{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,6]],"date-time":"2026-05-06T15:26:06Z","timestamp":1778081166048,"version":"3.51.4"},"reference-count":62,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2023,3,9]],"date-time":"2023-03-09T00:00:00Z","timestamp":1678320000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Andrology"],"published-print":{"date-parts":[[2023,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background<\/jats:title><jats:p>Previously, we reported that cysteine\u2010rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine\u2010rich secretory protein 2protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning.<\/jats:p><\/jats:sec><jats:sec><jats:title>Objectives<\/jats:title><jats:p>This study aimed to identify cysteine\u2010rich secretory protein 2 interacting partners.\u00a0These binding partner interactions were investigated under different conditions, namely, non\u2010capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187\u2010induced acrosome reaction.<\/jats:p><\/jats:sec><jats:sec><jats:title>Materials and Methods<\/jats:title><jats:p>The incubated pig sperm samples were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands were excised and subjected to liquid chromatography\u2013mass spectrometry (LC\u2010MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC\u2010MS for protein identification. The most prominent cystein\u2010rich secretory protein 2 interacting proteins that appeared in both independent LC\u2010MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with cystein\u2010rich secretory protein 2\u00a0in pig sperm.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Blue native gel electrophoresis and native immunoblots revealed that cystein\u2010rich secretory protein 2\u00a0was present within a \u223c150\u00a0kDa protein complex under all three conditions. Interrogation of cystein\u2010rich secretory\u2010protein 2\u2010immunoreactive bands from blue native gels as well as cystein\u2010rich secretory protein 2\u00a0immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein\u2010rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co\u2010immunoprecipitation and immunoblotting indicated that cystein\u2010rich secretory protein 2 interacted with pro\u2010acrosin (\u223c53\u00a0kDa) and Aacrosin binding protein\u00a0under all three conditions and additionally to acrosin (\u223c35\u00a0kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein\u2010rich secretory protein 2\u00a0was assessed via in situ proximity ligation assays. The colocalization signal of cystein\u2010rich secretory protein 2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of cystein\u2010rich secretory protein 2\u00a0and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post\u2010acrosomal sheath region upon capacitation.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion and Conclusion<\/jats:title><jats:p>These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.<\/jats:p><\/jats:sec>","DOI":"10.1111\/andr.13413","type":"journal-article","created":{"date-parts":[[2023,2,23]],"date-time":"2023-02-23T10:55:00Z","timestamp":1677149700000},"page":"1460-1471","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Characterization of acrosin and acrosin binding protein as novel CRISP2 interacting proteins in boar spermatozoa"],"prefix":"10.1111","volume":"11","author":[{"given":"Min","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Biomolecular Health Sciences Faculty of Veterinary Medicine, Utrecht University  Utrecht The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Riccardo Zenezini","family":"Chiozzi","sequence":"additional","affiliation":[{"name":"Biomolecular Mass Spectrometry and Proteomics Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences Utrecht University  Utrecht The Netherlands"},{"name":"Netherlands Proteomics Centre  Utrecht The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elizabeth G","family":"Bromfield","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Health Sciences Faculty of Veterinary Medicine, Utrecht University  Utrecht The Netherlands"},{"name":"Priority Research Centre for Reproductive Science School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle  Callaghan New South Wales Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Albert JR","family":"Heck","sequence":"additional","affiliation":[{"name":"Biomolecular Mass Spectrometry and Proteomics Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences Utrecht University  Utrecht The Netherlands"},{"name":"Netherlands Proteomics Centre  Utrecht The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"J Bernd","family":"Helms","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Health Sciences Faculty of Veterinary Medicine, Utrecht University  Utrecht The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9618-1261","authenticated-orcid":false,"given":"Bart M","family":"Gadella","sequence":"additional","affiliation":[{"name":"Department of 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