Lightning Talk + Poster ESA-SRB-ANZBMS 2024 in conjunction with ENSA

Phosphoproteomic analysis of human sperm capacitation and in silico kinase prediction reveals polo-like kinase 1 (PLK1) as a new target for male contraceptive development (#416)

Nathan D Burke 1 2 3 , David A Skerrett-Byrne 2 3 4 , Georgia E Blackley 1 , John E Schjenken 2 3 , Shaun D Roman 5 , Amanda L Anderson 2 3 , Brett Nixon 2 3 , Elizabeth G Bromfield 1 2 3
  1. School of BioSciences, Faculty of Science, Bio21 Institute, The University of Melbourne, Parkville, VIC, Australia
  2. Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW, Australia
  3. Infertility and Reproduction Research Program , Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
  4. Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany
  5. NSW Health Pathology, Newcastle, NSW, Australia

Due to their transcriptionally and translationally suppressed state, spermatozoa are reliant on post translational modifications to drive the generation of new proteoforms, such as phosphorylated proteins, that support their function and survival. Importantly, during transit of the female reproductive tract (termed capacitation), sperm fertilisation competency is established through phosphorylation-mediated signalling. With some kinases considered synonymous with successful sperm capacitation (e.g. protein kinase A), protein phosphorylation forms a dynamic and essential component of the fertilisation cascade. Despite the fundamental nature of phosphorylation to sperm function and fertility, a comprehensive analysis of the phosphoproteomic landscape of capacitating human spermatozoa has yet to be reported.

To characterise the phosphorylation events underpinning human sperm capacitation we performed EasyPhos phosphopeptide enrichment and high-resolution tandem mass spectrometry to quantify protein phosphorylation events in non-capacitated and capacitated human sperm. This strategy successfully identified 2,350 site specific phosphorylation events mapped across 902 unique sperm proteins. In congruence with previous findings indicating the importance of tyrosine phosphorylation to fertilisation, a 2-fold increase (representing a 104% gain) in tyrosine phosphorylated sites was observed following capacitation, compared to a modest 5% gain in the phosphorylation of serine residues under the same conditions. Mapping of phospho-residues to upstream kinases revealed a suite of novel sperm kinases with unexplored functions. Of particular interest, pharmacological inhibition of one of these targets, polo-like kinase 1 (PLK1), hampered sperm progressive motility and prevented tyrosine phosphorylation in human sperm. In vitro validation of these results in mice confirmed equivalent outcomes. These findings provide credence for in vivo proof of concept studies substantiating the utility of PLK1 as a potential non-hormonal contraceptive target.