Successful fertilisation requires sperm to adapt to the changing environment of the female reproductive tract despite their severely limited capacity for gene expression (1). During maturation, sperm chromatin becomes tightly compacted, most cytoplasm is lost, and most of the transcriptional machinery is eliminated (2). We previously showed that viscosity alters sperm swimming behaviour and dynamics, including the transition from three-dimensional (3D) swimming to two-dimensional (2D) slithering motion (3). This raises the question of whether the effect of viscosity extend beyond altering swimming behaviour to induce changes in the molecular profile of motile sperm.
To investigate the effect of viscosity on the sperm proteome, split bovine sperm samples (n=3) were subjected to swim-up for 45 min at 37°C in selection medium supplemented with methylcellulose to achieve viscosities of 1, 5, 20 and 250 cP. Selected motile fractions were analysed by LC-MS/MS proteomics. BODIPY-FL-tRNA incorporation was used to assess nascent protein synthesis under low- and high-viscosity conditions.
Across the four viscosity conditions, 1,657 proteins were shared, indicating that viscosity-conditioned selection acts within a largely conserved sperm proteome rather than isolating distinct proteomes. However, abundance profiles were not uniform. The 1 cP condition was dominated by membrane- and glycosylation-associated annotations, while 5 cP was enriched for structural and regulatory protein functions consistent with a transition state. At 20 cP, where sperm swim in 2D, enriched annotations converged on fertilisation, sperm-egg recognition and zona pellucida binding. In contrast, 250 cP showed stress/MAPK regulatory and aminoacyl-tRNA/ligase-related protein-handling signatures. The nascent protein synthesis signal was stronger at 250 cP than at 1 cP, supporting investigation of viscosity-associated protein-state dynamics.
These findings suggest that viscosity is not merely a mechanical input to sperm motility. They may inform more physiologically relevant sperm-selection methods and help identify molecular pathways involved in fertilisation and potential targets for non-hormonal male contraception.