Abstract
IZUMO1 is a sperm acrosomal membrane protein that is essential for mammalian fertilization through recognition of JUNO on the oocyte surface and accompanying IZUMO1-JUNO complex formation. Here, we report a new Izumo1 gene splicing variant (IZUMO1_v2) with a unique 52-amino-acid-long signal sequence transcribed from Exon 1b. Although the mRNA amount of Izumo1_v2 is 76 times lower than that of the original Izumo1 (IZUMO1_v1) in the testis, the cell-oocyte assay indicates that IZUMO1_v2-expressing COS-7 cells have the ability to attach to the oocyte equivalent of IZUMO1_v1. To clarify the physiological function of IZUMO1_v2, we produced an IZUMO1_v1-specific knockout mouse line with a nine-base deletion adjacent to the initial methionine codon of IZUMO1_v1 by the CRISPR/Cas9 system. The IZUMO1_v1 knockout male mice carry 0.19-fold lower level of IZUMO1 protein in the spermatozoon; however, reduction in fertility was only minimally affected compared to the wild-type mice, suggesting that only a small fraction of IZUMO1 is sufficient for triggering sperm-egg fusion. We propose that the alternative splicing generating IZUMO1_v2 might function as a fail-safe in mouse for when splicing is disturbed.
Highlights
In fertilization, two kinds of haploid cells, spermatozoa and oocytes, merge with each other to generate a new individual creature
We identified a novel transcript of the Izumo[1] gene with a longer signal sequence generated by alternative splicing, and named it IZUMO1 variant 2 (IZUMO1_v2)
Exon 1b includes the start codon of IZUMO1_v2 and is bound to the common Exon 2 (Fig. 1b). This Izumo1_v2 was translated exclusively in mouse (Mus musculus) because we could not find the in-frame transcript among other species including other rodents, in the latest NCBI database (Fig. 1b shows an example of rat [Wistar], accession number: LC426750)
Summary
Two kinds of haploid cells, spermatozoa and oocytes, merge with each other to generate a new individual creature. We have established an in vitro cell-oocyte binding system, in which cultured cells expressing the Izumo[1] gene, such as COS-7 cells, become adhesion-competent towards oocytes[8]. A reconstituted assay revealed that JUNO is excluded from the contact site once it recognizes IZUMO19, which robustly establishes firm adhesion of the two cells[8]. These studies strongly implied that there has to be a secondary receptor for IZUMO1. Since COS-7 cells solely expressing the Izumo[1] gene never acquire membrane fusion activity with oocytes[8], sperm-egg fusion is considered to consist of multiple steps. In order to clarify the function of IZUMO1_v2, we generated an IZUMO1_v1-specific knockout mouse line using the CRISPR/Cas[9] system, and investigated the detailed reproductive phenotype in vitro and in vivo
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