RNA modifications by paternal exposure to arsenic and intergenerational effects on sperm quality

PROJECT SUMMARY Inorganic arsenic (iAs) produces significant reproductive toxicity in adult males leading to decreased sperm quality. Aside from workplace exposure, individuals are exposed to high levels of iAs near hazardous waste sites and in geographic areas enriched with iAs. A recent study revealed that transient prenatal exposure to a high dose of iAs impaired sperm quality in multiple generations. However, it is not known whether paternal exposures to environmentally relevant dose of iAs during adolescence or early-life (gestation to weaning) produce adverse inheritable reproductive outcomes. We posit that adolescence and early-life are windows of susceptibility during which exposure to iAs negatively impacts not only the individuals being exposed but also their offspring. However, the molecular mechanisms mediating paternal intergenerational transmission of exposure-induced traits remain unclear. Recently, sperm-borne small-RNAs and their specific 5'-methylcytosine (m5C) modifications were shown to mediate the paternal transmission of diet-induced disorders. Yet, similar studies on environmental toxicants such as iAs are absent. In our pilot study, we discovered iAs-induced changes in pseudouridine (Ψ) and m5C abundance in sperm small-RNAs. Ψ and m5C were found to be the most abundant RNA modifications in sperm small-RNAs, and we hypothesize that these modifications mediate the paternal inheritance of poor sperm quality associated with iAs exposure, particularly during the developmental windows of adolescence (Aim 1) and early life (gestation to weaning) (Aim 2). We will determine if adolescent (Aim 1A) and early-life (Aim 2A) iAs exposure are windows of susceptibility conferring the intergenerational inheritance of impaired sperm quality. We will identify the mediating role of sperm small-RNAs and their modifications, Ψ and m5C, in adolescent (Aim 1B) and early-life (Aim 2B) exposure-induced paternal inheritance of impaired sperm quality by performing zygotic microinjection (ZI) of sperm small-RNA isolated from exposed or control mice to generate offspring from naïve zygotes. We expect the offspring of the adolescent exposure group to have poorer sperm quality, as in exposed fathers and sons produced by natural mating. To validate the functional role of specific modifications in our sperm phenotype inheritance model, we will isolate Ψ- and m5C-enriched sperm small-RNA fractions by RNA immunoprecipitation for zygotic microinjection. We will determine if microinjection of specific modification-enriched sperm small-RNAs during adolescent (Aim 1C) and early-life (Aim 2C) can reproduce the paternal sperm phenotype. We expect the ZI-produced offspring exposed to Ψ- or m5C-enriched sperm small-RNAs from adolescent and/or early-life exposure groups can recapitulate the poor sperm quality phenotypes. We will use Nanopore native RNAseq to map sperm Ψ and m5C modifications and identify small-RNA populations associated with the exposure window- specific intergenerational inheritance. Finally, we will correlate RNA epitranscriptomic and transcriptomic changes with the intergenerational effects of adolescent/early-life iAs exposure on sperm quality.