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The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs

Naokazu Inoue, Masahito Ikawa, Ayako Isotani & Masaru Okabe

Nature Vol 434, 10 March 2005, p. 234-237

Introduction

Although sperm-egg fusion is a vital step in the process of fertilization, there is very little information on what specific factors and proteins are involved. It have been determined an egg membrane protein, CD9, is functionally necessary for sperm-egg fusion. Previously, no sperm proteins have been identified as having a specific function in sperm-egg fusion, but the authors of this study propose that they have isolated and identified the first sperm related fusion protein, which they call Izumo. The findings of this study are summarized below and appropriately support the authors' proposal.

Figure 1:

Figure 1a. Shows the amino acid sequences of the Izumo protein in mouse (upper) and human (lower). Shared residues between the two sequences is marked by an asterick. The sequences shown in red are those that were obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS). The putative signal peptide is shown in orange, the transmembrane region is shown in blue, and the immunoglobulin-like domain is boxed in green. The arrowheads show cysteine residues that may form a disulphide bridge in the protein structure. As shown by the astericks, there is significant conservation of amino acid residues between the mouse and human Izumo protein. However, there is a gap of amino acids in the human Izumo sequence (residues 289-316) compared with the mouse sequence. The human sequence is also a shorter polypetide, having 350 residues compared with 397 in the mouse sequence.

Figure 1b. This model shows the simple stucture of Izumo as a membrane glycoprotein. As shown, the protein seems to make a hairloop structure, and is connected by a disulphide bridge between two cysteine residues. The outter membrane of the sperm head is shown at the bottom of the model, as a phospholipid bilayer, and the boxed region within the membrane is the transmembrane domain, as labeled blue in the previous figure. As expected, most of the protein is on the outter part (extracellular) of the sperm, enabling it to interact with the egg. The black dot off of the ring is a putative N-glycoside link motif (Asn 204).

Figure 1c. This is a western blot that detects for the presence of Izumo in different mouse tissues, specifically brain, heart, thymus, spleen, lung, liver, muscle, kidney, ovary, testis and sperm. An anti-mouse Izumo antibody derived from the gene sequence was used to detect the presence of mouse Izumo protein in these tissues. The mouse Izumo protein was detected only in the sperm and testis (lanes 10 and 11) as shown by a dark band in each of the two lanes, and labeled by the black arrowhead as the Izumo protein. It also seems that there is a greater presence of Izumo protein in sperm than in testis (each lane loaded at 30 ug). The molecular weight of mouse Izumo protein is 56.4 kDa.

Figure 1d. This is also a western blot, but detected for the presence of human Izumo protein in human sperm. Human Izumo protein was detected in the sperm as shown by the dark band, and labeled by the arrow. The protein is approximately 37.2 kDa.

Figure 1e. This is an immunostaining comparing the presence of Izumo protein in fresh mouse sperm (acrosome reaction has not occurred) vs. acrosome reacted sperm. The acrosome in both types of sperm was enhanced with green florescent protein. The upper panel clearly shows both the fresh sperm (labeled by green arrows) and acrosome reacted sperm (labeled by white arrowheads). The middle panel shows sperm that were stained red with a polyclonal anti-mouse Izumo antibody. When compared with the upper panel, only the acrosome reacted sperm were labeled with red, and hence these sperm were detected to have the Izumo protein. The lower panel shows fresh sperm that are not acrosome-reacted, as marked by the expression of GFP, and when compared with the middle panel, the red sperm do not express GFP because they are acrosome reacted. This figure is trying to show that Izumo is detectable on sperm only after the acrosomal reaction has occured. Once the acrosomal reaction has occured, the Izumo protein is detected by the red-staining antibodies (middle panel). The authors note that the Izumo protein is not a sperm membrane surface protein, but rather lies underneath the plasma membrane, and hence suggest that it is accessible by antibodies only after the acrosomal reaction has occured. Thus, the anti-Izumo antibodies cannot detect the presence of Izumo in fresh sperm because they cannot penetrate the plasma membrane of the sperm. These suggestions also apply for Figure 1f.

Figure 1f. This is an immunostaining very similar to Figure 1e, comparing the presence of Izumo protein in fresh sperm vs. acrosome reacted sperm of humans. Again the upper panel clearly shows both the fresh sperm (not labeled) and acrosome reacted sperm (labeled by black arrowheads). The middle panel shows sperm that were stained red with a polyclonal anti-human Izumo antibody. When compared with the upper panel, only the acrosome reacted sperm were labeled with red, and hence these sperm were detected to have the Izumo protein. The lower panel shows sperm that were acrosome reacted as detected by a green stain with anti-CD46 antibody. When compared with the middle panel, the sperm that were detected having Izumo (labeled red) are the same sperm that are shown to be acrosome reacted in the lower panel (labeled green). This figure is also trying to show that Izumo is detectable on sperm only after the acrosomal reaction has occured, except this figure is analyzing human sperm instead of mouse sperm.

Figure 2:

Figure 2a. This figure shows how the Izumo gene was disrupted. The top line is the wild type Izumo allele, the middle line is the targeting vector, and the bottom line is the targeted alle. The wild type allele shows restriction sites marked by E's. The vertical bars are exons and the horizontal bars are introns. The white box is a phosphoglycerine kinase (PGK) promotor. The middle line shows all introns with a neomycin-resistance gene (marked neo-r) and a diphtheria toxin A chain driven by an MC1 promotor (marked DT) are both shown as white boxes. The two large X's between the top and middle lines represent homologous recombination taking place. The dotted lines that connect the top and middle line show how the two DNA fragments are aligned during homologous recombination, and hence all the exons of the wild type allele are replaced with junk DNA including the neomycin-resistance gene. A probe is also included on the 3' end of the Izumo gene, and conserved in the mutant allele so that both genes can be detected during Southern blotting. The final product is shown on the bottom line, which includes four restriction sites (for EcoRI), the PGK promotor, and the neomycin resistance gene. The bottom line also shows that the wild type allele would be 15.9 kb in length when digested with EcoRI, whereas the targeted allele would be only 6.9 kb in length when digested with EcoRI. This figure shows how the Izumo wild type allele is being disrupted, specifically by replacing all the exons with a targeting construct that includes the neomycin resistance gene. The neomycin resistance gene serves as a positive selection marker while the diphtheria toxin A chain serves as a negative selection marker to show that correct homologous recombination took place and the desired mutant allele was produced.

Figure 2b. This figure is a southern blot that shows the Izumo gene was disrupted through homologous recombination, and shows the genotype of wild type, heterozygous, and mutant alleles. The mutant allele and the wild type allele were digested with EcoRI and then hybridized with the same probe that binds to the 3' end. As shown by Figure 1a, when the wild type allele is digested with EcoRI, the probe will bind to a 15 kb piece, whereas when the mutant allele is digested with EcoRI, the probe will bind to a 6.9 kb piece. This southern blot shows that lane 1 is homozgous for the wild type, showing one dark band for a 15 kb piece, and no other band. Lane 2 is heterozygous, showing a 15 kb band (wild type) and a 6.9 kb band (mutant). Lane 3 is homozyous mutant, showing one band for a 6.9 kb piece, and no other band.

Figure 2c. This is a northern blot of total testis RNA from wild type, heterozygous, and homozygous mutant mice, and detected with an Izumo probe. There is a band showing in the wild type and heterozyous lanes, but no band in the homozygous mutant lane. This shows that Izumo mRNA is being transcribed in wild type and heterozygous mice testis, but not in homozygous mutant. The GAPDH blot is shown as a loading control, and because there are bands in all the lanes, we can assume that RNA was correctly loaded and detected.

Figure 2d. This is a western blot that shows the Izumo protein is detected in wild type and heterozygous mice, but not in homozgyous mutant. To be sure that the disrupted Izumo gene was only inhibiting the Izumo protein, other sperm proteins were tested as well in homozygous mutant mice, specifically ADAM2, CD147, and sp56. All three of these sperm proteins were detected in wild type, heterozygous, and homozygous mutant mice.

Figure 3:

Figure 3a. This graph shows the infertility of male mice is due to Izumo disruption. Heterozygous males mated with wild type females show successful mating and fertility. However, homozygous mutant males mated with wild type females were not able to produce any fertilization. As a control, heterozygous males were mated with homozygous mutant females to show that Izumo is necessary for proper sperm function, and that female eggs do not need to produce Izumo to be fertilized. Hence, the control mating had signifcant fertilization.

Figure 3b. This graph shows that eggs inseminated with heterozygous mice sperm were fertilized while eggs inseminated with homozygous mutant mice sperm were not fertilized. Fertilzation of eggs was measured by the formation of a pronucleus.

Figure 3c. These two pictures show an egg with heterozygous mouse sperm (upper) and an egg with homozygous mutant mouse sperm (lower). In both pictures, the clear ring around the egg is the zona pellucida. The egg in the upper photo was successfully fertilized by heterozygous sperm. Multiple sperm are shown binding to the surface of the zona pellucida in the lower photo, showing that homozygous mutant sperm did not successfully fertilize the egg.

Figure 3d. Both the upper and lower pictures are showing accumulation of homozygous mutant sperm in the perivitelline space of the egg (past zona pellucida barrier), but as shown in previous figures, are not able to fertilize the egg. In both pictures, the sperm are labeled by white arrowheads, and the sperm are stained red in the lower picture with an acrosome reacted, sperm specific monoclonal antibody MN9. This figure is trying to show that over time, homozygous mutant sperm are able to penetrate the zona pellucida of the egg, but are still not able to fertilize the egg. Hence, it can be suggested that Izumo is functionally necessary for sperm-egg fusion and not zona pellucida penetration.

Figure 3e. This graph shows the number of eggs that were fertilized by heterozygous and homozygous mutant sperm after 2 hours and after 6 hours. The zona pellucida of the eggs in this experiment were experimentally removed. Heterozygous sperm were still able to fertilize the eggs after 2 hours and 6 hours, but the homozygous mutant sperm were not able to fertilize an eggs.

Figure 3f. This figure shows that even in the absence of the zona pellucida, heterozygous sperm were able to bind and fuse with the egg membrane, fertilizing it, while homozygous mutant sperm were only able to bind to the egg membrane. The Hoechst 33342 dye was used to stain any DNA that penetrated the egg membrane. As shown by blue lights and labeled with white arrowheads in the top right picture, heterozygous sperm fused with the egg. In the lower left picture, several homozygous mutant sperm can be seen around the surface of the egg, showing that these sperm are still able to bind to the membrane, but are not able to fuse with and fertilize the egg. If the mutant sperm had fused with the egg membrane and fertilized the egg, then it's DNA should have been stained by the Hoechst 33342 dye in the lower right picture. There is a faint blue mark in the lower right picture, but the author does not mention it, and therefore it is assumed to be ignored.

Table 1.

It has been determined by previous figures that homozygous mutant sperm were not able to fertilize eggs, but it was not clear whether this was due to the fusion process, or extended to later developmental stages. To address this question, the authors experimentally implanted homozygous mutant sperm directly into the cytoplasm (intracytoplasmic sperm injection) of wild type eggs, bypassing the fusion process. These eggs were then transplanted into pseudopregant females. This table summarizes the results of that experiment, showing the number of eggs used, how many survived after sperm injection, the number of eggs that developed, and the number of actual pups that were born from the fertilized eggs. The results show that the eggs directly inseminated and fertilized by homozygous mutant sperm were able to develop successfully at very similar rates as eggs fertilized by wild type sperm (the table even shows more pups born from mutant sperm than wild type sperm). These results support the notion that Izumo is in fact necessary for sperm to successfully fuse with an egg to then fertilize it. The inability of mutant Izumo sperm to fertilize eggs is not because it lacks the functional ability to fertilize the egg, but it is unable to specifally fuse with the egg membrane.

Figure 4.

Figure 4a. It was previously known that sperm-egg fusion is less species specific than sperm-zona fusion. This figure shows hamster eggs that lack a zona pellucida being inseminated with heterozygous and homozygous mutant mouse sperm. Heterogzyous Izumo sperm were able to fertilize the egg as shown by the Hoechst 33342 stain (labels DNA) whereas the homozygous mutant Izumo sperm were not able to fertilize the egg. The heterozygous mouse sperm heads fertilizing the hamster egg is shown by white arrow heads. The figure shows that mouse Izumo is necessary for sperm-egg fusion even in heterologous fusion with hamster eggs.

Figure 4b. The upper panel of this figure shows zona free hamster egg being fertilized by human sperm incubated with control IgG. The successful fertilzation is shown by the strong blue spots which are swelling sperm heads stained with the Hoechst 33342 dye (labels DNA) as shown by white arrowheads. The lower panel shows a zona free hamster egg incubated with human sperm and anti-Izumo antibody. Fertilization of this egg did not occur, as shown by a lack of significant blue spots when labeled with the Hoechst 33342 dye.

Critique

The results of this paper give a lot of support that the authors have in fact found a novel immunoglobulin protein that is involved in sperm-egg fusion. The authors were able to find the novel gene using a monoclonal antibody, OBF13, against mouse sperm that specifically inhibits the fusion process, and then were able to use several methods (LC-MS/MS, RT-PCR, database comparisons) to clone and sequence the Izumo gene. The interesting thing was how they were able to find a human homologue of the Izumo gene in the NCBI database as an unverified gene. Hence, this sperm-fusion protein had already been found, but had not yet been identified as such.

The gene sequence was then used to make polyclonal antibodies against human and mice Izumo proteins. The authors believed that they had found a novel protein that was specifically involved in sperm-egg fusion, and using these anti-Izumo antibodies, the authors were able to identifiy the Izumo protein as the likely candidate. The immunostaining experiment was able to show that Izumo is probably an acrosome related protein (possible within the acrosome) of the sperm, and can be accessed only after the acrosomal reaction has occurred. In the Western blot, the authors were able to identify the Izumo protein as being located only in testis and sperm cells, and showed the molecular weight of the protein as 56.4 kDa in mice and 37.2 kDa in humans. The Southern blot showed that the authors were successfully able to disrupt the Izumo gene through homologous recombination. Because they confirmed that they had successfully created a Izumo mutant, they could then test the function of the Izumo protein. The Northern blot also shows that the gene was successfully disrupted as it shows that mutant Izumo mRNA is not being transcribed in the testis. Once the authors confirmed that they had created a disrupted Izumo gene, the real beauty of the study began to show.

Once the authors had successfully identified the Izumo protein as being in the right location for a sperm-egg fusion protein, and developed a mutant version of the protein, the next step was to specifically test the fuction of Izumo. In Figure 3, heterozygous Izumo was tested against mutant Izumo to compare the success of fertilization. It was interesting that the authors chose to use heterozygous Izumo instead of wild type, because in the Southern blot, there seemed be more of the mutant 6.9 kb piece than the wild type 15.9 piece in heterozygous sperm. In contrast to heterozygous Izumo, the sperm with mutant Izumo were not able to fertilize eggs, although they were able to penetrate the zona pellucida. Specifically, sperm with mutant Izumo had a zero success rate at fertilizing eggs. Because the mutant Izumo sperm were shown to still penetrate the zona pellucida, the zona pellucida was mechanically removed to determine if Izumo was specifically necessary for sperm-egg fusion. In this experiment (ZP removed), Izumo heterozygous sperm were able to fertilize the egg while mutant were still not able. This experiment supported that Izumo is specifically involved in sperm-egg fusion. To further support this proposal, mutant sperm were experimentally injected into the cytoplasm of the egg, bypassing the sperm-egg fusion process. These mutant Izumo sperm were able to fertilize the egg and a significant number of pups were successfully born. From these experiments, the authors were able to determine that the Izumo protein is located in beneath the sperm membrane, and is specifically necessary for sperm-egg fusion in order for fertilization to occur. Hence, the authors have shown that Izumo is a very likely candidate as a sperm-related fusion protein.

The final experiments tried to determine how species specific Izumo was in the sperm-fusion process. Heterozygous Izumo sperm were still able to fertilize eggs, while mutant sperm were not. However, compared with the positive control (IgG), human Izumo sperm were not able to fertilize eggs. The authors note that the anti-human Izumo antibody could have interfered with the process. The one problem I had with the experiment was that in the Hoechst stain of the mouse-hamster Izumo mutant sperm, there were several blue spots although the authors note that no fusion occurred. Although they do seem to be smaller spots than those were fusion did occur, there does seem to be an unusually large number of them in that particular figure panel.

Overall, I feel the authors have shown very credible and supporting results that the Izumo protein is in fact necessary for sperm-egg fusion to occur. As always, the best way to determine whether or not you have found the right protein is to show its function, and that is exactly what this study was able to do. Through homologous recombination and gene disruption, the authors have shown that a mutant version of the Izumo gene causes a loss of function in sperm-egg fusion.

Future Work

Although the authors have determined that it is probable that the Izumo protein is located underneat the sperm membrane, they are assuming this based upon data showing that Izumo is only detected after the acrosome reaction has occurred. It would be nice to see an immunostaining in which the anti-Izumo antibodies were allowed to penetrate the membrane of the sperm before acrosome reaction had occurred. One possible way to allow this is to use Streptolysin O to make holes in the outer membrane, allowing tagged Ab's to enter the cell. Another method would be to engineer GFP into the Izumo gene. By doing this, you could see where the Izumo protein is before and after acrosomal reaction occurred, and have a better idea of specifically where it is localized within the cell..

Although Izumo is the first specific sperm-egg fusion protein to be identified, it is probable that it is not the only one. What are the other proteins and factors that are directly involved in sperm-egg fusion? One way of doing this is to continue to do what the authors did to find Izumo--using monoclonal antibodies and gene cloning to isolate and identify more sperm-fusion related proteins. Once these genes are identified and sequenced, much more manipulation of the protein and protein markers can occur to determine the specific functions of these sperm-fusion proteins.

From this study, Izumo was found only in mice and human species. A good question to address is if Izumo is a conserved sperm-fusion protein in other species as well. Because this study showed that Izumo is necessary for mice sperm to fuse with hamster eggs, it is probable that hamster sperm also carry the Izumo protein. Common motifs or conserved sequences of the Izumo gene could potentially be used to design probes to find Izumo in several other species. Furthermore, the human Izumo gene was already in the NCBI databank but was unverified. There could still be other unverified Izumo proteins in the database, and running a blast search could be one way of verifying them, if there are any.

In this study, through gene disruption, the authors have shown that Izumo is functionally necessary for sperm-egg fusion. What they do not know, however, is if Izumo is specifically interacting with the CD9 egg membrane protein. Using a morphological approach, one method to test for this would be to create anti-idiotype antibodies for Izumo and CD9 and see if these proteins interact with their respective anti-ideotype antibodies. In this experiment, you would probably not even need the egg or the sperm, but once you have created your anti-ideotypes, you could then simply extract the Izumo and CD9 proteins and see how they react with the anti-ideotype antibodies (Western blot). A positiive result in such an experiment would give support that Izumo and CD9 directly interact during sperm-egg fusion.

Finally, as the authors note, the discovery of Izumo can certainly be beneficial to the medical field, helping doctors to treat infertility and develop new contraceptive strategies. However, continued research and study of Izumo localization and function is necessary before any of these medical developments can become a reality.

Works Consulted

Naokazu I, Masahito I, Ayako I, Masaru O. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature. 10 March 2005.

Campbell AM, Bernd K, Serie, J. Introductory Biology 111: Cell and Molecular Biology Study Guide. 1999. Davidson College.

Campbell AM. <http://bio.davidson.edu/courses/Molbio/alttails/SLOmethod.html> Accessed 27 April 2005.

Campbell AM. <http://bio.davidson.edu/courses/genomics/method/homolrecomb.html> Accessed 27 April 2005.

Davidson College Molecular Biology Homepage

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