Deciphering the Sperm RNA Code of Aging with PANDORA-seq

Sperm aging impacts male fertility and offspring health, highlighting the need for reliable aging biomarkers to guide reproductive decisions. Using PANDORA-seq, a novel method to comprehensively profile small non-coding RNAs (sncRNAs), we identified an "aging cliff" in mouse sperm RNA profiles-a sharp age-specific transition marked by significant shifts in genomic and mitochondrial tRNA-derived small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNAs). Notably, rsRNAs embedded in sperm heads exhibited a transformative length shift, with longer rsRNAs increasing and shorter ones decreasing with age, suggesting altered biogenesis with age. Remarkably, this sperm rsRNA length shift was consistently observed in two independent human aging cohorts, highlighting its evolutionary significance. Moreover, a combination of tsRNAs and rsRNAs (tsRNA/rsRNA cocktails) resembling those in aged sperm induced transcriptomic changes in embryonic stem cells, impacting metabolism and neurodegeneration pathways, mirroring the phenotypes observed in offspring of aged sperm. These results reveal new insights of sperm aging, highlighting the conserved rsRNA length shift in mice and humans, with translational potential for fertility and intergenerational health Overall design: Mature sperm and sperm heads were collected from the cauda epididymis of male C57BL/6J mice aged 10, 30, 50, 70, and 90 weeks. Between 3 and 4 replicates were sequenced for each sample using PANDORA-seq and traditional sncRNA-seq. For paried human sperm heads, 8 donors provided two semen samples each over a span of 6 to 23 years. All human sperm head samples were sequenced using PANDORA-seq. For non-paired 47 human sperm head(cross-sectional cohort), 47 donors provide semen samples, and the sperm heads were only sequenced using PANDORA-seq. For the RNA-seq experiment, mESCs were transfected with vector, young_comb sncRNA mixture and old_comb sncRNA mixture seperately, After 24 hours of transfection, the total RNA were harvested by Trizol reagent for transcriptome RNA sequencing through polyA enrichment strategy.