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

项目总结 无机砷(IAS)对成年男性产生显著的生殖毒性，导致精子减少 质量。除了工作场所暴露外，个人还暴露在危险废物场地附近的高水平ias中。 以及在IAS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水平的 在多代人中，IAS剂量会损害精子质量。然而，目前尚不清楚父亲是否会接触到 在青春期或早期(孕期到断奶)服用与环境相关的IAS会产生不良影响 可遗传的生殖结果。我们假设青春期和早期生活是在 暴露于IAS不仅对受影响的个人产生负面影响，而且对他们的后代产生负面影响。然而， 调节暴露诱导性状父系代际传递的分子机制仍然存在 不清楚。最近，精子携带的小RNA及其特异性的5‘-甲基胞嘧啶(M5C)修饰被发现 以调节饮食引起的疾病的父系传播。然而，对环境毒物的类似研究，如 因为国际会计协会缺席了。在我们的初步研究中，我们发现免疫球蛋白诱导了假性尿苷(Ψ)和M5C丰度的变化 在精子小RNA中。Ψ和M5C被发现是精子小RNA中最丰富的RNA修饰， 我们推测，这些基因修饰与父系遗传的低精子质量相关。 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 到断奶)(目标2)。我们将确定青少年(目标1A)和早期(目标2A)IAS暴露是否是窗口 导致精子质量受损的代际遗传的易感性。我们将确定 精子小RNA及其修饰物Ψ和M5C在青春期(目标1B)和早期生活中的中介作用 (目标2B)通过合子显微注射诱导父亲遗传精子质量受损 (Zi)从暴露或对照小鼠中分离的精子小RNA，以从天真的受精卵中产生后代。我们 预计青少年接触组的后代精子质量较差，就像接触过的父亲和 通过自然交配产生的儿子。验证特定修饰在我们的精子表型中的功能作用 遗传模型，我们将通过核糖核酸免疫沉淀分离富含Ψ和M5C的精子小RNA组分 用于受精卵微量注射。我们将确定显微注射特定修饰的精子小RNA是否 在青春期(目标1C)和早期(目标2C)可以复制父亲的精子表型。我们期待着 ZI产生的后代暴露于青春期和/或早期富含Ψ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原生RNAseq 绘制精子Ψ和M5C修饰图，并识别与暴露窗口相关的小RNA群- 特定的代际遗传。最后，我们将关联RNA表位转录和转录 青春期/早期IAS暴露对精子质量的代际影响的变化

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.