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Review By: R. Cory Maluski
In this paper, the authors investigate possible sperm proteins that could be essential for fertilization. Due to recent discoveries that have revealed there are specific proteins located on the membrane of the egg that are essential for sperm and egg fusion, they wish to determine if there are any "sperm-related fusion factors." To identify the proteins involved with sperm-egg fusion, they introduced a monoclonal antibody, OBF13, that was known to prevent sperm-egg fusion, and then they extracted the protein to which it bound. The protein which they extracted was dubbed Izumo. Eventually, they discovered that this novel immunoglobulin superfamily protein was essential for the fusion of sperm and egg. In this review I intend to explain the results of the experiments performed by Inoue et al, critique some of their methods, and provide ideas for future experimentation regarding Izumo and sperm, egg fusion.
Figure 1 shows how the group identified and characterized the Izumo protein.
1A shows the amino acid sequences of both mouse and human Izumo, and it predicts the possible structure of the protein. It shows that the protein contains a possible transmembrane region and an immunoglobulin domain.
1B is a cartoon representation of what the protein is predicted to look like within the cellular membrane of the sperm.
1C is a simple western plot with 11 different mouse tissues samples being tested for the presence of mouse Izumo. According to the blot, which was labeled with an anti-mouse Izumo antibody, the Izumo protein is only present in the testis and sperm. The figure shows that the Izumo protein in the testis is 56.4 kDa in size.
1D is similar to figure 1C except it is a western blot of the human Izumo protein, and it only includes the data from human sperm cells, not from any other tissues. It shows that the human Izumo protein is smaller than the mouse Izumo protein, at approximately 37.2 kDa.
1E is a polyclonal antibody staining of mouse sperm cells. The sperm cells fluorescing green are those that still have intact acrosome structures; these cells were engineered to express EGFP 30 in their acrosome membranes. The sperm cells stained red are those that have already undergone the acromosomal reaction, and the anti-Izumo polyclonal antibodies have bound to the Izumo expressed in the newly exposed region of the sperm cells. This figure shows that the Izumo protein is only expressed on the exterior of the cell after the acromosomal reaction takes place.
1F is very similar to 1E, except this figure shows that the anti-Izumo antibody binds to human sperm cells only after the acromosomal reaction occurs. In this figure, however, the cells that fluoresce green are those which have undergone the acromosomal reaction, whereas in the previous figure, the green cells represented those which had not undergone the acromosomal reaction.
Figure 2 shows how the authors targeted the Izumo gene specifically to create an Izumo knockout, and it shows that they successfully created the knockout mouse.
2A shows a cartoon representation of the Izumo allele along with the targeting vector and the targeted allele during homologous recombination.
2B is a southern blot that confirms the homologous recombination process did in fact remove the Izumo gene. The blot shows that the wild type (+/+) contains only the normal Izumo gene which is 15 kb in size, while the heterozygous (+/-) animals contain both the normal Izumo gene and the recombinant gene of only 6.9 kb, and finally the knockout (-/-) for the Izumo gene contains only the recombinant gene.
2C is a northern blot of total testis RNA from the wild type, heterozygote, and knockout mice. The figure shows that the Izumo RNA is present in all mice except the knockout. GAPDH was used as a loading control in this experiment.
2D is a western blot analysis of the wild type, heterozygote, and knockout mice. It shows that Izumo was likely the only sperm protein affected by the knockout procedure. All proteins (Izumo, ADAM2, CD147, and sp56) were present in each of the tested genotypes, except for Izumo which was not resent in the knockout mouse.
Figure 3 shows the male infertility caused by the removal of the functioning Izumo gene; it also provides visual data explaining why the male Izumo knockouts are unable to fertilize eggs.
3A is a simple bar graph representing the litter size of that resulted from the mating of mice with different genotypes. It shows that the male Izumo knockout mice were unable to produce sperm that could fuse with eggs from any genotype of female mice. It also shows that female Izumo knockouts were still fertile.
3B is another bar graph that depicts the ability of the Izumo knockout sperm to cause pronucleus formation in the egg. The figure shows that sperm from Izumo knockout mice was not able to cause pronucleus formation in the egg. Nearly 100 percent of the eggs exposed to heterozygous sperm (+/-), on the other hand, underwent pronucleus formation.
3C is an actual image of the data represented in 3B, and it shows that the Izumo knockout sperm were not able to cause pronucleus formation due to their inability to penetrate the egg completely.
3D shows that the Izumo knockout sperm did in fact penetrate the zona pellucida, but they were unable to penetrate the perivitelline envelope, and thus they became stuck in the perivitelline space. The lower portion of the figure also reveals that the sperm within the perivitelline space had undergone the acromosomal reaction (the sperm were stained with MN9, an acrosome reacted sperm specific polyclonal antibody.
3E is yet another bar graph that shows the number of sperm that fused to eggs with respect to time. After four hours, there were approximately 4.25 heterozygous sperm bound to each egg, and no Izumo knockout sperm bound to any eggs. After six hours there were approximately 6 heterozygous sperm bound to each egg, whereas there were still no Izumo knockout sperm bound to any eggs.
3F is an actual image of the numerical data presented in 3E. It shows that while multiple heterozygous sperm have bound to an egg, no sperm from the Izumo knockout mice have bound to an egg.
Figure 4 shows the involvement of the Izumo protein in a xeno-species fusion system.
4A shows the ability of both heterozygous mouse sperm and Izumo knockout mouse sperm to fuse with zona-free hamster eggs. The data show that multiple heterozygous sperm were able to fuse with the hamster egg, whereas no Izumo knockout sperm were able to fuse with the hamster egg.
4B shows that Izumo is essential for sperm-egg fusion in human cells as well as in mouse cells. In this figure, some sperm were exposed to anti-Izumo antibodies, while others were exposed to IgG, a control substance. Those sperm that were exposed to anti-Izumo antibodies were unable to bind to zona-free hamster eggs, while those exposed to the control were able to bind to the zona-free hamster eggs.
Overall, I would say that the authors of this paper did do a good job organizing and interpreting their data; however, there are several minor errors (mainly in the figures of the paper) that create some skepticism that could have been easily avoided. Figure 1, for example, contains a few errors that are worth mentioning.
First, the authors claim to know the structure of the protein based off of sequence analysis. Since the crystalline structure of the protein has not yet been found, the true structure of the protein cannot be determined. The structure can, however, be predicted. The authors should have clarified that the structure represented in figure 1B was a predicted structure. Furthermore, the authors do not clarify which methods were used to make this prediction. However, one can assume that they used a hydropathy plot to determine the location of the transmembrane domain, and that they found the immunoglobulin region of the protein by comparing it to other known immunoglobulin proteins.
Next, they did not include a simple loading control in the western blot shown in figure 1C. If the lab had included a positive loading control in each lane, the readers could be sure that they did in fact load each lane properly. However, without the positive control, the readers cannot be certain that no errors were made. Regardless of whether all the lanes were loaded properly, however, the figure does show that the mouse Izumo protein in the testis is 56.4 kDa. In figure 1D, a western blot of human Izumo protein, only testis tissue is shown. It is not necessary to show other tissue types here, especially sine the testis are the area of interest, but it would have been nice to see some consistency between data sets.
And finally, in figures 1E and 1F, the authors create some confusion by having GFP labeled proteins located within the intact acrosomes of the sperm in figure 1E and by having green fluorescing antibodies point out the sperm that had undergone the acromosomal reaction in 1F.
Figure 3 also contains some slightly confusing images. All of the phase images are cluttered looking and the reader cannot tell exactly what is going on. For instance, in the introduction of the paper, the authors claim that the Izumo knockout mice produce sperm that can penetrate the zona pellucida, but cannot fuse with the egg. Figure 3C creates confusion about this claim because it clearly shows many Izumo knockout sperm on the exterior of the zona pellucida, while there are almost no heterozygous sperm on the exterior of the zona pellucida in the image directly above. The next image does clarify that there are in fact Izumo knockout sperm that have penetrated the zona pellucida by providing fluorescing images that clearly show the sperm within the zona, but the images provided in 3C could have been more clear.
Aside from the minor problems pointed out above, there are no other flaws that I could find, and I do believe that the authors sufficiently show that Izumo is a protein that is essential for sperm to fuse with an egg.
One future experiment that I would like to see performed would be determining if Izumo is a protein that binds with the egg cell, and if so, what exactly it is on the surface of the egg to which Izumo binds. There are numerous methods that could be used to complete this experiment, but I would personally choose to attempt to clone the Izumo gene, add some sort of marker to it, and introduce the purified Izumo protein into a solution containing zona pellucida-free eggs, giving the purified Izumo protein time to its binding site on the egg.
In order to perform this experiment, I would first isolate the Izumo gene. This could be done by a number of methods; however, I would likely cleave the mouse DNA with restriction enzymes until I located a section that contained the Izumo gene. This section of DNA would then be integrated into a plasmid so that the Izumo protein could be hyper-produced. After integrating Izumo into the plasmid, I would label it so that it could be easily seen later in the experiment. This could be done by adding a GFP sequence onto the end of the gene.
After cloning the gene, I would simply extract purified Izumo protein from the plasmids and add it to a solution containing zona pellucida-free hamster eggs. Zona pellucida-free eggs would be used because the Izumo protein by itself would likely not be able to penetrate the zona pellucida membrane, and hamster eggs would be used because they have been proven to be able to be fertilized by mouse sperm in previous xeno-species fusion experiments.
I would observe the interactions between the isolated Izumo protein and the egg cells at different time intervals to see if the protein binds to the eggs quickly, slowly, or not at all. I would predict that if Izumo is a protein that binds to the egg cell, the isolated form would likely be bound to the egg cells within six hours because in the experiment conducted by Inoue et al, the concentration of sperm bound to the eggs was the highest at six hours.
With any luck, this experiment would allow me to see what protein it is to which Izumo binds. However, it is certainly very likely that this experment could yeild no results at all. It is very likely that Izumo does not bind to anything on the surface of the egg cells. From the Inoue et al paper, we know that Izumo has a binding site on it which is essential for sperm-egg fusion; however, we do not know if this binding site binds to a protein within the membrane of the egg, a protein secreted by the egg, or even a protein secreted by the sperm cell. Perhaps it just aids some other protein in performing its job so that it can bind to the egg cell. If that is the case, I would expect to see no results at all.
Another experiment that I would be interested in seeing performed would be to determine if the discovery of Izumo could offer any aid in the search for a male contraceptive drug. The exact proceedure behind determining the answer to this question would be incredibly complex, but it would involve determining the exact structure of the Izumo protein via crystalization, determining the active site on the protein, and then finding a way to inhibit the active site.
The tricky part to all of this would be getting the substance that would inhibit the Izumo protein from performing its function to penetrate the acrosome of the sperm cells. Since the acrosome is not depleated until the sperm cell comes into contact with the egg, the Izumo protein would have to be shut down while still within the testies in order for the male contraceptive drug to function.
Inoue N, Ikawa M, Isotani A, and Okabe M. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434 , 234-238 (2005). (Click here to see a copy of the paper).