Male preconception phthalates and offspring embryo and sperm allele-specific methylome programming

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Project Summary Phthalates, a class of endocrine disrupting compounds (EDCs) used in plastics and personal care products, are ubiquitous environmental contaminants resulting in widespread human exposure. Epidemiologic data implicate paternal phthalates with adverse reproductive health including poor sperm quality, and more recently, with longer time to pregnancy − the latter suggests a sperm-derived effect. A growing body of compelling data demonstrates that environmental exposures can be embodied within the developing male germ cell through epigenetic marks, which in turn, can impart information at fertilization to affect the trajectory of health and development of offspring. It is known that prenatal epigenetic reprogramming of male germ cells is a particularly susceptible window to environmental exposures such as phthalates. Research has now emerged demonstrating that other such susceptible windows exist during germ cell development. Our objective is to determine the effects of adult exposure to di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP) and the mixture of DEHP and DBP on sperm DNA methylation and the persistence of sperm-derived changes on DNA methylation programming of the conceptus and F1 adult germ cells. We will exposure adult reproductive age male C57BL/6J mice with 0, 2.5 mg/kg/day of DEHP, 2.5 mg/kg/day of DBP, and its mixture (DEHP and DBP each at 2.5 mg/kg/day) for 70 days – approximately two spermatogenesis cycles. Epididymal sperm will be collected from mice in each group and will be analyzed for whole genome bisulfite sequencing (WGBS). Next, exposed C57BL/6J males will be mated with unexposed DBA/2Jfemales, which will allow for allele- specific analysis across the genome in the F1 hybrid embryos produced. We will perform RNA-seq and WGBS in embryonic and placental cells of gastrulating E6.5 embryos to determine the effects of paternal DEHP and/or DBP on each distinct lineage. Finally, we will analyze sperm quality parameters, oocyte follicle staging and reproductive hormones in male and female F1 offspring. We will also perform RNA-seq and WGBS on F1 sperm and oocytes. This research proposed is expected to identify an epigenetic understanding via sperm methylation of the effects of preconception male EDC exposure and offspring reproductive health. Understanding the involvement of sperm epigenetics within the exposure-disease paradigm will inform avenues of translational research for the development of novel approaches for the treatment and prevention of adverse reproductive health.