Involvement of CRISP2 in the organization of dense protein structures in porcine sperm

Abstract

In this thesis, we study cysteine rich secretory protein 2 (CRISP2) in pig sperm. CRISP2, which is synthesized during spermatogenesis, was localized by confocal immunofluorescent imaging in the tail and in the post-acrosomal region of the sperm head. High resolution localization by immunogold labeling electron microscopy (EM) of ultrathin cryosections revealed that CRISP2 was present in the perinuclear theca (PT) and neck region of the sperm head, as well as in the outer dense fibers and the fibrous sheath of the sperm tail. CRISP2 was involved in different protein complexes and showed different biochemical properties in the sperm head and tail. Later, we investigated the fate of sperm head CRISP2 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. Further, we report the first in depth, label-free proteomic characterization of the PT of boar spermatozoa. Through Ingenuity Pathway Analysis, we found surprising enrichment of endoplasmic reticulum (ER) proteins and the ER-stress response in the PT. Using the String and Cytoscape tools to visualize protein-protein interactions revealed an intricate network of PT protein complexes, including numerous proteasome subunits. Collectively, these data suggest that the PT may be a unique site of cellular homeostasis that houses an abundance of protein degradation machinery. This fits with previous observations that the PT structure dissociates first within the oocyte post-fertilization. Finally, we elucidate CRISP2 interacting partners using proteomic technology. We identified acrosin and acrosin binding protein (ACRBP) as novel CRISP2 interacting proteins. Additionally, our data also revealed a list of potentially interacting candidates that are functionally categorized into spermadhesins, protease inhibitors, core histones, chaperons, proteins with a mitochondrial function and ubiquitin. Biochemical validation of these interacting protein candidates is required. Nonetheless, our data suggest that CRISP2 might play multiple roles in sperm function and the fertilization process. It is clear that the ejaculated sperm are showing most robust and condensed state of the dense protein structures. This is ergonomically important for creating a minimal volume of well protected chromatin in the sperm head, a concentrated enzyme matrix for sperm-zona penetration in the acrosome. Condensed structures are also present in the sperm flagellum which has unique flexibility and metabolic properties. These properties can be adjusted to proper sperm motility characteristics during sperm maturation in the epididymis as well as during capacitation in the oviduct (or during IVF treatment). Both the formation of the dense protein structures in the testis and in the epididymal environments as well as the decondensation of these dense protein structures during post-fertilization events deserve further attention with special emphasis on the role of CRISP2 in (de)condensation dynamics.