**this page was done as an assignment for Molecular Biology, an undergraduate course at Davidson College**
The Immunoglobulin Superfamily Protein Izumo is Required for Sperm to Fuse with Eggs
Naokazu Inoue, Masahito Ikawa, Ayako Isotani & Masaru Okabe
Nature 10 Mar 2005 Vol. 434, 234-238.
Before this paper, only a fusion factor on the surface of the egg cell had been indentified. The research in this article presents a new protein, Izumo, that resides in the sperm membrane. It is necessary for sperm-egg membrane fusion.
To isolate Izumo, the researchers used OBF13, an antibody that inhibits fusion. They performed an immunoblot on a gel of sperm DNA and named the antigen that bound OBF13, Izumo. The nucleotide sequence of Izumo was interpreted using liquid chromatography tandem mass spectrometry and ten peptides in a database matched. The sequence was confirmed by performing PCR followed by reverse transcription to testis RNA.
They first characterize the protein, show they have successfully produced a knockout strain, and then show the function of Izumo by characterizing the sperm of mice with no Izumo protein.
FIGURE 1 Basic Info About Izumo
(A) shows the amino acid conservation of Izumo between humans and mice. The sequence is highly conserved. The various functional portions of the sequence are labelled and shown as cartoon in (B).
(B) notes the integral membrane region, a glycosylation site, a disulphide bond, and the extracellular immunoglobulin domain.
(C) is a western blot. Brain, heart, thymus, spleen, lung, liver, muscle, kidney, ovary, testis and sperm tissues were all probed with anti- mouse Izumo. Although no loading control is shown, the legend states that 30 µg were loaded into each lane. Izumo protein was detected in sperm and testis tissues. The amount detected in sperm tissue was about four times the amount detected in testis tissue. The mouse Izumo protein is 56.4 kDa.
(D) is a western blot. Sperm tissue (only) was probed with anti-human Izumo. The human version of the protein is 37.2 kDa.
C & D - similar results were seen in mice and humans.
(E) is an immunostain. Izumo was not detected with anti-mouse Izumo in new sperm cells, which have acrosomes. Izumo was detected with anti-mouse Izumo in sperm that have undergone the acrosome reaction.
(F) is an immunostain. Izumo was not detected with anti-human Izumo is new sperm cells which have acrosomes. Izumo was detected with anti-human Izumo in sperm that have undergone the acrosome reaction.
E & F - the same results were seen in mice and humans.
FIGURE 2 Creating Izumo Negative Mice
(A) is a map that demonstrates how the gene for neomycin-resistance in the targeting vector replaces exons 2-10 in the wild type mouse Izumo allele by homologous recombination to form the mutant allele that lacks the Izumo gene. EcoR1 restriction sites are labeled.
(B) is a southern blot of the EcoR1 digested genomic DNA. The bands are not as clear as would be ideal. The wild type DNA is 15 kb (an EcoR1 site on each end). The mutant that lacks the Izumo gene is 6.9 kb long. A wild type mouse yeilded a 15 kb band. A heterozygous mouse yeilded a band of each size and the homozygous knockout mouse yeilded a 6.9 kb band. This figure demonstrates that the knockout strain has been created.
(C) is a northern blot of testis RNA. The loading control GAPDH shows equal amounts of RNA in each lane: wild type, heterozygous and homozygous knockout. Izumo RNA is detected in both the wild type and the heterozygote. The Izumo RNA is not detected in the mouse with no Izumo gene. Therefore, Izumo is not transcribed in the knockout mice.
(D) is a western blot. ADAM2, CD147 and sp56 are sperm proteins and all are detected in wild type sperm (Izumo +/+), sperm with one allele knocked out(Izumo+/-) and homozygous knockout sperm (Izumo -/-). the Izumo protein is detected in the wild type sperm and in the heterozygote sperm but not in the sperm of the mouse who lacks both copies of the Izumo gene. Therefore, Izumo is not translated in the knockout mice.
FIGURE 3 Are Male Izumo Knockout Mice (Izumo -/-) Infertile?
(A) - Males with one copy of the Izumo gene (Izumo +/-) are fertile; when mated with wild type females, a litter of 8 was produced. Females that lack both copies of the Izumo gene are fertile; when mated with males heterozygous for Izumo, a litter of seven was produced. Males with no Izumo protein (Izumo-/-), however, were infertile; when mated with wild type females, no offspring were produced.
(B) - Fertilization was attempted by Izumo-/- males. Of 252 eggs, no pronucleus formation was observed which is a sign that no fertilization occured.
(C) depicts the numerous Izumo-/- sperm that bind to the zona pellucida . There is no sperm-egg membrane fusion (no fertilization), consequently there is no zona reaction that prevents more sperm from binding. In contrast, Izumo+/- sperm do fertilize the egg and so a zona reaction prevents several sperm from binding. The Izumo+/- sperm show about a quarter of the amount of bindng compared to the Izumo-/- sperm.
(D) - After penetrating the zona pellucida, Izumo-/- sperm aggregated in the perivitelline space . The Izumo-/- sperm have, however, undergone the acrosome reaction although they have not fused with the egg.
(E) shows that at the 2 and 6 hour mark, no Izumo-/- sperm fused with eggs while about 4.5 Izumo +/- sperm have fused with the egg after 2 hours, and an average of 6 sperm per egg after 6 hours.
(F) - The Izumo-/- and Izumo+/- sperm were stained with Hoechst 33342 to show fused sperm. None were detected in the eggs mated with Izumo-/- sperm and 3 were detected in eggs mated with Izumo+/- sperm.
TABLE 1 Does Izumo affect development?
Using ICSI, Izumo-/- and Izumo +/- sperm was injected directly into the egg's cytoplasm (the fusion step is skipped). The eggs were put into females and of 95 eggs injected with Izumo-/- sperm, 59 survived ICSI and 12 pups were born. Of 85 eggs injected with Izumo+/- sperm, 54 survived ICSI and 6 pups were born. Clearly, Izumo does not affect the development of mice after fertilization occurs.
FIGURE 4 Is Izumo required for interspecies fertilization?
(A) - The zona-free hamster egg sperm penetration test is used to determine the fertility of human sperm. The same test was performed with Izumo+/- sperm and Izumo-/- sperm. The sperm was stained with Hoechst 33342 and no Izumo-/- sperm were detected in the hamster eggs; 3 Izumo +/- sperm were detected.
(B) - Human sperm plus anti-Izumo showed no fusion. When positive control IgG was used, human sperm fused with the hamster egg.
PAPER CRITIQUE: WAS I CONVINCED?
By conducting several experiments, they have cleary demonstrated that Izumo-/- males are infertile while Izumo+/- males are not. The only difference between these two males is one copy of the Izumo gene. Half of the dosage shows a normal phenotype, while missing both copies shows drastic differences. I am conviced that the Izumo gene is the variable causing any detected differences.
First they showed that when Izumo-/- are mated with wild type females, no young are produced.
Secondly, that Izumo-/- sperm prevent a zona reaction while Izumo+/- do not. No zona reaction means fusion has not occured.
Thirdly, that Izumo -/- sperm do not fuse with the egg, while Izumo+/- sperm do fuse.
Lastly, that Izumo-/- sperm do not fuse with zona-free hamster eggs and Izumo+/- sperm do fuse.
This paper is great because these researchers ask a question, and then answer it.
First they wonder if Izumo interferes with proper development, maybe it is not specific to interfering with sperm-egg fusion. By surpassing the fusion stage using ICSI, they find that embryos from Izumo-/- mice develop similarly to Izumo+/- mice. It would have been nice had the authors also included the development of embryos injected with wildtype sperm. The results could be compared to those of the Izumo+/- and -/- injected embryos to see if there is a difference in the number of eggs that develop to the two-cell stage. It could be determined if in development the number of copies of the Izumo allele, one or two, have any affect.
Next Inoue and his team ask if Izumo+/- and -/- show differences in fusion ability with another species: hamsters. Again the ability of Izumo+/- to fuse and the failure of Izumo-/- to fuse is demonstrated.
In figure 1d, the authors only show one lane. Different tissue samples should be shown from humans to show that it actaully is only in the sperm cells. In 1c and d, no loading controls are shown, although they do state the amount piptetted, a lane could have been missed. This possibility can be ruled out with the use of a loading controls. The connections to humans throughout the paper are helpful. .
SUGGESTIONS FOR FUTURE EXPERIMENTS
The researchers did a great job answering their own questions, however follow-up experiements could determine exactly what part of the Izumo protein is necessary for fusion. This would help to define the exact area of fusion on the smallest scale. Presumably the integral membrane portion is necessary so that the protein is in fact embedded in the sperm membrane. The target would be the extracellular portion to find out exactly what area is necessary for egg membrane fusion.
To do this, I would engineer a homologous recombination and instead of replacing exons 2-10 (as was done in this paper), I would create a variety of partially altered alleles. First I would start by replacing exons one by one. If all products were functional, I'd move to the next level and knock out exons by twos.
I would demonstrate that our knockouts were successfully created by running a gel of the altered protein and observing the different sizes. So a protein missing exons 1 and 2 would be smaller than a protein missing only exon 1.
It would also be necessary to show that nonfuncitonal products were not a result of folding that has been affected. I could not just knock out areas and if the sperm is nonfunctional, attribute it to the necessity of that portion. In that case, it could just be that the topolgy has been affected and misfolding has lead to malfunction. To work around this, the exons would not be simply removed, rather, chimeric proteins would be created. The homologous region of exon 1 that has the same folding pattern as the predicted topolgy of Izumo would be replaced with exon 1 from a different species that does not fuse with mouse eggs. In this way, the folding of the protein would remain the same as wild type, so that variable is controlled for, and the only difference would be the fusion ability of the various sections.
The comparison would be between Izumo+/+, the wild type sperm, and Izumo-/altered. If the altered version of the gene is still functional, functional tests such as observing if few sperm vs many sperm bind the zona pellucida will have the same results as wild type. When functional tests of Izumo-/altered have results like those of Izumo-/- then the exons necessary for function have been targeted.
To support conclusions, immunoprecipitations could also be performed. Although this could not be the method to make prelimiary conclusions, as the data would be based on negative results, it would help support various findings about the different portions of the allele. If it is found that without exon 2, Izumo is non functional, Immunoprecipitation could help confirm this. With exon 2 missing, if anti Izumo pulled out just Izumo then the results are supported, however, if other proteins are pulled out with Izumo then it would seem that it was still fusing to a protein. Again, this should not be the primary test to make conclusions, however it could be used for some level of confirmation.
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**this page was done as an assignment for Molecular Biology, an undergraduate course at Davidson College**