Intergenerational Effects of Early-Life Arsenic Exposure on Sperm Quality and Epididymal Transcriptome: Integrative Analysis of Large and Small RNAs

Inorganic arsenic (iAs) is a widespread environmental toxicant associated with male reproductive dysfunction, yet the epididymal molecular responses to developmental exposure and their potential intergenerational consequences remain poorly understood. We investigated sperm quality and epididymal large- and small RNA profiles in directly exposed male offspring (F1) and their unexposed sons (F2). Pregnant C57BL/6J dams received 1.15 ppm iAs in drinking water throughout gestation and lactation. Adult F1 and F2 males underwent sperm-quality assessment and epididymal transcriptomic profiling, followed by differential-expression and pathway enrichment analyses. Developmental iAs exposure significantly reduced sperm concentration, total sperm count, and motility in F1 males, whereas reduced sperm concentration and total sperm count were also observed in unexposed F2 males. In F1 epididymides, large-RNA analysis identified enrichment of epithelial remodeling, inflammatory, and stress-response pathways, together with suppression of oxidative phosphorylation and metabolic pathways. Small RNA profiling revealed a downward trend in mitochondrial tRNA abundance. In contrast, F2 epididymides exhibited suppression of spermatogenesis- and cell cycle-related pathways and differential expression of 56 small RNAs, including spliceosomal small nuclear RNAs, small nucleolar RNAs, and microRNAs. An independent transcriptomic comparison with Occludin-knockout (Ocln−/−) mice, a genetic model of epididymal infertility, demonstrated concordant enrichment directions for 18 of 21 F1-associated Hallmark pathways, providing additional pathway-level support for the F1 findings. Comparable concordance was not observed in F2. To our knowledge, this is the first integrated analysis of epididymal large- and small RNA responses to developmental iAs exposure across two generations. These findings identify distinct epididymal molecular signatures associated with persistent and intergenerational reproductive abnormalities and highlight the epididymis as a potentially important site of developmental environmental responses.