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：主要目的是确定父系环境暴露导致生殖后遗症的分子机制。我们假设，父亲暴露导致AHR激活将导致共同的行动机制，将损害生殖能力。我们预测，将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，男性暴露于多环芳烃将损害精子污染，并导致甲基化的印记基因的变化，传递给后代。最后，我们提出，父系生殖表型造成的复杂混合物的多环芳烃和PM暴露是剂量和时间依赖性。目前尚不清楚这些影响在多大程度上是可逆的。研究设计：对经济合作与发展组织（OECD）生殖毒性试验的两代指南进行了修改，以得出父亲暴露于化石燃料和可燃物副产品对生精和生殖终点的影响。为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。将小鼠体外受精（IVF）与自然交配进行比较，以分离精液中的毒素对雄性生殖细胞的影响。将对CSC或BP暴露的雄性C57小鼠给药，以引起生精和偶联介导终点的分级，并将与未暴露的C57雌性小鼠交配。暴露后，将保留暴露的雄性动物35天洗脱期，再次交配，并根据需要在T+ 70天和T+105天重复（直至6月龄），以确定效应是否可逆。研究方法：由于暴露于燃烧产物和化石燃料是产生协同效应的复杂混合物，因此很难将各个因素的作用分开。因此，设计实验以检测AHR活化，同时强调强烈和/或连续的AHR配体活化的机制和功能后果。来自2.5日龄幼仔的睾丸片段将建立活细胞精子发生培养物，所述幼仔由在Pou 5 f1（A型精原细胞）、Acr（中期减数分裂）和Gsg 2（精子细胞）的启动子控制下表达细胞阶段特异性GFP的转基因小鼠产生。具有GFP细胞阶段鉴定的活细胞LMD将分离纯细胞阶段群体用于传代培养和细胞阶段特异性毒性试验。在体外和体内应激反应，异源反应，结构和功能途径将通过AHR基因组和非基因组途径的范围内的哨兵“标记”基因和蛋白质的快速筛选进行查询。qRT-PCR将用于尾部精子的鱼精蛋白mRNA。将提取核蛋白，并进行醋酸-尿素凝胶电泳，以定量鱼精蛋白含量，这将进一步补充定量免疫荧光显微镜。Mitotracker将与PRM 1和PRM 2抗体一起用于识别精子。将使用白色血细胞作为阴性对照。在合并的E7（原肠胚形成）胚胎上的RNAseq将确定精子中的鱼精蛋白比率和CSC暴露是否与小鼠胚胎中胚胎基因表达的任何变化相关。将评价计算机辅助精液分析（CASA）、形态学、组织学、雄性生殖器官重量和组织病理学。男性生殖细胞因子通过IVF从精液因子中分离出来。将完成随机选择的近期胚胎和产后幼仔的组织学检查。终点包括受精率、囊胚率、胚胎第16.5天和足月窝仔数、体重、身长、结构评估和内脏器官缺陷。采用qRT-PCR的激光显微切割（LMD）、microRNA、微阵列、采用共聚焦的免疫荧光（IF）、蛋白质印迹（WB）和TUNEL是主要的遗传学、基因组学和蛋白质组学测定。将使用C57 BL 6小鼠和芳烃受体（AHR）敲除小鼠。