Gulf War Exposures and the Molecular Mechanisms of Paternal Reproductive Risk

描述(由申请人提供)： 目的：本研究的主要目的是确定父方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激活将导致共同的作用机制，这将损害生殖能力。我们预测，将需要跨越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会损害精子的净化作用，并导致印记基因的甲基化变化，这些甲基化变化会传递给后代。最后，我们提出，暴露于多环芳烃和PM的复杂混合物所导致的父本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：修改了经济合作与发展组织(OECD)两代人生殖毒性测试指南，以得出父亲暴露于化石燃料副产品和可燃物对生精和生殖终点的影响。为了向海湾战争退伍军人提供暴露信息，香烟烟雾冷凝物(CSC)和苯并(A)芘(BP)将是具有代表性的多芳烃(PAH)和颗粒物(PM)的混合物。小鼠体外受精(IVF)将与自然交配进行比较，以分离精液中的毒素，从而发挥雄性生殖细胞的作用。CSC或BP暴露的雄性C57小鼠将被剂量诱导产生一定程度的生精和偶联介导的终点，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天的洗涤期，再次交配，并将根据需要在T+70天和T+105天重复(直到6个月大)，以确定是否有可逆的效果。方法：由于接触到燃烧和化石燃料的产物是产生协同效应的复杂混合物，因此很难将单独的作用分离开来。因此，实验被设计来检测AHR的激活，同时强调强烈和/或持续的AHR配体激活的机制和功能后果。在Pou5f1(A型精原细胞)、ACR(中期减数分裂)和Gsg2(精子细胞)启动子的控制下，表达细胞期特异性GFP的转基因小鼠2.5日龄幼鼠的睾丸片段将建立活细胞生精培养。活细胞LMD结合GFP细胞阶段鉴定将分离出纯细胞阶段群体，用于传代培养和细胞阶段特异性毒性测试。在体外和体内，将通过快速筛选AHR基因组和非基因组途径中的前哨“标记”基因和蛋白质来询问STRSS反应、异种反应、结构和功能途径。尾部精子的鱼精蛋白基因将采用定量逆转录聚合酶链式反应。核蛋白将被提取并经醋酸-尿素凝胶电泳法定量测定鱼精蛋白的含量，这将由定量免疫荧光显微镜进一步补充。MitoTracker将与PRM1和PRM2抗体一起用于识别精子。白血球将作为阴性对照。对汇集的E7(原肠形成)胚胎的RNAseq将确定精子中的鱼精蛋白比例和CSC暴露是否与小鼠胚胎中胚胎基因表达的任何变化相关。将对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进行评估。男性生殖细胞因子通过试管受精从精液因子中分离出来。随机选择的近期胚胎和出生后幼崽的组织病理学将完成。终点包括受精率、囊胚率、胚胎天数16.5天和足月产仔数、体重、长度、结构评估和内部器官缺陷。激光显微切割(LMD)、微RNA、微阵列、共聚焦免疫荧光(IF)、蛋白质印迹(WB)和原位末端标记法(TUNEL)是主要的遗传学、基因组和蛋白质组学分析方法。将使用C57BL6小鼠和芳香烃受体(AHR)基因敲除小鼠。

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.