Characterization of acrosin and acrosin binding protein as novel CRISP2 interacting proteins in boar spermatozoa

ABSTRACT: Previously we have reported that cysteine rich secretory protein 2 (CRISP2) is involved in high molecular weight complexes in boar spermatozoa under a native state. In the current study, CRISP2 interacting partners were identified using proteomic technology and their interaction was investigated under non-capacitating conditions, after the induction of in vitro sperm capacitation and then subsequently during an ionophore A23187 induced acrosome reaction. 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 acrosin and acrosin binding protein (ACRBP) were among the most abundant proteins and present under the 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. These results suggest that CRISP2 may act as a scaffold for protein complexes formation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Spermatozoon

SUBMITTER: Riccardo Zenezini Chiozzi  

LAB HEAD: Albert JR Heck

PROVIDER: PXD037181 | Pride | 2023-03-09

REPOSITORIES: Pride