenotype; Plastics; Play; Polychlorinated Biphenyls; Predisposition; Pregnancy; Probability; Process; Property; Rattus; Research; Sampling; Signal Transduction; Small RNA; Sperm Maturation; Testing; Transfer RNA; Untranslated RNA; Work; androgenic; bisulfite sequencing; deep sequencing; diphenyl; dosage; egg; epigenome; estrogenic; exposed human population; extracellular vesicles; feeding; fungicide; intergenerational; male; next generation sequencing; novel; offspring; pregnant; programs; reproductive tract; sperm cell; stem cells; success; transcriptome; vinclozolin

ABSTRACT Exposures to environmental endocrine-disrupting chemicals (EDCs), especially during early life, are strongly linked to adverse health outcomes. Furthermore, EDC exposures to the fetus, which also expose the germ cells (precursors to sperm and egg) within the fetus, can cause heritable epigenetic changes that are passed to future generations. Previous work has implicated epimutations of DNA CpG methylation as one molecular mechanism by which EDCs program the germline. Proposed work will explore this mechanism, which is under-studied, and in addition, seek to understand how external factors from the male reproductive tract involved in sperm maturation may themselves contribute to epimutations. More specifically, the proximal portion (caput) of the epididymis has a substantial influence on sperm during their final steps of maturation. Extracellular vesicles (EVs) from the epididymis – epididymosomes - released from the epithelium fuse and transfer their diverse molecular contents to sperm, including small noncoding RNAs (sncRNA), thereby contributing to the functional competency of sperm and to the developmental trajectory of a fertilized embryo. These sncRNAs have the capacity to regulate gene expression and direct DNA methylation in sperm. In the current proposal, we will establish whether and how two different EDC classes that signal via differing hormonal mechanisms cause direct CpG methylation changes to DNA, and/or change the transfer of sncRNA content of epididymosomes to sperm to indirectly cause epimutations. The two EDC classes studied here are the polychlorinated biphenyl mix Aroclor 1221 (A1221), an industrial mixture that has weakly estrogenic properties; and vinclozolin (VIN), an anti- androgenic fungicide. Both EDCs are still environmentally relevant. Rats are exposed to human relevant dosages of A1221 or VIN, or vehicle, through feeding the dams during gestation and lactation. In adult male rats, sncRNA cargo of EVs from caput epididymis and mature sperm from the cauda epididymis will be extracted and used for deep sequencing and bioinformatics. DNA from sperm in the same samples will be subjected to reduced representation bisulfite sequencing to identify epimutations in CpG islands. These data will establish a definitive epigenetic profile that will allow us to pinpoint the origin of EDC induced epimutations. This proposal is biomedically relevant, as all humans are exposed to EDCs, with mechanisms of sperm maturation highly conserved across mammals.

摘要 暴露于环境内分泌干扰化学品（EDCs），特别是在生命早期， 与不良健康结果有关。此外，EDC暴露于胎儿，这也暴露了生殖细胞， （精子和卵子的前体）在胎儿内，可以引起遗传的表观遗传变化，传递给未来 代以前的工作已经暗示DNA CpG甲基化的表位突变是一种分子机制 内分泌干扰物对生殖细胞进行编程。拟议的工作将探讨这一机制，这是研究不足， 此外，力求了解外部因素如何从男性生殖道参与精子 成熟本身可能有助于表突变。更具体地，所述第一部分的近侧部分（头）被构造成与所述第二部分的近侧部分（头）接触。 在精子成熟的最后阶段，附睾对精子具有实质性的影响。细胞外囊泡 (EVs)从上皮细胞释放的附睾-附睾体-融合并转移它们的多样性， 分子内容物的精子，包括小的非编码RNA（sncRNA），从而有助于功能 精子的能力和受精胚胎的发育轨迹。这些sncRNA具有 调节基因表达和指导精子DNA甲基化的能力。在目前的提案中，我们将 建立是否以及如何通过不同的激素机制信号的两种不同的EDC类引起直接 CpG甲基化改变DNA，和/或改变附睾体的sncRNA含量向精子的转移 间接导致表位突变这里研究的两种EDC类是多氯联苯混合物Aroclor 1221（A1221），一种具有弱雌激素性质的工业混合物;以及乙烯基氯唑啉（VIN），一种抗- 雄激素杀菌剂这两种EDC仍然与环境相关。大鼠暴露于人类相关剂量 A1221或VIN或溶剂，通过在妊娠期和哺乳期饲喂母鼠。在成年雄性大鼠中，sncRNA 将提取来自附睾头的EV货物和来自附睾尾的成熟精子，并用于 深度测序和生物信息学。来自相同样本中精子的DNA将进行还原分析。 代表性亚硫酸氢盐测序以鉴定CpG岛中的表突变。这些数据将建立一个明确的 表观遗传谱，这将使我们能够查明EDC诱导的表突变的起源。这项建议是 生物医学相关，因为所有人类都暴露于EDCs，精子成熟机制高度 在哺乳动物中是保守的。