Cell-free seminal mRNA and microRNA exist in different forms

Area of Science:

• Reproductive biology
• Molecular biology
• Biomarker discovery

Background:

• Cell-free mRNA and miRNA are of significant interest as biomarkers and intercellular signaling molecules.
• Understanding the physical nature of these extracellular RNAs is crucial for their application.
• This study investigates cell-free seminal mRNA (cfs-mRNA) and miRNA (cfs-miRNA) in human semen.

Purpose of the Study:

• To elucidate the physical nature and localization of cell-free mRNA and miRNA in human seminal plasma.
• To understand the stability and origins of extracellular RNAs in the male reproductive tract.
• To inform the development of reproductive biomarkers based on RNA characteristics.

Main Methods:

• Quantitative real-time PCR was used to measure specific mRNAs, miRNAs, and piRNAs.
• Time-course analysis assessed the stability of cfs-miRNA and cfs-mRNA.
• Filtration and Triton X-100 treatment were employed to differentiate RNA properties.
• Seminal microvesicles (SMVs) were isolated and analyzed for RNA content.
• Protease K digestion and filtration explored the form of cfs-miRNA in the supernatant.

Main Results:

• cfs-miRNA and cfs-mRNA showed similar stability but differed significantly after filtration and detergent treatment, indicating distinct physical properties.
• The majority of cfs-mRNA was found within seminal microvesicles (SMVs), with amounts varying by SMV size.
• Most cfs-miRNA was found in the supernatant, independent of SMVs.
• cfs-miRNA in the supernatant passed through a 0.10-µm filter but was degraded by protease K, suggesting a protein-bound form.

Conclusions:

• Predominant cfs-mRNA is encapsulated within SMVs, whereas most cfs-miRNA exists in protein complexes.
• These findings explain the stability of extracellular RNAs in semen.
• The data provide insights into the origins and functions of seminal RNAs and guide strategies for developing male reproductive biomarkers.