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The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs
Naokazu Inoue, Masahito Ikawa, Ayako Isotani , and Masaru Okabe
Nature. 2005 Mar 10;434(7030):234-8.
Summary of Results:
Prior to this paper, it was known that CD9 on the egg’s membrane, a cell surface protein involved in cell-cell adhesion, is essential for egg-sperm fusion, however any fusion-related proteins on the sperm’s membrane remained unknown. Through their research, published in this paper, Inoue et al. identified a protein on the surface of mice (Mus musculus) sperm, named Izumo, required for egg-sperm fusion. The authors identified this protein using monoclonal antibodies and gene cloning techniques. The results of this paper, which analyze the fusion and fertilizing abilities of Izumo -/- knockout mice (and other transgenic mice), confirm their findings that Izumo is indeed required for egg-sperm fusion.
In 1987, the authors that produced this paper discovered OBF13, an anti-mouse sperm monoclonal antibody that bound to an unknown molecule on the sperm head. When experiments were performed using zona-free mice eggs, OBF 13 significantly reduced fertilization rates. Upon performing two-dimensional gel-electrophoresis and immunoblotting using OBF13, Inoue et al. identified the unknown molecule as the antigen Izumo.
Confirming Izumo’s Identity
Upon examination of the of the unknown antigen bound to OBF13, Inoue et al. identified 10 proteins corresponding to part of a registered DNA sequence. Indeed, RT-PCR confirmed that the amino acid sequences of the resulting cDNA matched that of the database. Using the peptide sequences obtained from the RIKEN database, Inoue et al. compared mouse and human orthologs of Izumo. As a result, they identified putative signal peptide and transmembrane regions, an immunoglobulin-like domain, and cysteine residues that they speculate form a disulphide bridge (Figure 1a). Given these data and using their knowledge of structure/function relationships, Inoue et al. depicted the possible structure of the membrane-bound Izumo (Figure 1b). Subsequently, they performed two Western blot analyses. In one Western blot using tissues isolated from various tissues and a polyclonal antibody raised against recombinant mouse Izumo, they identified a 56.4-kDa protein located exclusively in the testis and sperm (Figure 1c). In a second Western blot analysis using human sperm and an anti-human Izumo antibody, they identified a 37.2-kDa protein (Figure 1d).
Izumo is only detectable in acrosome-reacted sperm
In an effort to determine whether Izumo was detectable on the surface of sperm with or without the presence of the acrosome, Inoue et al. created a transgenic mouse line that expresses green fluorescent protein (GFP) in the acrosome. Using immunostaining with GFP and red-fluorescing Izumo they found that Izumo was only detectable in acrosome-reacted sperm (no- GFP) (Figure 1e). The same conclusion was observed for human sperm in which a green anti-CD4 antibody was used to detect a lack of acrosome activity (as transgenic humans could not be created) (Figure 1f).
Recombination Experiments Successful
In an effort to determine the physiological function of this novel protein, Inoue et al. created transgenic mice through the process of homologous recombination. To create an Izumo deficient allele, they used a targeting vector containing a gene for neomycin-resistance (neo r) and a chain (diptheria toxin A) driven by a promoter (DT). In the mutant allele the sequence for Izumo protein (in black boxes) was replaced by that of neo r, shortening normal Izumo from a 15kb strand to a 6.9kb strand (Figure 2a). These fragments were confirmed by DNA Southern blot analysis using external probes (E). As expected, wild type mice had two 15kb alleles, heterozygous mice had one 15kb and one 6.9kb allele, and homozygous knockout mice had two 6.9kb alleles (Figure 2b).
Deletion of Izumo does not affect expression levels of related proteins
Next, the authors asked themselves if the deletion of Izumo in knockout mice affected subsequent levels of related proteins. To answer this question, they compared expression levels of three related proteins, ADAM2, CD147, and sp56 (all involved in sperm-egg interactions), among wild type, heterozygous, and homozygous knockout mice. Western blots confirmed that each of the proteins was expressed in equal amounts in all transgenic mice lending support to the conclusion that Izumo deletion did not affect expression levels of related proteins (Figures 2c and 2d). As expected, Izumo protein and mRNA was undetectable in knockout (Izumo -/-) mice.
Izumo -/- mice are sterile
After confirming the success of their recombination techniques, the authors put the knockout sperm to the test. Figure 3a compares litter sizes resulting from heterozygous and homozygous knockout mice paired with wild type and Izumo knockout females. Of the nine wild type females paired with knockout mice none produced litters, suggesting that Izumo is required for fertilization. Figure 3a also confirms not only that Izumo +/- fertilize normally, but also that the presence or absence of Izumo in females has no affect on fertility (as standard error bars overlap).
Izumo -/- sperm fail to initiate pronucleus formation
To rule out the possibility that an event occurred after fertilization to impair Izumo -/- offspring production, Inoue et al. examined eggs for pronucleus formation. Indeed, in 252 eggs, pronuclei were not formed in the presence of knockout sperm indicating that fertilization never occurred (Figure 3b).
Izumo -/- sperm fail to fuse with eggs, fail to trigger ZP reaction
To exclude the possibility that sperm-egg fusion occurred but another event prevented pronucleus formation, the authors directly examined the number of fused sperm per egg. Inoue et al. observed that while Izumo -/+ sperm successfully fused with eggs, none of the 187 mice eggs paired with Izumo -/- sperm were able to fuse (Figure 3e). Failure of Izumo -/- sperm to fuse was also confirmed microscopically as Hoechst 33342, an antibody that identifies fused sperm, was only detected in Izumo +/- sperm (Figure 3f). The same results were obtained when Izumo -/- sperm were paired with zona-free hamster eggs (Figure 4a).
Using high powered microscopes, the authors observed that eggs in the presence of Izumo -/- sperm had many more sperm on their zona pellucida (ZP) than Izumo +/- sperm (Figure 3c). They noted that normally, when sperm and egg fuse, the ZP reacts by immediately inhibiting further sperm-binding ability. Therefore, it is likely that Izumo -/- sperm do not trigger this ZP reaction because they lacked the ability to fuse with eggs.
To exclude the possibility that Izumo -/- sperm had failed to undergo acrosomal reactions, Inoue et al. labeled sperm with MN9, an antibody that reacts with acrosome reacted sperm. Figure 3d indicates that sperm did undergo acrosomal reactions and appeared to accumulate in the perivitelline space.
Izumo -/- mice successfully reproduce after ICSI Injection
In order to ensure that Izumo -/- sperm would have had the capacity to produce surviving embryos, given that they were able to fertilize, knockout sperm were injected into eggs using intracytoplasmic sperm injection (ICSI). Upon injection, the resulting embryos developed normally and, in fact, twice as many pups were born to Izumo -/- sperm as Izumo +/- sperm (Table 1).
Human Izumo is required for fertilization
To determine whether human Izumo was required in heterologous sperm-egg fusion systems, human sperm were paired with zona-free hamster eggs. In the presence of a control antibody, IgG, human-hamster fusion was successful, as indicated by Hoechst 33342 (Figure 3b). However, when human Izumo was inhibited by anti-human Izumo antibodies, sperm-egg fusion was unsuccessful.
Upon thorough examination of the results and discussion presented by Inoue et al, the conclusions of this paper do seem to be well supported by the data. Like every scientific publication, however, even those published in nature, this paper does show room for improvement.
To begin, their results would have been better supported if they had included loading controls for the Western blots illustrated in figures 1c and 1d. The identification of positive controls in each lane would have confirmed that detectable amounts of protein extracted from tissues were loaded onto the gel. Furthermore, with reference to the western blot of anti-human Izumo, the inclusion of other tissue extracts, as in the mice, would have confirmed that in humans, as well, Izumo is localized in the testis and sperm. For each of the western blots, they also failed to include lanes demonstrating molecular weight markers.
At times in this paper, a more detailed explanation of some of the figures would have helped to clarify potentially misleading results. For example, it is interesting that what they claim is a loading control, GAPDH, does not appear to be located on the same gel as Izumo. Other loading controls for western blots in figure 2d would have confirmed that detectable amounts of protein were loaded onto the gel. They also could have explained in more detail exactly why ADAM2, CD147, and sp56 proteins were chosen as proof that Izumo deletion did not affect expression levels of related proteins. They mention that these proteins are ‘involved in sperm-egg interactions,’ however it is highly likely that a multitude of proteins are involved in these interactions. Why did they choose these proteins over those reported in the discussion section to function in sperm-egg fusion such as CD46, equatorin, or SAMP32?
It is also intriguing that, throughout the paper, Inoue et al. fail to explain why they consistently compare Izumo -/- sperm with Izumo +/- sperm, as opposed to wild type sperm. While figures 2c and 2d indicate that wild type and Izumo +/- mice produce mRNA and protein in equal amounts, these data do not necessarily imply that their fertilization capacities are equal. Readers would have been more easily convinced if wild type male and female mice were paired to determine their average litter size. These results would then be compared to those in Figure 3a.
Inoue et al. did not sufficiently satisfy my curiosity regarding Table 1. For example, why was it that Izumo -/- males fathered twice as many pups through ICSI as Izumo +/- males? Was this difference statistically significant? Could there be another protein involved whose expression was influenced by the mutant allele? Did the up regulation or down regulation of this unknown protein lead to increased probability of the embryo’s survival to birth? These are intriguing questions that could have been addressed in the discussion section.
In addition, with regards to figure 4, it is a mystery as to how it was possible to inhibit human Izumo in presumably non-acrosome-reacted human sperm with anti-human Izumo antibodies. As we learned from figures 1e and 1f, Izumo is only detectable on acrosome reacted sperm, as it most probably lies between the sperm’s plasma membrane and the outer acrosomal layer. Therefore we would expect that anti-human Izumo antibodies (like mouse) would be unable to label Izumo due to the intact acrosome. It is possible that this fact was implied but never explicitly stated.
Lastly, the authors underemphasized the importance of their rescue experiment. This experiment is critical because it demonstrates that the mutation is reversible for subsequent generations. In other words, this disruption of the genome does not seem to affect other areas of the genome yielding similar results to figure 2d. The effectiveness of their rescue experiments would have been demonstrated through a comparison of fertility rates of wild type and rescued mice.
Overall, the authors of this publication support their claims very effectively through figures and discussion. Explanation in some areas would have clarified the readers’ doubts and satisfied their curiosities. With regards to experimental design, they did an excellent job of asking hard questions and testing them in order to rule out the possibility that other events may have accounted for their results.
There is no doubt that the discovery of Izumo, a fusion-related protein on the sperm cell membrane, will have a significant impact in the field of reproductive technologies, especially in relation to human contraceptives. This sperm-surface protein is the first of its kind to be identified and therefore lends itself to significant analysis of function and physiology in the future.
It is first critical to identify other proteins that interact with Izumo that may contribute to the sperm-egg fusion system. As of yet, scientists have only identified a minority of proteins involved in sperm-egg interactions, some which include ADAM2, CD147, and sp56. Now, using experimental techniques such as the yeast 2 hybrid system, scientists can isolate and classify proteins that may interact with Izumo to aid in the fusion/fertilization process. It then would be beneficial to perform Northern and Western blot analyses of these novel proteins in Izumo knockout mice to verify that their expression levels are unaltered by the mutated alleles. These novel proteins could then be knocked out via homologous recombination in mice to examine their function and determine their importance in fertilization.
The identification of essential sperm fusion proteins could influence the development of diagnostic tests to determine the source of infertility in males. Currently, semen analysis focuses on sperm volume, density, motility, and morphology, however with these data and innovative technology, it is possible to determine the source of male infertility on a molecular level. For example, Inoue et al. demonstrate in this paper that it is possible to determine a sperm’s ability to undergo acrosomal reaction and fuse with the egg through the use of CD4 and Hoechst 33342 antibodies, respectively. Now, with the discovery of Izumo, it may be possible to use fluorescently labeled anti-Izumo antibodies to confirm the presence of normal Izumo in acrosomally reacted sperm. Similar diagnostic tests could be created using newly identified proteins from the yeast 2-hybrid system.
In the future, we may use these data to create male contraceptives, whether temporary or permanent. For example, when scientists perfect RNAi technology, it may be possible to inject double stranded RNA (using Izumo mRNA isolated from the testes) into a male’s testis. The injection of ds Izumo RNA would presumably result in decreased Izumo production and gene inhibition, leading to male infertility. This process would most likely cause permanent infertility, however, because RNAi interference effects are known to persist into the next generation.
The development of temporary contraceptive devices might also be possible. Recall that the heterologous sperm-egg fusion systems demonstrated that the function of normal human Izumo could be inhibited by the presence of anti-human Izumo antibodies. Given these data, new monoclonal antibodies could be created, for example using novel fusion proteins (specifically those that don’t require removal of the acrosome or sperm manipulation). These antibodies could somehow be introduced, possibly into the semen, to inhibit fertilization through fusion inhibition, although this technology will likely not be available in the near future.
Lastly, a number of future experiments could be designed to address some of the questions explored in the discussion section. They begin by reporting that other sperm surface proteins candidates have been identified. For example, they suggest that CD9 on the egg’s plasma membrane may interact with Izumo or other fusion related proteins. Immunoprecipitation experiments could be performed using antibodies against CD9 or Izumo, for example, to indicate what proteins may interact with them. They also noted in the discussion section that after the acrosome reaction, Izumo is no longer localized to the equatorial segment where fusion first took place. These findings may indicate that Izumo performs multiple functions in the fertilization process. For example, after aiding in the fusion process, Izumo may contribute to pronucleus formation or another event in fertilization. This may explain their findings that Izumo -/- sperm failed to initiate pronucleus formation. In order to track the movement of Izumo following fusion, an expression vector, for example GFP, could be inserted into the genome between the Izumo promoter and gene so that the protein’s location can be monitored at varying stages during fertilization.
Only time will tell how the findings of Inoue et al. will influence our understanding of cell adhesion proteins- especially those involved in fertilization- and, ultimately, the development of male contraceptive methods. I predict that Izumo itself will not be the target of these methods, however, because of its proximity between the sperm’s plasma membrane and the outer acrosomal layer. This location makes it difficult to target externally, as would be necessary for impermanent contraceptive methods. In fact, other proteins are currently being studied whose proximities make them easier contraceptive targets than Izumo. For example, for the past 10 years, scientists have been working to develop a contraceptive that inhibits P34H, a protein located on the acrosomal cap of mature sperm that appears to play a role in zona pellucida binding (Boue et al., 1994). In addition, the recent creation of male ‘birth control’ (currently in clinical trials) will likely be a cheaper and more effective contraceptive method than one that would directly inhibit proteins on a molecular level.
Boue, F., Berube, B., DeLamirande, E., Gagnon, C., and R. Sullivan. 1994. “Human Sperm-Zona Pellucida Interaction Is Inhibited by an Antiserum against a Hamster Sperm Protein.” Biology of Reproduction. 51: 577-587.
Inoue, N., Ikawa, M., Isotani, A., and M. Okabe. 2005. “The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs.” Nature. 34(7030):234-8.
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