Oral Presentation ESA-SRB 2023 in conjunction with ENSA

Phosphoproteomic analysis of human sperm capacitation reveals roles for novel kinases in regulating sperm motility and acrosomal exocytosis (#152)

Nathan Burke 1 2 3 , David A Skerrett-Byrne 1 2 , Amanda L Anderson 1 2 , Shaun D Roman 4 , John E Schjenken 1 3 , Brett Nixon 1 2 5 , Elizabeth G Bromfield 1 2 3 5
  1. The Priority Research Centre for Reproductive Biology, The University of Newcastle, Callaghan, New South Wales 2308, Australia
  2. Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, New South Wales, Australia
  3. School of BioSciences, Faculty of Science, Bio21 Institute, The University of Melbourne, VIC, Australia
  4. NSW Health Pathology, NSW Health, Newcastle, NSW, Australia
  5. These authors contributed equally to this work,

Extrinsic microenvironments provide crucial stimuli to transcriptionally and translationally silent human spermatozoa priming them for fertilisation. Residency in the female reproductive tract is one such microenvironment that establishes fertilisation competency by promoting capacitation associated phosphorylation signalling cascades. With some kinases considered synonymous with successful sperm capacitation (e.g. protein kinase A), protein phosphorylation forms a dynamic and essential component of sperm maturation. Despite the essential nature of phosphorylation to mammalian fertilisation, a comprehensive analysis of the phosphoproteomic landscape of capacitating human spermatozoa has yet to be reported.


To characterise the cellular signalling events underpinning sperm capacitation we performed phosphopeptide enrichment and high-resolution tandem mass spectrometry to quantify protein phosphorylation in populations of non-capacitated human spermatozoa as well as those subjected to capacitation stimuli in vitro. 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. Capacitation significantly upregulated phosphorylation in 124 proteins (1.5-fold change, p<0.05) and stimulated phosphorylation of a further 40 proteins. Of this subset of capacitation-sensitive phosphoproteins, 44% had a previously characterised role in sperm function, including A-kinase anchoring protein 4 (AKAP4) and heat shock protein A2 (HSPA2), which are critical contributors to motility and sperm-egg binding. Mapping of phospho-residues to upstream kinases revealed a suite of novel sperm kinases with previously unappreciated functions. Pharmacological inhibition of p21 activated kinase 1 (PAK1) and polo-like kinase 1 (PLK1) during capacitation hampered sperm progressive-motility, while AKT serine/threonine kinase 1 (AKT1) inhibition suppressed acrosomal exocytosis. These findings permit a new understanding of key kinases that act as functional regulators of human spermatozoa.