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Abstract Representing the 60 trillion cells that build a humanbody, a sperm and an egg meet, recognize each other, and fuse toform a new generation of life. The factors involved in thisimportant membrane fusion event, fertilization, have been soughtfor a long time. Recently, CD9 on the egg membrane was found to beessential for fusion [1], but sperm-related fusion factors remainunknown. Here, by using a fusion-inhibiting monoclonal antibody [2]and gene cloning, we identify a mouse sperm fusion related antigenand show that the antigen is a novel immunoglobulin superfamilyprotein. We have termed the gene Izumo and produced a genedisruptedmouse line. Izumo −/− mice were healthy but males were sterile.They produced normal-looking sperm that bound to and penetrated thezona pellucida but were incapable of fusing with eggs. Human spermalso contain Izumo and addition of the antibody against human Izumoleft the sperm unable to fuse with zonafree hamster eggs.Identification of Izumo To identify factors involved in sperm–eggfusion, we used a monoclonal antibody, OBF13, against mouse spermthat specifically inhibits the fusion process [2]. The antigen wasidentified by separation of the crude extracts from mouse sperm bytwo-dimensional gel electrophoresis and subsequent immunoblottingwith the monoclonal antibody. We named the antigen ‘Izumo’ after aJapanese shrine dedicated to marriage. The identified spot wasanalyzed by liquid chromatography tandem mass spectrometry(LC–MS/MS), and ten peptides that were 100% identical to a part ofthe sequence listed in the RIKEN full-length database were found.The registered DNA sequence was confirmed by sequencing afterpolymerase chain reaction with reverse transcription (RT–PCR) withtotal RNA prepared from the testis. A human homologue was found asan unverified gene in the NCBI database. The gene encodes a novelimmunoglobulin superfamily (IgSF), type I membrane protein with anextracellular immunoglobulin-like domain that contains one putativeglycosylation site (Fig. 1a). Mouse Izumo was shown to be a testis(sperm)-specific 56.4-kDa antigen by western blotting with apolyclonal antibody raised against recombinant mouse Izumo (Fig.1b). Izumo was also detectable as a 37.2-kDa protein by westernblotting of human sperm with anti-human Izumo antibody (Fig. 1c).Izumo was not detectable on the surface of fresh sperm. Coincidingwith the fact that mammalian sperm are incapable of fertilizingeggs when ejaculated and that fertilization occurs only after anexocytotic process called the acrosome reaction, both mouse andhuman Izumo became detectable on sperm surface only after theacrosome reaction (Fig. 1d, e). This would probably be becauseIzumo is not localized on plasma membrane of fresh spermatozoa butis hidden under plasma membrane and accessible after the acrosomereaction, as occurs with CD46 on mouse sperm [3]. Figure 1Identification and characterization of Izumo. a, Izumo is a typicaltype I membrane glycoprotein with one immunoglobulin-like domainand a putative N-glycoside link motif (Asn 204). b, Izumo wasdetected exclusively in testis and sperm by western blotting. Thetissues examined are, from left to right: brain, heart, thymus,spleen, lung, liver, muscle, kidney, ovary, testis and sperm. Thearrowhead indicates mouse Izumo protein. c, Western blottinganalysis of human Izumo protein from human sperm. The arrowindicates human Izumo protein. d, Immunostaining of Izumo in spermfrom an acrosin-promoter-driven transgenic mouse line that hasenhanced green fluorescent protein in the acrosome. Izumo was notdetected in fresh sperm with intact acrosomes expressing EGFP(indicated by green arrows), but was revealed on acrosome-reacted(non-green fluorescent) sperm (stained red, shown by whitearrowheads), when stained with the polyclonal antibody againstmouse Izumo. e, Human sperm were also stained with polyclonalanti-human Izumo antibody (red). Acrosome-reacted human sperm(stained green with anti-CD46 antibody) were reactive to theantibody against human Izumo but the same antibody did not react toacrosome-intact (CD46-negative) sperm. Scale bar, 10 mm. 40 ANNUALREPORT OF OSAKA UNIVERSITY—Academic Achievement—2004-2005Establishment of Izumo-deficient mice To address the physiologicalrole of Izumo in vivo we generated Izumo-deficient mice byhomologous recombination. An Izumo targeting construct was designedto replace exons 2–10 with a neomycin-resistant gene (neor ). Boththe targeting event in D3 embryonic stem cells and the germlinetransmission of targeted genes were confirmed by Southern blotanalysis. In the homozygous mutant mice, the full-length messengerRNA and the Izumo protein were not detected. Because the disruptionof a gene can cause a concomitant increase or decrease in somerelated genes, we examined CD46, sp56, CD55, CD147, and ADAM2,which were reported to be involved in sperm–egg interactions. Wecould not find a significant change in these protein levels insperm after the deletion of Izumo gene. The fecundity ofIzumo-deficient males Izumo −/−mutant mice were healthy and showedno overt developmental abnormalities. Izumo -/- femalesdemonstrated normal fecundity. Izumo +/− males also showed normalfertilizing ability. However, Izumo −/− males were sterile despitenormal mating behaviour and ejaculation, with normal vaginal plugformations. After observation of 28 plugs, nine pairs of Izumo −/−male and wild-type females were kept for another 4 months but nopregnancies were observed. In at least four different cases of geneknockouts that resulted in male sterility attributed to impairedzonabinding ability, the sperm also failed to migrate into theoviduct. However, disruption of Izumo did not cause any defect insperm migration into the oviduct (data not shown, and there was noreduction of sperm motility in Izumo −/− sperm motility wasmeasured 120 min after incubation by computer-aided sperm analysis(CASA; mean; s.e.m.=81.7±7.7% in Izumo +/− sperm and 77±8.9% inIzumo −/− sperm)). The sterile nature of Izumo −/− sperm was shownin the in vitro fertilization system (Fig. 2a). The impairedfertilization step undoubtedly followed zona penetration becausesperm penetrated the zona pellucida and accumulated in theperivitelline space of the eggs (Fig. 2b). Fusion ability inIzumo-deficient sperm Syngamy can be considered to occur to twostages: binding of the sperm plasma membrane to that of the egg,and actual membrane fusion. Izumo −/− sperm were capable of bindingto the plasma membranes of eggs whose zona pellucida had beenmechanically removed [4] (Fig. 2c). In this system, the Izumo +/−sperm incubated for 2 and 6 h fused to eggs in approximate ratiosof 4.5 and 6 sperm per egg, respectively, but no Izumo −/− spermfused with eggs (Fig. 2c). Sperm can not fuse with eggs unless theformer have undergone the acrosome reaction. To verify theacrosomal status of Izumo −/− sperm, we stained the spermaccumulated in perivitelline spaces with the MN9 monoclonalantibody, which immunoreacts only to the equatorial segment ofacrosomereacted sperm [5]. The staining indicated that the Izumo−/− sperm had undergone the acrosome reaction (Fig. 2b) but failedto fuse with eggs. Development of eggs after intracytoplasmic sperminjection (ICSI) with Izumo-deficient sperm Because no offspringwere fathered by Izumo −/− male mice, it was unclear whether thedefect was limited to fusion or extended to later developmentalstages. To address this question, we used ICSI to insert Izumo −/−sperm directly into the cytoplasm of wild-type eggs and bypass thefusion step. Eggs injected with Izumo −/− sperm were successfullyactivated and the fertilized eggs were transplanted into theoviducts of pseudopregnant females. The eggs implanted normally andthe resulting embryos developed appropriately to term with ratessimilar to those of heterozygous mice. Human Izumo is also involvedin sperm-egg fusion Sperm–egg fusion is known to be lessspecies-specific than sperm–zona interaction. For example, humansperm can not penetrate the hamster zona pellucida but they canfuse with zona-free hamster eggs, and this system (zona-freehamster-egg sperm penetration test) has been used for theassessment of human sperm fertility. We first examined thecontribution of mouse Izumo in a zona-free hamster-egg spermpenetration assay. As indicated in Fig. 3a, the mouse Izumo wasessential not only in the homologous fusion system but also forheterologous fusion with hamster eggs. Similarly, when theanti-human Izumo polyclonal antibody was added to the incubationmixture, no fusion was observed, whereas the sperm treated withcontrol IgG fused with eggs at an average of 5.9±0.7 sperm per egg.The total numbers of eggs observed were 23 and 29, respectively(n=3). These results indicated that human Izumo is involved in thefertilization process in human sperm (Fig. 3b). Rescued fertilityof Izumo-deficient male by transgene The phenotypes of geneknockout mice are not always related Figure 2 Male infertilitycaused by Izumo disruption. a, In vitro fertilization of sperm fromIzumo +/−and Izumo −/−mice. Unlike Izumo +/−, the eggs inseminatedwith Izumo −/− sperm had many sperm on their zona pellucida, owingto the failure of sperm–egg fusion that probably leads to theabsence of zona-reaction to lessen the sperm-binding ability of thezona pellucida. b,Upper panel, accumulation of many sperm in theperivitelline space of the eggs recovered from the females matedwith Izumo −/− males. Lower panel, sperm in perivitelline spacelabelled with acrosome reacted, spermspecific monoclonal antibodyMN9. c, Fused sperm stained by Hoechst 33342 preloaded into theegg. The arrowheads show the fused sperm. to the disrupted genesbut are sometimes caused by disruption of a neighbouring gene. Toexamine whether the phenotype was directly derived from the lack ofIzumo on sperm, we performed a rescue experiment by crossing Izumo−/− mice with transgenic mouse lines generated to express Izumo byusing the testis-specific calmegin promoter [6]. The sterilephenotype was rescued with the transgenically expressed Izumo onmouse sperm (Fig. 4). Discussion In the search for sperm surfaceproteins that function in sperm–egg plasma-membrane binding andfusion, various candidates such as DE, CD46, equatorin, Sperad andSAMP32 have been reported. ADAM family proteins are given the mostattention for their possession of a putative fusion peptide (ADAM1)and disintegrin domain (ADAM2 and ADAM3). None of the micepossessing disrupted ADAM1a, ADAM2 and ADAM3 show a significantdefect in the ability to fuse with eggs [7-9], but do show animpairment of sperm–zona binding ability. Similarly, CD46disruption does not diminish fusion [3]. In contrast, CD9 on theegg surface is essential for the fusing ability of eggs [1] andsome indications for the involvement of the binding of integrins toCD9 are postulated in reference to sperm–egg fusion. However, thedisruptions of the most probable candidate integrin α6β1 cause nomajor influence on the fusing ability of eggs. Thus, for severalyears, postulated fertilization mechanisms were repeatedly changedas a result of gene disruption experiments. This suggests that theessential nature of the candidate gene must be judged afterobserving the phenotype of the gene-disrupted mice. In thiscontext, Izumo is the first sperm membrane protein shown to beessential for fusion. It is not yet known whether sperm Izumointeracts with egg CD9, as occurs with placental IgSF proteinPSG17; neither do we know why the localization of Izumo afteracrosome reaction is not limited to the 41 Osaka University 100Papers : 10 Selected Papers ANNUAL REPORT OF OSAKAUNIVERSITY—Academic Achievement—2004-2005 Figure 4 Transgene toexpress mouse Izumo under the control of calmegin promoter. a, Thelocations of primers A to E were indicated in this figure. b, lane1; Izumo +/− mouse with intrinsic Izumo, lane 2 and 3; Izumo −/−mouse with transgenically expressed Izumo and Izumo His-tag,respectively. c, Litter size obtained by mating male mice withC57BL/6 wild-type mice. The group numbers are equal to those shownin b. The numbers in parentheses indicate the numbers of matings.References 1. Miyado, K. et al., Requirement of CD9 on the eggplasma membrane for fertilization. Science, 287, 321-4 (2000). 2.Okabe, M. et al., Capacitation-related changes in antigendistribution on mouse sperm heads and its relation to fertilizationrate in vitro. J Reprod Immunol, 11, 91-100 (1987). 3. Inoue, N. etal., Disruption of mouse CD46 causes an accelerated spontaneousacrosome reaction in sperm. Mol Cell Biol, 23, 2614-22 (2003). 4.Yamagata, K. et al., Sperm from the calmegin-deficient mouse havenormal abilities for binding and fusion to the egg plasma membrane.Dev Biol, 250, 348-57 (2002). 5. Manandhar, G. & Toshimori, K.,Exposure of sperm head equatorin after acrosome reaction and itsfate after fertilization in mice. Biol Reprod, 65, 1425-36 (2001).6. Ikawa, M. et al., Calmegin is required for fertilin alpha/betaheterodimerization and sperm fertility. Dev Biol, 240, 254-61(2001). 7. Cho, C. et al., Fertilization defects in sperm from micelacking fertilin beta. Science, 281, 1857-9 (1998). 8. Nishimura,H., Cho, C., Branciforte, D. R., Myles, D. G. & Primakoff, P.,Analysis of loss of adhesive function in sperm lacking cyritestinor fertilin beta. Dev Biol, 233, 204-13 (2001). 9. Nishimura, H.,Kim, E., Nakanishi, T. & Baba, T., Possible Function of theADAM1a/ADAM2 Fertilin Complex in the Appearance of ADAM3 on theSperm Surface. J Biol Chem, 279, 34957-62 (2004). equatorialsegment where fusion initially takes place. All we can say now isthat continued study of this protein’s function will undoubtedlylead to a fuller understanding of the cell–cell fusion process infertilization and perhaps in other somatic systems such as musclecells or trophoblasts. The finding not only provides insight intothe enigmatic fusion mechanism but also promises benefits in theclinical treatment of infertility and the potential development ofnew contraceptive strategies