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

Abstract

Background: Previously, we reported that cysteine-rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine-rich 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.

Objectives: This study aimed to identify cysteine-rich secretory protein 2 interacting partners. These binding partner 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 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 cystein-rich secretory protein 2 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 cystein-rich secretory protein 2 in pig sperm.

Results: Blue native gel electrophoresis and native immunoblots revealed that cystein-rich secretory protein 2 was present within a ∼150 kDa protein complex under all three conditions. Interrogation of cystein-rich secretory-protein 2-immunoreactive bands from blue native gels as well as cystein-rich secretory protein 2 immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein-rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co-immunoprecipitation and immunoblotting indicated that cystein-rich secretory protein 2 interacted with pro-acrosin (∼53 kDa) and Aacrosin binding protein under all three conditions and additionally to acrosin (∼35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein-rich secretory protein 2 was assessed via in situ proximity ligation assays. The colocalization signal of cystein-rich 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-rich secretory protein 2 and acrsin binding protein 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.

Keywords: CRISP2; boar spermatozoa; capacitation; colocalization; interacting proteins; protein complexes.

FIGURE 1

Cysteine‐rich secretory protein 2 (CRISP2) is involved in a ∼150 kDa protein complex under native conditions. After incubation and washing, non‐capacitated (NC), capacitated (CAP), and ionophore‐induced (II) sperm cells were lysed and prepared for blue native polyacrylamide gel electrophoresis (PAGE) and SDS‐PAGE. (A) Native blots on the lysates from NC, CAP, and II sperm cells probed with anti‐CRISP2 antibody. (B) Lysates from NC, CAP, and II sperm cells were mixed with 4X SDS sample buffer, cooked and analyzed by western blots. Three ejaculates from different boar were mixed as one biological replicate and this experiment was replicated three times.

FIGURE 2

Validation of acrosin and acrosin binding protein (ACRBP) as CRISP2 interacting partners by western blots analysis of CRISP2 immunoprecipitation (IP). CRISP2 antibody was cross‐linked to protein A/G magnetic beads and incubated with lysates from NC, CAP, and II sperm cells overnight at 4°C. After intensive washing, CRISP2 and CRISP2‐associated proteins were disassociated from the magnetic beads. (A) Sperm lysates before incubation with the beads were saved and analyzed by western blots. (B) CRISP2 pull‐down products were analyzed by western blots probed with anti‐acrosin, anti‐ACRBP, and anti‐CRISP2 antibodies. Full western blots images are shown in Supplementary Figure S3.

FIGURE 3

Subcellular localization of acrosin and ACRBP in boar spermatozoa. Western blots of acrosin (A) and ACRBP (B) on the lysates from whole sperm, sperm heads, and tails. (C) Percoll‐washed sperm cells were fixed in −20°C methanol for 5 min, incubated with anti‐acrosin followed by Alexa Flour 568 conjugated secondary antibody (red), counterstained with Hoechst 33342 (blue). (D) Sperm cells were fixed in 4% paraformaldehyde (PFA) and permeabilized in cold acetone for 10 min, incubated with anti‐ACRBP followed by Alexa Flour 488 conjugated secondary antibody (green) and counterstained with Hoechst 33342 (blue). This experiment was replicated three times. Scale bar = 10 µm.

FIGURE 4

Immunolocalization of CRISP2. (A) NC, CAP, and II sperm cells were fixed in 4% PFA and permeabilized in cold acetone for 10 min, incubated with anti‐CRISP2 followed by Alexa Flour 568 conjugated secondary antibody (red) and counterstained with Hoechst 33342 (blue). (B) Immunofluorescent staining of CRISP2 on sperm cells fixed in −20°C methanol for 5 min, incubated with anti‐CRISP2 followed by Alexa Flour 488 conjugated secondary antibody (green) and counterstained with Hoechst 33342 (blue). Arrows indicated additional CRISP2 signals were observed. Three ejaculates from different boar were mixed as one biological replicate, and this experiment was replicated three times. Scale bar = 10 µm.

FIGURE 5

Colocalization of acrosin and CRISP2 via an in situ proximity ligation assay (PLA). NC, CAP, and II sperm cells were permeabilized in −20°C methanol for 5 min, incubated with anti‐acrosin and anti‐CRISP2 antibodies, followed by appropriate secondary antibodies conjugated with oligonucleotides. Sperm cells were counterstained with peanut agglutinin lectin (PNA; green) and Hoechst 33342 (blue). Primary antibodies were omitted in control. Three ejaculates from different boar were mixed as one biological replicate, and this experiment was replicated twice. Scale bar = 10 µm.

FIGURE 6

Colocalization of ACRBP and CRISP2 via PLA. Sperm cells were fixed in 4% PFA and permeabilized in cold acetone for 10 min, incubated with primary antibodies conjugated with oligonucleotides, and counterstained with Hoechst 33342 (blue). Three ejaculates from different boar were mixed as one biological replicate, and this experiment was replicated twice. Primary antibodies were omitted in control. Scale bar = 10 µm.