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）： 目的：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，将需要多种模态和终点来检测转世的效应。此外，我们建议男性接触PAHS会损害精子的精神分子，并导致印刷基因的甲基化变化，这些基因传播到后代。最后，我们提出，由PAH和PM的复杂混合物暴露导致的父亲生殖表型是剂量和时间依赖性的。效果在多大程度上是可逆的。研究设计：对经济合作与发展组织（OECD）的生殖毒性测试指南进行了修改，以引起父亲暴露对化石燃料的副产品的影响以及可加压对精子原性和生殖端点的影响。为了告知海湾战争退伍军人的暴露，香烟烟雾凝结物（CSC）和苯并（a）pyrene（BP）将是多氨基烃（PAH）和颗粒物（PM）的代表性复杂混合物。将在雄性生殖细胞作用中进行鼠类体外受精（IVF）与天然交配，以将精液中的毒素隔离。 CSC或BP暴露的雄性C57小鼠将受到剂量，以引起精子生成和夫妇介导的终点的渐变，并将与非暴露的C57雌性配对。暴露于暴露后的35天冲洗期将保留暴露的男性，并再次交配，并在t+ 70天和t+ 105天（直到6个月大）时重复进行，以确定效果是否具有可逆性。方法：由于对燃烧和化石燃料产物的暴露是作为产生协同作用的复杂混合物而发生的，因此很难隔离单个代理的作用。因此，设计实验以检测AHR激活，同时强调强烈和/或连续AHR配体激活的机械和功能后果。在POU5F1（A型A型），ACR（中减去性）和GSG2（精子）（精子）的启动子控制下，由转基因小鼠染料的2.5天幼崽的睾丸片段在启动子的控制下表达细胞期特异性GFP。具有GFP细胞阶段鉴定的活细胞LMD将隔离纯细胞阶段种群，用于亚培养和细胞阶段特异性毒性测试。在AHR基因组和非基因组途径的背景下，将通过Sentinel的“标记”基因和蛋白质的快速筛选来询问体外和体内STRS的反应，异种生物反应，结构和功能途径。 QRT-PCR将用于尾精子的精蛋白mRNA。将提取核蛋白并进行乙酸 - 尿素凝胶电泳，以量化精蛋白蛋白含量，这将通过定量免疫荧光显微镜进一步补充。 Mitotracker将与PRM1和PRM2抗体一起识别精子。白细胞将用作阴性对照。 RNASEQ在合并的E7（胃组织）胚胎上的RNASEQ将确定精子和CSC暴露中的精素比是否与小鼠胚胎中胚胎基因表达的任何变化相关。将评估计算机化辅助精液分析（CASA），形态学，组织学，男性生殖器官重量和组织病理学。通过IVF，雄性生殖细胞因子与精液液因子分离。随机选择的近期胚胎和产后幼崽的组织病理学将完成。终点包括受精率，胚泡率；胚胎第16.5天和期限垃圾的大小，重量，长度，结构评估和内脏器官缺陷。带有QRT-PCR，microRNA，微阵列，免疫荧光（IF）的激光显微解剖（LMD），带有共聚焦，蛋白质印迹（WB）和TUNEL是主要的遗传，基因组和蛋白质组学分析。将使用C57BL6小鼠和芳基烃受体（AHR）敲除小鼠。