Project 5: miRNAs as mediators of developmental toxicity following paternal germ cell toxicant exposure

Brown University, Providence RI

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT The objective of this project, miRNAs as Mediators of Developmental Toxicity following Paternal Germ Cell Toxicant Exposure, is to test how a non-genotoxic exposure that alters sperm sncRNA levels can disrupt regulation of embryonic development. There is evidence that paternal exposure to germ cell toxicants during the preconception window can result in adverse effects on embryonic development. However, it is largely unknown how paternal preconception exposures lead to adverse effects on offspring development or what roles specific small non-coding RNAs in sperm play in the mechanisms of developmental toxicity following paternal exposures. In this project, we will test how paternal preconception exposure to ethylene glycol monomethyl ether (EGME) disrupts the sperm RNome, the consequences for embryo development, and the roles of sperm RNAs in EGME- mediated testicular germ cell apoptosis. EGME is a selective testicular toxicant that targets pachytene spermatocytes, resulting in spermatocyte apoptosis. Pachytene spermatocytes synthesize more RNA than any other testicular germ cell stage, and undergo stage-specific patterns of alternative splicing. Our preliminary data shows that exposure to EGME at doses below those that cause germ cell death disrupts expression of sperm miRNAs, and that the specific miRNAs altered by EGME exposure are important for embryonic development. To build on these preliminary findings, this project will test the working hypothesis that EGME adversely affects early embryo development by altering the small RNA profile of sperm, leading to disrupted embryonic transcriptome regulation. This hypothesis will be tested by fulfilling the following Specific Aims. Aim 1: Test the impact of EGME-induced disruption of sperm RNA on differentiation of embryonic stem cells and success of embryonic development. Aim 2: Define cell-type specific changes that mediate EGME-driven sperm RNome changes. The proposed research is innovative because it aims to discover specific epigenetic mechanisms responsible for intergenerational effects of a toxicant. It is also innovative because we will use a well-established toxicant model to overcome limitations with genetic approaches to studying meiosis. The proposed research is significant because it will address the persistent knowledge gap of identifying specific sperm sncRNA regulatory functions in the embryo that are disrupted by paternal toxicant exposure, leading to disruption of embryo development. The project will be highly synergistic with the COBRE for RNA Biology. We will utilize the RNA Biology Core to implement novel research strategies including single-cell RNA-seq to identify lineage-specific changes in embryonic cell development, spatial multiomics to identify the cell types responsible for altered sperm RNome following EGME exposure, and a combination of long-read sequencing and RNA mass spectrometry to identify changes in meiosis-critical RNAs that lead to EGME-induced spermatocyte apoptosis. We have collaborated with the COBRE leadership to plan experimental strategies, and we will utilize analysis approaches developed in Research Project 2.