Epididymal extracellular vesicles harbor and convey mRNA to sperm for transfer to zygotes

The epididymis plays a critical role in promoting sperm maturation, including remodeling the sperm RNA payload. While small RNAs have been extensively studied in this context, the epididymal contribution to larger sperm RNAs, such as messenger RNAs (mRNAs), remains underexplored. This is largely due to the translational quiescence of mature spermatozoa and the hypothesis that these RNAs are residual by-products of spermatogenesis. Yet, mRNAs carried by sperm have been detected in the zygote, indicating they may act beyond fertilization. However, whether epididymal somatic cells contribute mRNAs to sperm, as they do small RNAs, has not been experimentally examined. Here, we provide a comprehensive analysis of the mRNA landscape of mouse sperm, epithelial cells, and extracellular vesicles (EVs) isolated from the proximal (caput) and distal (cauda) epididymis. Through this analysis and sperm-EV co-incubation experiments, we demonstrate the transfer of mRNAs from epididymal EVs to sperm. Further, through sperm RNA microinjection into zygotes, we uncover gene regulation in the early embryo driven by the introduction of sperm RNAs, specific to >200-nucleotide RNA species. These findings reveal the dynamic mRNA profile of sperm that is delivered to the egg and demonstrate that RNA species beyond small RNAs are capable of influencing preimplantation embryo gene expression.