Gulf War Exposures and the Molecular Mechanisms of Paternal Reproductive Risk

海湾战争暴露与父亲生殖风险的分子机制

• 批准号: 8660889
• 负责人: Deborah Ann Hansen
• 金额: --
• 依托单位: ST. LOUIS VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8660889
• 关键词: Accounting; Acetic Acids; Affinity; Age-Months; Antibodies; Aortic Valve Stenosis; Aromatic Amines; Aromatic Hydrocarbons; Aryl Hydrocarbon Receptor; Benzene; Benzo(a)pyrene; Binding; Biological Assay; Birth; Birth Certificates; Breathing; Burn injury; Cells; Cessation of life; Competence; Complement; Complex; Complex Mixtures; Congenital Abnormality; DNA; DNA biosynthesis; Data; Defect; Development; Dose; Economics; Embryo; Embryonic Development; Environmental Exposure; Event; Excision; Exposure to; Female; Fertility; Fertilization; Fertilization in Vitro; Fire - disasters; Fossil Fuels; Functional RNA; Gene Expression; Gene Proteins; Generations; Genes; Genetic; Genomics; Germ Cells; Guidelines; Gulf War; High Prevalence; Histology; Histones; Histopathology; Hydrocarbons; Immunofluorescence Immunologic; Immunofluorescence Microscopy; In Situ Nick-End Labeling; In Vitro; Incineration; Individual; Infant; Kidney; Knock-out; Knockout Mice; Lasers; Length; Leukocytes; Life; Link; Litter Size; Maternal Exposure; Mediating; Meiosis; Messenger RNA; Methods; Methylation; MicroRNAs; Microdissection; Military Personnel; Modality; Molecular; Morphology; Mus; Nature; Nuclear; Nuclear Proteins; Oils; Organ; Organ Weight; Outcome; Oxidative Stress; Particulate Matter; Partner in relationship; Paternal Exposure; Pathway interactions; Petroleum; Phenotype; Physical condensation; Physiological Processes; Population; Positioning Attribute; Prevalence Study; Process; Protamines; Proteins; Proteomics; Protocols documentation; Receptor Activation; Records; Reporting; Research Design; Risk; Seminal fluid; Sentinel; Specificity; Spermatids; Spermatocytes; Spermatogenesis; Spermatogonia; Staging; Stem cells; Structural Congenital Anomalies; Testing; Testis; Time; Tissues; Toxicity Tests; Toxicology; Toxin; Transgenic Mice; Tricuspid Valve Insufficiency; Urea; Veterans; Weight; Western Blotting; Wood material; Work; Xenobiotic Metabolism; Xenobiotics; aryl hydrocarbon receptor ligand; base; biological adaptation to stress; blastocyst; cell growth; cell type; cigarette smoking; computerized; design; environmental particulate; exhaust; gel electrophoresis; imprint; in vivo; male; malformation; mouse Ahr protein; non-genomic; promoter; protamine 1; public health relevance; pup; reproductive; research study; response; sperm cell; stomach cardia; toxicant

DESCRIPTION (provided by applicant): Objectives: The primary objective is to identify the molecular mechanisms whereby paternal environmental exposures result in reproductive sequelae. We hypothesize that paternal exposures resulting in AHR activation will result in common mechanisms of action that will compromise reproductive competence. We predict that multiple modalities and endpoints, across multiple cell types and tissues, will be needed to detect the transgenerational effects. Moreover we suggest that male exposure to PAHs will compromise sperm protamination and result in methylation changes of imprinted genes that are transmitted to successive generations. Finally we propose that the paternal reproductive phenotype resulting from exposures to complex mixtures of PAHs and PM is dose and time dependent. It is unknown to what extent the effects are reversible. Research design: The Organization for Economic Cooperation and Development (OECD) two-generation guideline for reproductive toxicity testing was modified to elicit the effects of paternal exposures to the byproducts of fossil fuels and combustibles on spermatogenic and reproductive endpoints. To inform the exposures of Gulf War Veterans, cigarette smoke condensate (CSC) and benzo(a)pyrene (BP) will be the representative complex mixture of polyaromatic hydrocarbons (PAH) and particulate matter (PM). Murine in vitro fertilization (IVF) will be compared to natural mating to segregate toxins in the seminal fluid fro male germ cell effects. CSC or BP exposed male C57 mice will be dosed to elicit a gradation of spermatogenic and couple mediated endpoints, and will be mated with non-exposed C57 females. Exposed males will be retained for a 35-day washout period post exposure, again mated, and will be repeated at T+ 70 days and T+105 days as needed (until 6 months of age) to determine if there is reversibility of effect. Methods: Because the exposures to the products of combustion and fossil fuels occur as complex mixtures producing synergistic effects, it is difficul to segregate the actions of individual agents. Experiments were therefore designed to detect AHR activation while emphasizing the mechanistic and functional consequences of intense and/or continuous AHR ligand activation. Testis fragments from 2.5 day pups sired by transgenic mice expressing cell stage specific GFP under the control of promoters for Pou5f1 (type A spermatogonia), Acr (mid meiosis), and Gsg2 (spermatids) will establish live cell spermatogenesis cultures. Live cell LMD with GFP cell stage identification will isolate pure cell stage populations for subculture and cell stage specific toxicity testing. In vitro and in vivo strss response, xenobiotic response, structural, and functional pathways will be interrogated through a rapid screen of sentinel "marker" genes and proteins within the context of AHR genomic and non-genomic pathways. qRT-PCR will be used for protamine mRNA of caudal sperm. Nuclear proteins will be extracted and subjected to acetic acid-urea gel electrophoresis to quantify the protamine protein content, which will be further complemented by quantitative immunofluorescence microscopy. Mitotracker will be used with the PRM1 and PRM2 antibodies to identify sperm. White blood cells will be used as negative controls. RNAseq on pooled E7 (gastrulating) embryos will determine whether protamine ratios in sperm and CSC exposure correlate with any changes in embryonic gene expression in the mouse embryo. Computerized assisted semen analysis (CASA), morphology, histology, male reproductive organ weights, and histopathology will be evaluated. Male germ cell factors are separated from seminal fluid factors through IVF. Histopathology of randomly selected near-term embryos and post-natal pups will be completed. Endpoints include fertilization rates, blastocyst rates; embryo day 16.5 and term litter size, weight, length, structural assessment, and internal organ defects. Laser microdissection (LMD) with qRT-PCR, microRNA, microarray, immunofluorescence (IF) with Confocal, western blot (WB), and TUNEL are the primary genetic, genomic, and proteomic assays. C57BL6 mice and aryl hydrocarbon receptor (AHR) knockout mice will be used.

描述（由申请人提供）： 目标：主要目标是确定父亲环境暴露导致生殖后遗症的分子机制。我们假设，导致 AHR 激活的父亲暴露将导致共同的作用机制，从而损害生殖能力。我们预测，需要跨多种细胞类型和组织的多种模式和终点来检测跨代效应。此外，我们认为男性接触多环芳烃会损害精子的精精蛋白，并导致遗传给后代的印记基因甲基化变化。最后，我们提出，由于暴露于 PAH 和 PM 的复杂混合物而产生的父系生殖表型具有剂量和时间依赖性。目前尚不清楚这些影响在多大程度上是可逆的。研究设计：经济合作与发展组织（OECD）的两代生殖毒性测试指南进行了修改，以得出父亲接触化石燃料和可燃物副产品对生精和生殖终点的影响。为了了解海湾战争退伍军人的暴露情况，香烟烟雾冷凝物（CSC）和苯并（a）芘（BP）将是多环芳烃（PAH）和颗粒物（PM）的代表性复杂混合物。小鼠体外受精（IVF）将与自然交配进行比较，以隔离精液中的毒素免受雄性生殖细胞的影响。将给暴露于 CSC 或 BP 的雄性 C57 小鼠给药，以引发生精和偶介导的终点分级，并将与未暴露的 C57 雌性小鼠交配。暴露的雄性将在暴露后保留 35 天的清洗期，再次交配，并根据需要在 T+ 70 天和 T+105 天重复（直到 6 个月龄），以确定效果是否可逆。方法：由于接触燃烧和化石燃料的产物是作为复杂混合物产生的，会产生协同效应，因此很难区分各个因素的作用。因此，实验被设计为检测 AHR 激活，同时强调强烈和/或连续 AHR 配体激活的机制和功能后果。由在 Pou5f1（A 型精原细胞）、Acr（减数分裂中期）和 Gsg2（精子细胞）启动子控制下表达细胞阶段特异性 GFP 的转基因小鼠产生的 2.5 日龄幼崽的睾丸片段将建立活细胞精子发生培养物。具有 GFP 细胞阶段识别功能的活细胞 LMD 将分离纯细胞阶段群体，用于传代培养和细胞阶段特定毒性测试。将通过在 AHR 基因组和非基因组途径的背景下快速筛选前哨“标记”基因和蛋白质来询问体外和体内应激反应、异生物质反应、结构和功能途径。 qRT-PCR 将用于尾部精子的鱼精蛋白 mRNA。提取核蛋白并进行乙酸-尿素凝胶电泳以定量鱼精蛋白含量，并通过定量免疫荧光显微镜进一步补充。 Mitotracker 将与 PRM1 和 PRM2 抗体一起使用来识别精子。白细胞将用作阴性对照。对合并的 E7（原肠胚形成）胚胎进行 RNAseq 将确定精子和 CSC 暴露中的鱼精蛋白比率是否与小鼠胚胎中胚胎基因表达的任何变化相关。将评估计算机辅助精液分析（CASA）、形态学、组织学、男性生殖器官重量和组织病理学。通过 IVF 将男性生殖细胞因子与精液因子分离。将完成随机选择的近期胚胎和产后幼崽的组织病理学检查。终点包括受精率、囊胚率；胚胎第 16.5 天和足月产仔数、体重、身长、结构评估和内脏器官缺陷。激光显微切割 (LMD)、qRT-PCR、microRNA、微阵列、共聚焦免疫荧光 (IF)、蛋白质印迹 (WB) 和 TUNEL 是主要的遗传、基因组和蛋白质组检测。将使用 C57BL6 小鼠和芳基碳氢化合物受体 (AHR) 敲除小鼠。