Sperm surface proteases are emerging as important players in the maturation and functional competence of spermatozoa. Numerous cytotoxic compounds, such as bacterial toxins, require cleavage by cell surface proteases to enter the cell and exert cytotoxic activity. This could be exploited in developing novel contraceptive approaches, whereby enzymatically active sperm-specific proteases could facilitate the targeted internalisation and activation of protease-activated toxins.
Amongst a panel of protease inhibitors, AEBSF, a serine protease inhibitor, reduced the capacity of stallion spermatozoa to undergo spontaneous capacitation and acrosome reaction (AR) in a dose-dependent manner. Screening of our stallion sperm proteome for AEBSF-susceptible proteases revealed a putative testis-specific serine protease, PRSS21 (testisin). Sequence alignment and 3D protein modelling predicted a species-unique structure of equine testisin, with an apparent domain duplication and likely additional transmembrane domains as compared to human and mouse testisin. Immunofluorescent labelling of surface testisin shifted from the anterior sperm head to the equatorial region throughout capacitation and AR, concurrent with an increase in labelling intensity as quantified by flow cytometry. Blue Native PAGE and western blotting indicated that testisin forms part of at least two large protein complexes, one of which disperses upon capacitation/AR. Co-immunoprecipitation revealed association of testisin primarily with zonadhesin, along with several other proteins specific to the acrosomal vesicle, justifying the protein’s distinct localisation in the anterior sperm head and surface exposure upon AR.
This study presents the first characterisation of testisin and its protein-protein interactions in stallion spermatozoa. Our results confirm the presence of testisin on the sperm surface and support a functional role for this protein in the final stages of sperm maturation. Further identification of equine testisin’s proteolytic substrates will facilitate engineering of a testisin-activated cytotoxin. We propose that testisin may participate in cleavage and activation of zonadhesin, a protein responsible for species-specificity of sperm-zona binding.