PROJECT ABSTRACT The goal of this proposal is to establish how exposures to ubiquitous plastics and prenatal stress interact to confer epigenetic changes to developing germ cells, and how such changes lead to reproductive and metabolic phenotypes in later life and potentially in subsequent generations. Numerous studies involving consumer and agricultural chemicals demonstrate effects that appear in the progeny and even grandchildren of individuals exposed in utero. Correspondence between period of susceptibility and epigenetic reprogramming suggest that epigenetic changes to germ cells are involved. However, as the majority of studies utilize oral gavage for dosing, the effects of the chemical and prenatal stress are difficult to resolve. Phthalates are the major chemical in household dust and have been detected in 100% of pregnant women in California. Our preliminary studies in mice find that prenatal exposures to two different phthalates during the period of germ cell epigenetic programming leads in each case to decreased repression of retrotransposons and germ cell developmental defects; evidence from our lab suggests that this occurs through disruption of the piRNA pathway, which silences endogenous transposable elements through epigenetic and post-translational mechanisms. In this proposal, we will interrogate the combined and separate effects of biologically relevant doses of phthalate and early gestational stress on the epigenetic state of mouse germ cells and relate these changes to the extent of retrotransposon activity and reproductive phenotypes in the progeny. Studies in Aim 1 will determine how early gestational stress and phthalate exposure interact to alter the epigenetic state of fetal germ cells and the potential for inheritance of that state. This will disentangle the effects of stress and chemical exposure that accompany oral gavage in many prior studies with quantifiable, molecular readouts as well as testing potential mechanisms of direct and multigenerational epigenetic dysregulation. Aim 2 will examine phenotypic consequences of early gestational stress and phthalate exposure on germ cells and female reproductive aging in the next generation. By connecting adult reproductive phenotypes to fetal exposures, this will begin to disentangle the effects of plastics versus prenatal stress. Pharmacologic modulation of key nodes in the stress response pathway will potentially provide insight on appropriate therapies for women in high-risk pregnancies. Aim 3 will determine the extent of genetic disruptions via transposon insertions induced by early gestational stress and phthalates in directly exposed germ cells and subsequent generations. These studies will identify environmental factors that pose greatest risk to genomic integrity and potentially a drug therapy to mitigate those effects.