RNA modifications by paternal exposure to arsenic and intergenerational effects on sperm quality

项目摘要 无机砷（iAs）对成年男性产生显著的生殖毒性，导致精子数量减少 质量.除了工作场所暴露外，个人在危险废物场地附近也暴露于高水平的iAs 和在国际协定丰富的地理区域。最近的一项研究表明，短暂的产前暴露于高浓度的 iAs剂量在多代中损害精子质量。然而，尚不清楚父亲的暴露是否 在青春期或生命早期（妊娠期至断奶期）环境相关剂量的iAs会产生不良反应， 可遗传的生殖结果。我们认为，青春期和早期生活是易感性的窗口， 暴露于iAs不仅会对暴露的个体产生负面影响，还会对他们的后代产生负面影响。然而，在这方面， 介导父代间遗传的遗传诱导性状的分子机制仍然存在 不清楚最近，精子携带的小RNA及其特异性5'-甲基胞嘧啶（m5C）修饰被证明是 介导饮食引起的疾病的父系传播。然而，对环境毒物的类似研究， 因为iAs不存在。在我们的初步研究中，我们发现iAs诱导的假尿苷（pseudouridine，IUD）和m5C丰度的变化 精子中的小RNA发现m5C和m5C是精子小RNA中最丰富的RNA修饰， 我们假设这些修饰介导了与精子质量相关的父亲遗传， iAs暴露，特别是在青春期（目标1）和生命早期（妊娠）的发育窗口期 （目标2）。我们将确定青少年（目标1A）和早期生活（目标2A）iAs暴露是否是窗口 这种易感性导致精子质量受损的代际遗传。我们将确定 精子小RNA及其修饰物，m5C，在青春期（Aim 1B）和早期生活中的介导作用 (Aim 2B）通过进行合子显微注射，确定性诱导的受损精子质量的父系遗传 (ZI)从暴露或对照小鼠中分离的精子小RNA，从幼稚受精卵中产生后代。我们 预计青少年暴露组的后代精子质量较差，就像暴露的父亲一样， 自然交配产生的儿子。验证精子表型中特定修饰的功能作用 遗传模型，我们将通过RNA免疫沉淀法分离富含α-和m5C的精子小RNA组分 用于合子显微注射。我们将确定显微注射特定修饰富集的精子小RNA 在青春期（Aim 1C）和早期（Aim 2C）可以复制父系精子表型。我们预计 ZI产生的后代暴露于青春期和/或早期生活中富含β-或m5C的精子小RNA 暴露组可以概括精子质量差的表型。我们将使用Nanopore天然RNAseq来 绘制精子DNA和m5C修饰图，并识别与暴露窗口相关的小RNA群体- 具体的代际传承。最后，我们将把RNA表观转录组学和转录组学联系起来， 随着青少年/早期iAs暴露对精子质量的代际影响而变化。

PROJECT SUMMARY Inorganic arsenic (iAs) produces significant reproductive toxicity in adult males leading to decreased sperm quality. Aside from workplace exposure, individuals are exposed to high levels of iAs near hazardous waste sites and in geographic areas enriched with iAs. A recent study revealed that transient prenatal exposure to a high dose of iAs impaired sperm quality in multiple generations. However, it is not known whether paternal exposures to environmentally relevant dose of iAs during adolescence or early-life (gestation to weaning) produce adverse inheritable reproductive outcomes. We posit that adolescence and early-life are windows of susceptibility during which exposure to iAs negatively impacts not only the individuals being exposed but also their offspring. However, the molecular mechanisms mediating paternal intergenerational transmission of exposure-induced traits remain unclear. Recently, sperm-borne small-RNAs and their specific 5'-methylcytosine (m5C) modifications were shown to mediate the paternal transmission of diet-induced disorders. Yet, similar studies on environmental toxicants such as iAs are absent. In our pilot study, we discovered iAs-induced changes in pseudouridine (Ψ) and m5C abundance in sperm small-RNAs. Ψ and m5C were found to be the most abundant RNA modifications in sperm small-RNAs, and we hypothesize that these modifications mediate the paternal inheritance of poor sperm quality associated with iAs exposure, particularly during the developmental windows of adolescence (Aim 1) and early life (gestation to weaning) (Aim 2). We will determine if adolescent (Aim 1A) and early-life (Aim 2A) iAs exposure are windows of susceptibility conferring the intergenerational inheritance of impaired sperm quality. We will identify the mediating role of sperm small-RNAs and their modifications, Ψ and m5C, in adolescent (Aim 1B) and early-life (Aim 2B) exposure-induced paternal inheritance of impaired sperm quality by performing zygotic microinjection (ZI) of sperm small-RNA isolated from exposed or control mice to generate offspring from naïve zygotes. We expect the offspring of the adolescent exposure group to have poorer sperm quality, as in exposed fathers and sons produced by natural mating. To validate the functional role of specific modifications in our sperm phenotype inheritance model, we will isolate Ψ- and m5C-enriched sperm small-RNA fractions by RNA immunoprecipitation for zygotic microinjection. We will determine if microinjection of specific modification-enriched sperm small-RNAs during adolescent (Aim 1C) and early-life (Aim 2C) can reproduce the paternal sperm phenotype. We expect the ZI-produced offspring exposed to Ψ- or m5C-enriched sperm small-RNAs from adolescent and/or early-life exposure groups can recapitulate the poor sperm quality phenotypes. We will use Nanopore native RNAseq to map sperm Ψ and m5C modifications and identify small-RNA populations associated with the exposure window- specific intergenerational inheritance. Finally, we will correlate RNA epitranscriptomic and transcriptomic changes with the intergenerational effects of adolescent/early-life iAs exposure on sperm quality.