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This study aimed to characterize the serine protease testisin (<jats:styled-content style=\"fixed-case\">PRSS<\/jats:styled-content>21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins.<\/jats:p><\/jats:sec><jats:sec><jats:title>Materials and Methods<\/jats:title><jats:p>The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibitor, <jats:styled-content style=\"fixed-case\">AEBSF<\/jats:styled-content>. Testisin localization, before and after exposure of stallion spermatozoa to capacitating conditions and calcium ionophore, was examined using live cell immunofluorescence and flow cytometry. Immunohistochemistry of testicular and epididymal tissues was used to further dissect the origins of sperm testisin. Testisin's participation in high molecular weight protein complexes and identification of its interacting partner proteins were investigated using Blue Native <jats:styled-content style=\"fixed-case\">PAGE<\/jats:styled-content>, co\u2010immunoprecipitation, and mass spectrometry, with interrogation of protein\u2013protein interaction databases and gene ontology analysis of partner proteins used to further explore the potential roles of the testisin\u2010containing complex in sperm function.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Testisin surface expression increased significantly in capacitated spermatozoa (<jats:italic>p<\/jats:italic>\u00a0&lt;\u00a00.001), increased further following acrosome reaction (<jats:italic>p<\/jats:italic>\u00a0&lt;\u00a00.01), and was localized to the equatorial region of the sperm head. Testisin was also detected in luminal fluid within the caput and corpus regions of the epididymis, epididymal spermatozoa, and epididymal epithelial cells. Testisin formed several multiprotein complexes; co\u2010immunoprecipitation revealed interactions of testisin with a multitude of zona pellucida\u2010binding proteins, including <jats:styled-content style=\"fixed-case\">ZPBP<\/jats:styled-content>,<jats:styled-content style=\"fixed-case\"> ZAN<\/jats:styled-content>, acrosin, several heat\u2010shock proteins, and components of the <jats:styled-content style=\"fixed-case\">TCP<\/jats:styled-content>1 complex.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>Testisin appears to form part of the zona pellucida\u2010binding complex in stallion spermatozoa and may be involved in the proteolytic cascade that prepares the sperm surface for interaction with the oocyte.<\/jats:p><\/jats:sec>","DOI":"10.1111\/andr.12569","type":"journal-article","created":{"date-parts":[[2018,12,14]],"date-time":"2018-12-14T05:25:50Z","timestamp":1544765150000},"page":"199-212","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["The serine protease testisin is present on the surface of capacitated stallion spermatozoa and interacts with key zona pellucida binding proteins"],"prefix":"10.1111","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7371-5400","authenticated-orcid":false,"given":"A.","family":"Swegen","sequence":"first","affiliation":[{"name":"Priority Research Centre for Reproductive Science University of Newcastle Callaghan NSW Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"N. 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