Seminal extracellular vesicle–mediated NF-κB signalling at the male–female reproductive tract interface — ASN Events
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Seminal extracellular vesicle–mediated NF-κB signalling at the male–female reproductive tract interface (#521)

Ellen L Stonestreet 1 2 , Cottrell T Tamessar 1 2 3 , Shanu Parameswaran 1 2 , Elizabeth Torres-Arce 1 2 , Piper Miller 1 2 , Heidi J Gilchrist 1 2 , Sarah A Robertson 4 , David J Sharkey 4 , John E Schjenken 1 2
  1. Centre for Reproductive Science, School of Science, College of Engineering, Science and Environment, The University of Newcastle, Callaghan, NSW, Australia
  2. Reproductive and Family Health Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
  3. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde, Sweden
  4. Robinson Research Institute, and School of Pharmacy and Biomedical Science, College of Health, Adelaide University, Adelaide, South Australia, Australia

Background: Components of seminal plasma, including seminal extracellular vesicles (SEVs), influence epithelial cells lining the female reproductive tract to promote an immune environment supportive of pregnancy. We have previously identified NF-κB-associated molecules as potential SEV-derived immune signalling mediators. In this study, we characterised proteins and microRNAs carried by SEVs that are associated with the regulation of NF-κB signalling.

Methods: SEVs from normozoospermic semen samples (n=8) were enriched and characterised using established approaches. Immunoblotting and densitometry were used to identify NF-κB proteins carried by SEVs using ectocervical (Ect1) epithelial cells as a positive control. SEV-associated microRNAs were identified from published miRNA-sequencing data and assessed for their capacity to target NF‐κB signalling using DIANA-miRPath.

Results: NF‐κB associated proteins were found to be carried by SEVs, with the post-translationally processed NF‐κB2 subunit p52, its unprocessed precursor, p100 and the p65 subunit of NF‐κB, RELA found to be carried by SEVs. In contrast, the NF‐κB1 precursor subunit p105, NF‐κB1 processed subunit p50, RELB, REL, NF‐κBIA, or NF‐κBIE were not detected. Amongst detectable proteins in SEVs, p52 was the most highly abundant NF‐κB protein detected, being on average 2.6-fold more abundant than in Ect1 cells. In contrast, p100, and RELA were lowly abundant in SEVs, >99% lower than in Ect1 cells. Notably, substantial variation in the abundance of p52 (range=0.264-7.066), p100 (range=0.001-0.010) and RELA (0.0003-0.0069) in SEVs was observed across donors. In addition to signalling proteins, SEVs also carried the following NF‐κB associated microRNAs: hsa-miR-146a-5p, hsa-let-7b-5p, hsa-miR-181a-5p, hsa-miR-146b-5p, hsa-miR-221-3p, hsa-miR-10a-5p, hsa-miR-204-5p, hsa-let-7i-5p, hsa-miR-181b-5p, and hsa-miR-31-5p.

Conclusion: SEVs carry NF-κB-associated proteins and microRNAs that may influence maternal NF-κB signalling in Ect1 cells. Future studies will determine whether these bioactive cargo are transferred to epithelial cells and modulate epithelial immune responses within the female reproductive tract.