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.
Using military records linked to birth certificates, a six-state study found higher rates of cardia and renal defects among infants conceived by deployed Gulf War males. To date, there are limited data to explain the molecular mechanisms whereby paternal environmental exposures result in reproductive sequelae. Completion of these studies will: (a) identify the murine spermatogenic and reproductive phenotype resulting from paternal exposures to complex mixtures of polyaromatic hydrocarbons and particulate matter, (b) segregate seminal fluid factors from male germ cell effects, (c) elucidate the mechanisms whereby paternal exposures cause sub-fertility and birth defects;and (d) determine the reversibility of effects. Because the toxic components in cigarette smoke are similar to those formed during incineration, the incomplete combustion of fossil fuels, and diesel truck exhaust, this work will expand our understanding of the impact of Gulf War exposures on male fertility and the paternal contributions to embryo development.