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

The epididymis plays a critical role in 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 mRNAs, remains underexplored. mRNAs were among the first RNA species identified in sperm, yet their functional relevance has remained elusive, 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 could play a role beyond fertilization. However, if the soma, the epididymis, actively contributes mRNAs to sperm as it does small RNAs, has not been experimentally assessed. To investigate this, 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 large RNA species. These findings ultimately 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.