The fate of porcine sperm CRISP2 from the perinuclear theca before and after in vitro fertilization

Abstract

In a previous study we reported that porcine sperm cysteine rich secretory protein 2 (CRISP2) is localized in the post-acrosomal sheath (PAS)-perinuclear theca (PT) as reduction-sensitive oligomers. In the current study, the decondensation and removal of CRISP2 was investigated during in vitro sperm capacitation, both after induction of the acrosome reaction and after in vitro fertilization. Confocal immunofluorescent imaging revealed that additional CRISP2 fluorescence appeared on the apical ridge and on the equatorial segment (EqS) of the sperm head following capacitation, likely due to cholesterol removal. After an ionophore A23187 induced acrosome reaction, CRISP2 immunofluorescence disappeared from the apical ridge and the EqS area partly due to the removal of the acrosomal shroud vesicles but also partly due to its presence in a subdomain of EqS (EqSS). The fate of sperm head CRISP2 was further examined post-fertilization. In vitro matured porcine oocytes were co-incubated with boar sperm cells for 6-8 h and the zygotes were processed for CRISP2 immunofluorescent staining. Notably, decondensation of CRISP2, and thus of the sperm PT, occurred while the sperm nucleus was still fully condensed. CRISP2 was no longer detectable in fertilized oocytes in which sperm nuclear decondensation and paternal pronucleus formation were apparent. This rapid dispersal of CRISP2 in the PT is likely regulated by redox reactions for which its cysteine rich domain is sensitive. Reduction of disulfide bridges within CRISP2 oligomers may be instrumental for PT dispersal and PT elimination.

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

“…It shows that within a few hours post fertilization (before any signs of decondensation of the sperm nucleus), CRISP2 from the perinuclear theca is dissociated and degraded in the oocyte's cytosol. 62 The interactome with CRISP2 as identified in the current study can thus either during the Ca 2+ II acrosome reaction or after fertilization become rapidly released from CRISP2‐containing protein complexes and thus become functional. However, the possible functions of those proteins, from the CRISP2‐containing protein complexes in the fertilization cascade should be experimentally explored in the future.…”