Molecular Cloning, Characterization of Porcine IZUMO1, an IgSF Family Member

Abstract

IZUMO1, belonging to the family of mammalian immunoglobulin proteins, has been well characterized in the mouse. Here, we describe the molecular cloning and expression analysis of porcine IZUMO1 (pIZUMO1). Partial sequence information published in the National Center for Biotechnology Information (NCBI) database was used to generate the full-length sequence for IZUMO1 using rapid amplification of cDNA ends (RACE). A search of the porcine genomic sequence in the NCBI database identified a bacterial artificial chromosome (BAC) encoding the pIZUMO1 gene. This BAC is derived from porcine chromosome 6 and is syntenic with the corresponding regions of mouse, bovine, and human genomes encoding the IZUMO gene family. This BAC was found to encode an IZUMO1 protein with a predicted amino acid sequence having high similarity with mouse and human IZUMO1. Western blot analysis of proteins from porcine tissues indicated that pIZUMO1 was specifically expressed in the sperm. Furthermore, to confirm whether pIZUMO1 forms complexes, we overexpressed pIZUMO1 in HEK293 cells. The recombinant pIZUMO1 from cell extracts was found to form complexes. Our finding suggests that pIZUMO1 forms homodimeric complex on the sperm membrane. Furthermore, an IVF inhibition assay with an antibody for the porcine IZUMO1 Ig-like domain showed that Ig-like domain effectively prevented pig sperm-egg interactions.

CRISPR/Cas9-mediated mutation revealed cytoplasmic tail is dispensable for IZUMO1 function and male fertility

“…In the pig IZUMO1 still forms complexes; however, these are mainly homodimers of IZUMO1 (Kim et al 2013). As seen in the work by Ellerman et al (2009) complexes of this size are most likely formed by interactions between the extracellular portion of the protein, therefore it is unsurprising that pig IZUMO1 forms complexes of this size, even without a long cytoplasmic region.…”

“…55 aa), shorter in humans (approx. 40 aa) and very short in porcine species -only one amino acid in length (an alanine) (Kim et al 2013). In order to investigate the importance of this region, this study produced a mutated mouse line with a truncated version of IZUMO1.…”

Molecular cloning and characterization of Izumo1 gene from bovine testis

“…Second, we showed that bIZUMO1 is expressed on the surface of sperm as a homodimeric complex. Inter-IZUMO1 complex formation was recently described during mouse IZUMO1 immunoprecipitation experiments, and it was suggested that the protein forms a homodimeric complex in mouse sperm [ 13 , 14 ]. Indeed, most immunoglobulin superfamily members have been reported to exert their function in various biological processes, such as germ cell migration, transmigration of leucocytes and viral invasion, through homophilic and heterophilic interactions [ 15 , 16 ].…”

Roles of the zona pellucida and functional exposure of the sperm‐egg fusion factor ‘IZUMO’ during in vitro fertilization in pigs

“…Kim et al . () subsequently cloned and characterized the porcine IZUMO1 gene, raised an anti‐porcine IZUMO1 antibody, and analyzed the expression of IZUMO1 in porcine species. Furthermore, they found that IZUMO1 is well conserved across species (pig, crab‐eating macaque, mouse and bull).…”

“…The expression of IZUMO has also been analyzed in porcine species, and shown to be specific to boar sperm (Kim et al . ). In humans, the relationship between IZUMO and infertility has been studied, and anti‐IZUMO antibodies have been found in serum samples from immunoinfertile women (Clark & Naz ).…”