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

摘要 暴露于环境内分泌干扰物(EDCs)，特别是在生命早期， 与不利的健康后果有关。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，可以引起可遗传的表观遗传变化，并传递给未来 几代人。以前的工作表明，DNA CpG甲基化的表观突变是一种分子机制 EDC通过它对生殖系进行编程。拟议的工作将探讨这一正在研究中的机制，以及 此外，寻求了解来自男性生殖道的外部因素如何参与精子 成熟本身可能有助于表型突变。更具体地说，指骨的近端部分(头) 附睾在精子成熟的最后阶段对精子有很大的影响。胞外小泡 (EVS)从附睾体--附睾体--从上皮释放出来，融合并转移其不同的 精子的分子内容，包括小的非编码RNA(SncRNA)，从而有助于发挥功能 精子的能力和受精胚胎的发育轨迹。这些SncRNA具有 在精子中调节基因表达和直接DNA甲基化的能力。在目前的提案中，我们将 确定两个不同的EDC类别是否以及如何通过不同的激素机制发出信号导致直接 CpG甲基化改变DNA，和/或改变附睾体SncRNA含量转移到精子 以间接导致表型突变。这里研究的两类EDC是多氯联苯混合物Aroclor 1221(A1221)，一种具有微弱雌激素性质的工业混合物；以及长春新灵(VIN)，一种抗雌激素药物 雄激素杀菌剂。这两个环境保护中心仍然与环境有关。大鼠暴露于人类相关剂量 A1221或VIN，或交通工具，通过在怀孕和哺乳期间喂食水母。在成年雄性大鼠中，SncRNA 从附睾头提取的精液和从附睾尾提取的成熟精子将被提取并用于 深度测序和生物信息学。同一样本中精子的DNA将被减少 代表亚硫酸氢盐测序以确定CpG岛上的表位突变。这些数据将建立一个明确的 表观遗传学图谱将使我们能够精确定位EDC诱导的表观突变的起源。这项建议是 与生物医学相关，因为所有人类都暴露在内分泌细胞中，具有高度的精子成熟机制 在哺乳动物中是保守的。

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.