Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa

J Cell Physiol. 2020 Jun;235(6):5340-5352. doi: 10.1002/jcp.29421. Epub 2020 Jan 6.


Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca2+ -dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca2+ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin-regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca2+ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin-binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca2+ mediator. In the present study, sperm calmodulin-binding proteins were identified by mass spectrometry after Ca2+ -dependent biotinylated-calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca2+ -dependent manner by calmodulin-conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm-egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.

Keywords: biological process; calmodulin pull-down; gene ontology; mammalian spermatozoa; proteomics; sperm head proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acrosome Reaction / genetics
  • Animals
  • Calcium / metabolism*
  • Calmodulin / genetics*
  • Calmodulin-Binding Proteins / genetics*
  • Cattle
  • Fertilization / genetics
  • Humans
  • Male
  • Protein Binding / genetics
  • Sperm Capacitation
  • Sperm Motility / genetics
  • Spermatozoa / growth & development*
  • Spermatozoa / physiology
  • Zona Pellucida / metabolism


  • Calmodulin
  • Calmodulin-Binding Proteins
  • Calcium