Ethylene Glycol Monomethyl Ether Altered Rat Sperm Small RNAs with Critical Developmental Roles

Ethylene glycol monomethyl ether (EGME) is a testicular germ cell toxicant that selectively targets spermatocytes. In rats, male-only EGME exposure reduces mating success and can lead to an increase in resorbed fetuses. In a previous study, five-day exposure to 50, 60, or 75 mg/kg/d EGME in male rats led to a decrease in sperm motility and increase in retained spermatid heads with a lowest observed adverse effect level (LOAEL) of 75 mg/kg/d. At 60 mg/kg/d, EGME exposure altered the proportion of sperm small RNA reads mapped to different small RNA categories and the distribution of read lengths. To understand the possible role of sperm sncRNAs in EGME-mediated reduction in spermatogenesis, fertility, and embryonic development, we analyzed sperm sncRNA data from EGME-treated male rats to identify differential expression at the individual RNA level. EGME treatment resulted in dose-dependent increases in the expression levels of microRNAs (miRNAs), piRNAs, and tRNA-derived small RNAs (tsRNAs), and mixture of dose-dependent increases and decreases in abundance of rsRNA reads. We identified 12 miRNAs that were differentially expressed at all EGME doses, with a monotonic, dose-dependent increase. High-confidence targets of these 12 miRNAs are known to be expressed in pre-implantation embryos and statistically enriched for Gene Ontology (GO) biological processes related to early development, such as cell fate commitment and regulation of developmental growth. These results demonstrated that the EGME-induced changes in sperm sncRNA levels were reproducible, dose-dependent, and provided a putative mechanism of paternal EGME effects on embryonic development, which will be investigated in future studies.