*This website was produced as an assignment for an undergratuate course at Davidson College.*
Review of a Molecular Biology Paper
This purpose of this website is to present a thorough analysis of the molecular biology paper "The Immunoglobulin Superfamily Protein Izumo is Required for Sperm to Fuse With Eggs" that appeared in the magazine "Nature" in September of 2004. Although this paper does not have any relevance to CD4 or any of my previous websites, the style of the analysis presented in this website is critcal for the understanding of scholarly molecular biology papers.
The Immunoglobulin Superfamily Protein Izumo is Required for Sperm to Fuse With Eggs
Noakazu Inoue, Masahito Ikawa. Ayako Isotani & Masaru Okabe
As seen in Nature, Vol.434, 234-238, 10 March 2005
Goal of the Paper:
Not to long ago, scientists discovered that a protein found on the surface of the egg called CD9, was a critical player in sperm to egg fusion. However before this paper, little was known about the sperm-related fusion factors. In this paper the authors suggest a new immunoglobulin superfamily protein, that they called "Izumo," as one sperm-related factor that is necessary for sperm to egg fusion. To begin, the authors used a fusion-inhibiting monoclonal antibody along with gene cloning, in order to find the sperm-related fusion factor that they called OBF13. In their experiments, the authors present that through the use of the monoclonal antibody OBF13, the inhibition of the Izumo protein halted the fusion between and egg and a sperm. According to the data, they explain that the sperm is alive and healthy, can actually pass through the zona pellucida, but cannot fuse with the egg. Through these experiments the authors try to prove that the Izumo protein is sufficient for allowing, or disallowing, sperm to egg fusion.
The Data:
Figure 1. This figure illustrates the amino-sequence of Izumo, along with its expression in sperm cells.
(a). The authors use this figure to compare the amino-acid sequences of the Izumo gene as it appears in humans and mice. The regions that are shown in red are the peptide sequences that the scientist obtained by LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) described previously in the paper. The asterisks that appear below the amino-acid sequences are used to illustrate that these segments are identical in both human and mouse. The transmembrane region is shown in blue, while the putative signal region is shown in orange. The arrows are used to point out the cysteine residues that may form a disulphide bridge with one another.
(b). This figure is an illustration of the Izumo protein. In the illustration we can see the disulphide bond whose regions are mentioned in Figure 1. (a). The illustration shows that the Izumo gene is a type I membrane glycoprotein with one immunoglobulin-like domain. The immunoglobulin-like domain is illustrated as the extension off the main loop of the type I membrane glycoprotein. The authors state that there is also a putative N-glycoside link motif attached to the immunoglobulin-like domain. It is at this location where a sugar, or glucose, is attached to the nitrogen group of the amino acid. The basic function of this figure is to simply illustrate the structure of the Izumo protein.
(c). This figure is a Western blot that is used to determine what tissues the Izumo gene is expressed in. The authors detected the gene by using 1ug ml anti-mouse Izumo protein in a 30 ug protein solution for the 11 different tissues examined, and then blotted the results. The 11 tissues that the authors examined were the, brain, heart, liver, thymus, spleen, lung, muscle, kidney, ovary, testis, and sperm, and found that the Izumo gene is exclusively expressed in the male sex glands and sperm. This figure also illustrates that the mouse Izumo protein is approximately 56.2 kDa in size.
(d). This figure is also a Western blot but instead of the mouse Izumo protein, it illustrates the size of the human Izumo protein. This blot is used to show that Izumo is indeed expressed in humans as well as in mice. One interesting note about this figure is that the human Izumo is much smaller than that of the mouse, measuring about 37 kDa.
(e). This figure illustrates an immunostaining, or polyclonal antibody staining, from acrosin-promoter-driven mouse sperm cells that were engineered to express EGFP 30 (GFP) in their acrosome regions. Since the cells were engineered to express EGFP 30 in their intact acrosome regions, we can see that only the fresh sperm (indicated by the green arrows) express the green fluorescent property (the third slide particularly). When the Izumo protein reacts with an anti- Izumo antibody (as seen in the second slide) in the acrosome reacted sperm, the protein is seen expressed dyed red in the second slide (indicated by white arrows). Similarly the anti- Izumo monoclonal antibodies do not bind to anything on the sperm that has not undergone acrosomal reactions. This illustrates that the Izumo protein is only expressed on the surface, after the sperm cell has undergone an acrosomal reaction.
(f). This figure is very similar to Figure 1. (e) with a few exceptions. In this figure, instead of mouse sperm, human sperm is used. Another difference is that instead of engineering the sperm with an intact acrosome to express EGFP 30, the authors engineered the acrosome-reacted sperm to express the fluorescence (this time anti-CD46). This figure illustrates that, as in the mouse, the Izumo gene is only expressed in post acrosome-reacted sperm.
Figure 2. This figure illustrates the mutant Izumo allele and how it is expressed (or not expressed) in mice.
(a). This figure illustrates the complete structure of the wild type mouse Izumo allele as well as the targeted vector along with the targeted allele. The horizontal bars representi the introns within the allele while the exons are represented by vertical bars. Through the process of homologous recombination, the scientists created the mutant allele by replacing exons 2-10 and the inserting neomycin resistance gene (neo r) and the diphtheria toxin A chain labeled DT. This insertion creates an Izumo -deficient mutant mouse allele.
(b). This figure is a Southern blot used to illustrate the effectiveness of the engineered mutant allele in inhibiting the Izumo protein activity. Lane one contains the wild type allele (+/+), while the second and third contain the heterozygous mutant (+/-) and homozygous mutant (-/-), respectively. The lanes show that the wild type allele contains the wild type Izumo gene (15 kb when digested with EcoRI), while the heterozygous allele contains both the Izumo gene along with the recombinant gene (6 kb), while the homozygous mutant only contains the recombinant gene (6 kb). This shows that the homozygous mutant was effective in eliminating the expression of the Izumo gene.
(c). This figure is a Northern blot of the mouse RNA in the wild type, heterozygous, and homozygous Izumo mutant mouse. The authors use GAPDH as a positive control in this experiment. The data shows that both the wild type and the heterozygous mutant allele express the Izumo gene. However, the homozygous mutant does not transcribe Izumo RNA at all. This further illustrates that the mutant allele is able to knock out the expression of the Izumo gene.
(d). This figure is a Western blot created to make sure that the Izumo mutant allele did not effect the expression of other sperm proteins. In this figure we can see that the homozygous Izumo mutant allele only effects the expression of Izumo while it has no effect on CD147, sp56, or ADAM2. This figure continues the previous illustration that Izumo mutant mice are unable to produce the Izumo protein.
Figure 3. This figure illustrates how the mutant allele causes infertility in males and explains why these alleles cannot fertilize female eggs.
(a). This is a simple bar graph that illustrates the fecundity of wild type (+/+), heterozygous (+/-) Izumo mutant, and homozygous (-/-) Izumo mutant alleles in males and females. This diagram illustrates that heterozygous males and wild type females can produce offspring as well as heterozygous males and homozygous Izumo mutant females. However, homozygous mutant males are unable to reproduce presenting the idea that (-/-) male sperm cannot fuse with female eggs. Even thought (-/-) males appear to be infertile, (-/-) females are still able to produce offspring.
(b). This figure illustrates the reaction of (+/-) males and (-/-) during in vitro fertilization. In this graph we see that nearly 100% of the (+/-) male sperm participated in pronucleus formation while the (-/-) sperm were not able to perform this formation at all (0%). This diagram illustrates that these (-/-) individuals cannot fertilize the egg.
(c). This is an actual picture of the reactions tested in Figure 3. (b). We can see that the sperm with the (+/-) allele are able to fuse with the egg, while the (-/-) alleles did not penetrate the zona pellucida and are unable to promote pronucleus formation.
(d). This figure is an illustration of (-/-) mutant sperm that are trapped within the perivitellin space. This upper figure shows that the (-/-) allele sperm are in fact able to penetrate the zona pellucida but are unable to break through the perivitellin envelope of the egg. The lower figure shows sperm that have been labeled with the acrosome-reacted sperm-specific monoclonal antibody MN9, to show that in fact the (-/-) Izumo sperm are present in the perivitellin space, but are still unable to fertilize the egg.
(e). This figure illustrates the number of sperm, whether (+/-) or (-/-) Izumo mutant, that bind to the egg over a 2 hour and 6 hour period. We see that a number of sperm with the (+/-) allele fused with the egg at 2 hours (4.25 sperm per egg) and 6 hours (about 6 sperm per egg). On the other hand, we also see that no sperm with the (-/-) mutant Izumo allele fused with the eggs.
(f). In this figure the scientists stained the sperm/eggs with Hoechst 33342; a compound that targets fused sperm heads. In this illustration we can see that in the top panel it appears that the (+/-) sperm do bind to the egg (shown by the white arrows) while there is no such fusion in the (-/-) Izumo mutant alleles.
Table 1. This table was created to show that the Izumo gene is actually responsible for the fusion of the sperm to the egg. In this experiment the scientists use a method called intracytoplasmic sperm injection (ICSI) in which they injected the (-/-) Izumo mutant alleles directly into wild type eggs and then transplanted the eggs into "pregnant" females. In this experiment the researchers found that the sperm reacted normally and that the embryos developed similarly to those created from the (+/-) Izumo mutant alleles. The offspring's genotype was (-/-) suggesting that these individuals were sterile and would not pass on the mutation to future generations. This experiment further solidifies the idea that the Izumo protein is responsible for allowing/disallowing the sperm to fuse with the egg and that the Izumo (-/-) sperm are not dysfunctional if they do happen to cross the membrane.
Figure 4. This figure assesses the function of the Izumo in a xeno-species fusion system.
(a). This figure illustrates the ability (or lack there of) of both the heterozygous (-/+) Izumo mutant mouse sperm and the homozygous (-/-) mutant sperm to bind to zona-free hamster eggs. As before we see that the heterozygous mutant displays binding while the homologous (-/-) Izumo mutant does not display fusing activity. This further solidifies that the Izumo gene is essential for sperm/egg fusion.
(b). In this figure the scientist decide to observe the effects of the Izumo protein in human sperm. The procedure calling for the use of zona-free hamster cells was chosen because it is known that the zona pellucida cannot be penetrated through different species, yet human sperm can fuse with an egg that lacks this barrier. In the first picture we see that there is fusion of wild type human sperm (exposed to a control IgG) to the zona-free hamster egg as illustrated by the Hoechst 33342 staining. However in the lower photo, when anti-human Izumo was added, no fusion was observed. This data further supports the importance of the Izumo protein for the process of egg to sperm fusion.
Critique of the Paper:
Over all I feel that the authors did an adequate job of supporting their claim that the Izumo gene is critical for the fusion of a sperm cell to an egg. I feel that the authors took many different approaches in order to create a broad understanding of the gene in question.
Starting with Figure 1, I felt that the authors gave a good introduction to the Izumo gene and a good hypothesis of what the gene looks like and how it functions. However since the exact structure of the protein is not yet known, it would have been a good idea to explain that the cartoon is merely a prediction. Though this is true, their prediction is still feasible (through the usage of hydropathy graphs to determine the intramembrane properties, etc.). Secondly, it seems a little odd that a protein that is so similar in function between humans and mice has a 20 kDa difference in its protein size. On a more experimental level, I did not understand why the authors decided to use different methods when engineering their EGFP 30 sperm. For instance they used the EGFP 30 to mark sperm that did not undergo acrosomal reactions in (e) , yet they use a flourescent marker to do the exact opposite in (f) . Why wouldn't they just use the same procedure used in (f) as they did in (e) ? Never the less, by showing that the gene is only expressed in male gametes and sex organs, they were able to show that the Izumo is truly a gene that is specific to sexual reproduction in mammals. The authors also adequately demonstrated that the gene is only expressed after an acrosomal reaction.
In Figure 2, the authors clearly illustrated the Izumo allele as well as clearly explained how they planned to form the mutagenic allele. I felt that the only figure in this segment that was a little bit blurry was (b). Other than that the authors did a good job in proving information that supports that the mutant allele did not affect other vital sperm proteins in (d) .
I thought there was a little confusion in Figure 3 (c) in which it looks as if the sperm are not penetration the zona pellucida, yet in further experiments they explain that the sperm did in fact penetrate this layer. Furthermore, in figure (f) the authors state that the (-/-) mutant Izumo sperm do not fuse with the egg, yet there is a blue dot in the middle of the egg that looks very similar to the evidence they use in the figure above to prove that (+/-) Izumo mutant genes do bind. What is this dot? Perhaps an explanation of this may have been in order. Other than these few aspects, I feel that the graphs and other data do seem to support the author's suggestions.
In Figure 4 , I found the data to also be a little confusing. For example in part (a) in the bottom picture little white specks are seen but are not considered significant, while small white blurs in the picture above are. Secondly, much like my problem with Figure 3 (f) , there seems to be a white spot in the bottom right hand corner of the picture. Is this a possible fusion location? If so what caused it?
Other than these few details, I feel that the authors did a very good job in supporting their argument by using many different experimental approaches to proving proof that the Izumo protein is a significant player in sperm to egg fusion.
Future Experiments:
In the future I think it would be nice to crystallize the protein so that we can see the exact structure of the protein. If we have the structure we can begin to understand the exact mechanisms that cause the Izumo protein to bind to the egg cell. By studying the protein's structure we can further relate the protein's structure as it relates to its function. A more broad understanding of this protein may lead to finding that infertile men may have deficiencies in producing this Izumo protein. As discussed in class, phenotype recovery is a procedure that could be of use in this situation. By determining how large or small of a piece of the Izumo DNA is needed to recover the phenotype, we may be able to figure out how to recover the protein expression in males who do not express the gene. In theory, down the line we may be able make clinically safe versions of the Izumo protein that allow a person, who would normally be infertile, produce offspring.
Other than the structure of the protein, it would be nice to know what exactly the protein-protein interactions are between the Izumo protein and the proteins of the egg cell. The usage of the "yeast two-hybrid system" may give insight to these protein-protein interactions that occur between the two cells. To do this GAL4 activation domain plasmids would need to be constructed as well an Izumo reporter gene. This would consist of a promoter and a coding region. Through using this method and scanning the genomic libraries, we would hopefully further understand the protein-protein interactions of these two cells.
Similarly, it could be very interesting to see if the Izumo could be altered in a safe fashion in which it could be used as the first form of male birth control. If we can figure out the structure of protein and the active sites allow for sperm to egg binding, we could possibly develop a drug that alters the structure of the protein in a way that the active site becomes obsolete. Such an experiment would first involve the crystallization of the protein first so that we fully understand the structure. From there we could target certain areas within the protein with binding agents that may alter in the structure to suit our needs.
References:
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/misc/movies/Y2HS.mov > Accessed 28 April 2005
Comments, Questions, Concerns? Email me at oshernandez@davidson.edu.