Our goal in this study was to characterize domains of the IgSF sperm protein Izumo and identify paralogs, whose investigation might ultimately contribute to our knowledge of Izumo function in sperm-egg fusion. PSI-BLAST searches allowed us to identify novel proteins showing significant homology to the N-terminal domain of Izumo 1. Izumo 1, 2 and 3 have in common testis-specific expression and the presence of a transmembrane domain. On the other hand, Izumo 4 stands out from the other three members because it lacks a transmembrane domain and shows a wider tissue expression. Proteins that are testis-specific are reported to evolve more rapidly on average than proteins with maximal expression in other tissues (Turner et al., 2008
). This seems to be the case for Izumo 1, 2 and 3, which are testis-specific and show a greater divergence than Izumo 4 which is predicted to be expressed in other tissues in addition to testis. Proteins involved in sperm-egg interactions also often evolve rapidly (Swanson and Vacquier 2002
). That Izumo 1 has a required role in sperm-egg fusion and shows the lowest inter-species homology in the family is consistent with that idea. The other members show significantly higher inter-species homologies and their functions (if any) in fertilization remain to be analyzed.
Izumo was described by Inoue et al (2005)
as a single 56 kDa band on mouse sperm. In this work using both an antibody against the whole recombinant extracellular domain and an antibody against a cytoplasmic peptide we detected a similar ~56 kDa band and an additional ~60 kDa band. Dephosphorylation experiments indicated the ~60 kDa protein is a phosphorylated form of Izumo 1. Both Izumo 3 and Izumo 4 were also detected as two different forms, which remained after treatment with alkaline phosphatase. The two different forms of each likely represent predicted splice variants. All three Izumo members are predicted to be N-glycosylated proteins, and Inoue et al (2008)
recently demonstrated Izumo 1 indeed has N-linked carbohydrates. Whether the different forms of Izumo 3 and 4 correspond to splice variants, glycosylation variants, or other post-translational modifications is currently unknown.
Izumo 1 was shown by immunofluorescence studies to be localized exclusively on the sperm head (Inoue et al., 2005
). As described previously by others (Miranda et al., 2009
), we detected Izumo 1 on the anterior acrosome region of acrosome-intact sperm. However on acrosome-reacted sperm while Miranda et al (2009)
observed Izumo 1 localized on regions adjacent to the equatorial segment we observed Izumo 1 localizing on either the entire acrosomal region or on the anterior acrosome. The different results on localization of Izumo 1 on acrosome-reacted sperm using different antibodies raise the possibility of existence of separate subpopulations of the molecule, but further studies are required to clarify the localization of Izumo 1 on fusion-competent sperm. The Western blot experiments performed on purified sperm heads suggest that Izumo 3 and Izumo 4 are are expressed on the sperm head and the immunofluorescence experiments confirmed the presence of Izumo 3 on the post-acrosomal region of the sperm head. Even though Izumo 1, 3 and 4 were all expressed in the sperm head, the fates of specific Izumo members after the AR were different: The two forms of Izumo 3 and the ~56 kDa form of Izumo 1 were unchanged by the AR, whereas Izumo 4 and the ~60 kDa form of Izumo 1 were greatly diminished after the AR. The specific loss of the phosphorylated (~60 kDa) form of Izumo 1 could have been due to its localization on the outer acrosomal membrane, which is lost as vesicles after the AR, or alternatively, to the dephosphorylation of this form.
Gene disruption studies have shown that expression of some sperm proteins influences the expression of others (Nishimura et al., 2001
, Carlson et al., 2005
). Our results that the amounts of Izumo 3 and Izumo 4 in sperm were similar in wild type and Izumo 1 knockout animals indicate that expression of Izumo 3 and Izumo 4 is independent of Izumo 1. The possibility that Izumo 1 forms protein complexes was analyzed by extraction of proteins with the detergent PFO followed by electrophoresis under mildly denaturing conditions. We found that Izumo 1 was present in several larger complexes. We favor the model that the complexes represent homo-multimers of the Izumo proteins. The molecular masses of the complexes in sperm were multiples of the monomers (55–60 kDa). And, Izumo 1 formed complexes not only in mouse sperm, but also in rat and hamster sperm. Moreover, the masses of the several complexes in CHO cells expressing the several recombinant forms of the Izumo family members supported the idea that the complexes were homo-multimers. Formation of putative multimers of higher order was variable among species, but the presence of putative dimers was observed in mouse, rat and hamster sperm, suggesting that this ability is conserved among rodents. Additional experiments will be required to determine if other proteins are present in the complexes and whether the complexes are essential for the function of Izumo 1 in gamete fusion.
The dissection of the regions required for complex formation of Izumo 1 suggests the presence of two distinct regions, each involved in separate Izumo 1 functions. According to this model, a region in the Izumo domain participates in formation of smaller complexes, possibly dimers. And, and a region in the transmembrane domain and/or the cytoplasmic tail participates in formation of larger complexes, possibly Izumo 1 multimers. The significant reduction in complex formation observed when a His-tag was added to the C-terminal end of the cytoplasmic tail suggests that the cytoplasmic tail plays an important role in the formation of mouse Izumo 1 putative trimers and tetramers.
Izumo 3 and Izumo 4 were also observed to form complexes. Although a smear rather than a defined spot was observed for Izumo 3, the molecular masses of Izumo 3 and Izumo 4 complexes were in the range expected for the corresponding dimers (~74 kDa Izumo 3, 86–110 kDa for Izumo 4). Moreover, the recombinant Izumo domain of Izumo 3 was able to form complexes of the mass of dimers. Taken together, these results support the model that the Izumo domain possesses a conserved protein-protein interaction motif. The absence of a transmembrane domain in Izumo 4 coupled with its loss after the AR and its presence in several somatic tissues suggest that this member of the Izumo family does not have a central role in in sperm-egg fusion. The generation of knockout animal models will help elucidate the functions of the novel Izumo members, which perhaps might be in spermatogenesis or earlier steps in fertilization.
The co-immunoprecipitation studies suggested Izumo 1 associates with other, as of yet unidentified sperm surface proteins. Although Izumo 1 has been shown to be present in DRM in non-capacitated sperm (Sleight et al., 2005
), under the conditions we performed the co-immunoprecipitation experiments (acrosome-reacted sperm, detergent extraction peformed at RT) Izumo 1 did not partition in the detergent-insoluble, light buoyant fractions suggesting the co-precipitating proteins rather than linked to Izumo 1 by a lipid microdomain are part of a a multi-protein complex. Although the importance of Izumo 1 in gamete fusion is well established, its precise molecular function is unknown. We reported earlier that the tetraspanin CD9, an egg protein essential for gamete fusion (Kaji et al., 2000
; Le Naour et al., 2000
; Miyado et al., 2000
), is able to bind directly to pSG17, a member of the IgSF, raising the possibility that it could also bind other sperm IgSF members (Ellerman et al., 2003
). Since Izumo 1 has an Ig domain, it is conceivable that it could bind directly to CD9. We assayed all of the recombinant Izumo 1 constructs described here for their ability to bind to the oocyte and affect sperm-egg fusion in vitro, with negative results (data not shown). This argues against the idea that Izumo 1 interacts directly with a protein in the oocyte.
We favor the model that rather than directly mediating the interaction between the sperm and the egg, Izumo 1 is essential for formation of complexes in cis
with other sperm proteins that are required for gamete fusion. In other biological systems involving fusion between membranes, the participation of membrane protein complexes is well established. In intracellular vesicle fusion, SNARE proteins localized in the vesicles and in the target membrane zipper into an alpha-helical bundle that pulls the two membranes tightly together to exert the force required for fusion. Many other proteins interact with this SNARE complex to control its assembly and/or membrane fusion (Rizo and Rosenmund, 2008
; Sudhof and Rothman, 2009
). In addition, fusion of Herpes simplex with host cells requires four glycoproteins, gB, gD, gH and gL, and recent data indicate these proteins are part of a single complex (Atanasiu et al., 2007
; Avitabile et al., 2007
). One model is that the complex is required for the fusogen molecule to adopt the fusion-active configuration, or alternatively that the proteins are biologically active only when they are assembled into a complex (Campadelli-Fiume et al., 2007
). Given that Izumo 1 is essential for gamete fusion and that it is part of a protein complex, it is possible that the absence of Izumo 1 influences the organization or stability of a protein complex essential for gamete fusion. The identification of the sperm proteins associated with Izumo 1 will allow exploration of this possibility.