Phosphoproteomics reveals novel regulators of sperm function and fertility — ASN Events
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  2. Joint Poster Session
  3. Phosphoproteomics reveals novel regulators of sperm function and fertility

Phosphoproteomics reveals novel regulators of sperm function and fertility (#531)

Georgia E Blackley 1 , Nathan D Burke 1 , Brett Nixon 2 , David A Skerrett-Byrne 2 3 , Elizabeth G Bromfield 1 2
  1. School of BioSciences, The University of Melbourne, Parkville, Victoria, Australia
  2. Hunter Medical Research Institute, The University of Newcastle, Callaghan, New South Wales, Australia
  3. Helmholtz Zentrum München, German Research Centre for Environmental Health, Nauenberg, Germany

Background: During their transit of the female reproductive tract (termed capacitation), spermatozoa establish fertilisation competency through complex phosphorylation-mediated signalling. Given their transcriptional and translational quiescence, sperm rely heavily on post-translational mechanisms to generate new proteoforms, including phosphorylated proteins, that support their function and survival.  However, despite the fundamental role of phosphorylation in male fertility, the signalling networks governing this process in human sperm remain poorly defined. This study aimed to broaden therapeutic targets for sperm targeted contraceptives and infertility therapies by adapting a contemporary phosphoproteomic technique, termed EasyPhos, to investigate human sperm signalling.

Methods: Phosphorylation enrichment, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on non-capacitated and capacitated sperm populations (n=5). In silico approaches were then used to predict sperm kinases regulating the phosphosites detected by LC-MS/MS. Commercially available kinase inhibitors were used to examine the role of predicted kinases in sperm function and in vitro fertilization (IVF).  

Results: This approach yielded high depth phosphoproteomes of non-capacitated and capacitated human spermatozoa, revealing >2000 phosphosites and 52 kinases with previously uncharacterized roles in sperm capacitation. Targeted pharmacological inhibition confirmed novel roles for several kinases in the regulation of sperm function. Polo like kinase 1 (PLK1) inhibition in human sperm significantly reduced progressive sperm motility, attenuated capacitation-associated tyrosine phosphorylation and reduced the sperm acrosomal reaction, a requirement for fertilization. Inhibition of PLK1 in mouse sperm yielded similar results, with the additional effect of reducing sperm-fertilization capacity in IVF.

Conclusion: This study revealed that PLK1 is a conserved regulator of mouse and human sperm signalling. Our publicly available phosphoproteome substantially expands the candidate pool of sperm targets for contraceptive and infertility therapies.