This project, Retinoic Acid Signaling Disruption by Phthalates in Human and Rodent Fetal Testis, will improve male reproductive health by providing mechanistic information about disruption of a critical fetal testis developmental pathway by a ubiquitous class of environmental chemicals. Retinoic acid signaling regulates the entry of testicular germ cells into meiosis, and exogenous retinoic acid has dramatic effects on signaling pathways involved in gonadal sex determination and differentiation of the testis. Phthalates interact with retinoic acid signaling in the fetal testis in vitro. Phthalates and retinoic acid both cause adverse effects on the fetal seminiferous cord of multiple species. However, questions remain about the mechanisms responsible for the interaction and about the long-term effects of disrupting this signaling pathway. Fetal testis culture experiments have demonstrated that exogenous retinoic acid disrupts seminiferous cord development and signaling for sex determination, and that phthalates interact with retinoic acid to both enhance and inhibit some of these effects. R00 research will focus on comparisons between the rat fetal testis and both mouse and human fetal testis models (Specific Aim 1). This will allow for important cross-species comparison with experiments in fetal testis cultures and cultured Sertoli cells. These experiments will quantify the response of the fetal testis to retinoic acid and phthalates using histology, immunohistochemistry, and gene expression as endpoints. Additional experiments will measure the impact of fetal phthalate and retinoic acid exposure on the mouse testis in later life, including development of the testis, spermatogenesis in adult mice, and changes in DNA methylation (Specific Aim 2). This will be a first step toward clarifying the mechanisms by which phthalate toxicity exerts persistent and potentially transgenerational effects by disrupting retinoic acid signaling in the fetal testis. This project is guided by the working hypothesis: phthalates interfere with fetal testicular development through disruption of retinoic acid signaling in the seminiferous cord. The proposed experiments provide critical information about environmental exposures and effects on male reproductive health and serve the goal of this project: to describe the mechanism by which phthalates act on the retinoic acid signaling pathway to produce adverse outcomes during human fetal testicular development. This goal will be achieved by fulfilling the following Specific Aims. 1: Compare the impact of disrupted retinoic acid signaling on testis development across species. 2: Identify persistent adverse outcomes of phthalate exposure mediated by altered retinoic acid signaling.