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BACKGROUND Previously we reported that cysteine rich secretory protein 2 (CRISP2) is involved in high molecular weight complexes in boar spermatozoa. These CRISP2 protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning. OBJECTIVES To identify CRISP2 interacting partners whereby their interactions were investigated under different conditions, namely non-capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187 induced acrosome reaction. MATERIALS AND METHODS 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 from were excised and subjected to liquid chromatography mass spectrometry (LC-MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC-MS for protein identification. The most prominent CRISP2 interacting proteins that appeared in both independent LC-MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with CRISP2 in pig sperm. RESULTS Blue native gel electrophoresis and native immunoblots revealed that CRISP2 was present within a ∼150 kDa protein complex under all three conditions. Interrogation of CRISP2-immunoreactive bands from blue native gels as well as CRISP2 immunoprecipitated products using mass spectrometry consistently revealed that, beyond CRISP2, acrosin and acrosin binding protein (ACRBP) were among the most abundant interacting proteins and did interact under all three conditions. Co-immunoprecipitation and immunoblotting indicated that CRISP2 interacted with pro-acrosin (∼53 kDa) and ACRBP under all three conditions and additionally to acrosin (∼35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with CRISP2 was assessed via in situ proximity ligation assays. The colocalization signal of CRISP2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of CRISP2 and ACRBP colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post acrosomal sheath region upon capacitation. DISCUSSION AND CONCLUSION 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. This article is protected by copyright. All rights reserved.