SAT432 Interactions Of Endocrine-disrupting Chemical (EDC) Exposure And Fear Recall On Small Noncoding RNA (sncRNA) Contents Of Rat Caput Extracellular Vesicles

Abstract

Abstract Disclosure: R. Gillette: None. M. Aguirre: None. K. Bell: None. Y. Nadeem: None. L. Thompson: None. A.C. Gore: None. Extracellular vesicles (EVs) transfer a complex repertoire of small non-coding RNAs (sncRNA) to sperm as they transit the caput of the epididymis. These molecules are essential for the functional competence of sperm and are directly influenced by environmental factors such as stress, diet, and drug use. We hypothesized that prenatal exposure to EDCs would alter the sncRNA contents of caput extracellular vesicles, and that an exposure to a previously established contextual fear cue would exacerbate this effect. Pregnant dams were fed Nilla Wafers with either 3% DMSO (vehicle control) or Aroclor 1221 (1 mg/kg; a weakly estrogenic EDC) from embryonic day (E)8 until E18 and post-natal day (PND)1 to P21. In adulthood male rat offspring were subjected to a contextual fear conditioning paradigm in which each rat was exposed to an odorant paired with a shock (four 500 ms shocks at 0.7 mA) for three consecutive days. Two weeks later, all rats were returned to the conditioning chambers and exposed to the odorant for 5 minutes (no shocks). The next day, male rats were euthanized and the epididymis was dissected. EVs and total RNA isolated from the caput portion were sequenced on a NovaSeq 6000 SP in a single-end 75 bp format. Reads were checked for quality, trimmed for adapter sequences, aligned to the rat genome, and assigned an annotation designation across 10 different categories of sncRNA. We found that the majority of caput EV sncRNA were either tRNA fragments or piRNA and that unlike mice, miRNA accounted for a small portion of the total reads. The third largest category of aligned reads was in intergenic space and not associated with canonical small non-coding (snc) RNA loci. In-depth investigation determined these reads (∼19 nt) aligned strictly within the boundaries of known CpG islands (CpGi), which have not previously been reported to express any form of sncRNA. Re-exposure to a fear-associated scent 24 hours prior to sample collection substantially reduced the proportion tRNAs in EVs, which were supplanted by an increase in piRNA, CpGi sRNA, and lncRNA. This effect was exacerbated by prenatal EDC exposure and was particularly prevalent in the lncRNA and CpGi sRNA categories. Taken together our data show that the small RNA contents of caput epididymosomes of male rats is substantially different from previous observations reported in mice. We demonstrate that exposure to a contextual fear cue is a powerful influence on sncRNA composition of caput extracellular vesicles and that it is uniquely altered by prenatal EDC exposure. Finally, we provide evidence for what we believe may be a novel category of sncRNA. Presentation: Saturday, June 17, 2023