Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Structure of IZUMO1–JUNO reveals sperm–oocyte recognition during mammalian fertilization


Fertilization is a fundamental process in sexual reproduction, creating a new individual through the combination of male and female gametes1,2,3,4. The IZUMO1 sperm membrane protein5 and its counterpart oocyte receptor JUNO6 have been identified as essential factors for sperm–oocyte interaction and fusion. However, the mechanism underlying their specific recognition remains poorly defined. Here, we show the crystal structures of human IZUMO1, JUNO and the IZUMO1–JUNO complex, establishing the structural basis for the IZUMO1–JUNO-mediated sperm–oocyte interaction. IZUMO1 exhibits an elongated rod-shaped structure comprised of a helical bundle IZUMO domain and an immunoglobulin-like domain that are each firmly anchored to an intervening β-hairpin region through conserved disulfide bonds. The central β-hairpin region of IZUMO1 provides the main platform for JUNO binding, while the surface located behind the putative JUNO ligand binding pocket is involved in IZUMO1 binding. Structure-based mutagenesis analysis confirms the biological importance of the IZUMO1–JUNO interaction. This structure provides a major step towards elucidating an essential phase of fertilization and it will contribute to the development of new therapeutic interventions for fertility, such as contraceptive agents.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: IZUMO1–JUNO interaction.
Figure 2: Structure of IZUMO1 and JUNO.
Figure 3: Structure of the IZUMO1–JUNO complex.
Figure 4: IZUMO1 containing interface mutations abolishes cell–oocyte binding.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure-factor data of human IZUMO1, JUNO (form 1 and form 2), IZUMO1-JUNO complex (form 1, form 2, and form3) have been deposited in the Protein Data Bank under the accession numbers 5JK9, 5JKA, 5JKB, 5JKC, 5JKD and 5JKE, respectively.


  1. Klinovska, K., Sebkova, N. & Dvorakova-Hortova, K. Sperm–egg fusion: a molecular enigma of mammalian reproduction. Int. J. Mol. Sci. 15, 10652–10668 (2014)

    Article  CAS  Google Scholar 

  2. Okabe, M. The cell biology of mammalian fertilization. Development 140, 4471–4479 (2013)

    Article  CAS  Google Scholar 

  3. Evans, J. P. Sperm–egg interaction. Annu. Rev. Physiol. 74, 477–502 (2012)

    Article  CAS  Google Scholar 

  4. Knobil, E. & Neill, J. D. The Physiology of Reproduction. 2nd edn, (Raven Press, 1994)

  5. Inoue, N., Ikawa, M., Isotani, A. & Okabe, M. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434, 234–238 (2005)

    Article  ADS  CAS  Google Scholar 

  6. Bianchi, E., Doe, B., Goulding, D. & Wright, G. J. Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature 508, 483–487 (2014)

    Article  ADS  CAS  Google Scholar 

  7. Ellerman, D. A. et al. Izumo is part of a multiprotein family whose members form large complexes on mammalian sperm. Mol. Reprod. Dev. 76, 1188–1199 (2009)

    Article  CAS  Google Scholar 

  8. Inoue, N. et al. Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion. Development 140, 3221–3229 (2013)

    Article  CAS  Google Scholar 

  9. Han, L. et al. Divergent evolution of vitamin B9 binding underlies Juno-mediated adhesion of mammalian gametes. Curr. Biol. 26, R100–R101 (2016)

    Article  CAS  Google Scholar 

  10. Wibowo, A. S. et al. Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition. Proc. Natl Acad. Sci. USA 110, 15180–15188 (2013)

    Article  ADS  CAS  Google Scholar 

  11. Chen, C. et al. Structural basis for molecular recognition of folic acid by folate receptors. Nature 500, 486–489 (2013)

    Article  ADS  CAS  Google Scholar 

  12. Monaco, H. L. Crystal structure of chicken riboflavin-binding protein. EMBO J. 16, 1475–1483 (1997)

    Article  CAS  Google Scholar 

  13. Aydin, H., Sultana, A., Li, S., Thavalingam, A. & Lee, J. E. molecular architecture of the human sperm Izumo1 and egg Juno fertilization complex. Nature (this issue)

  14. Bianchi, E. & Wright, G. J. Cross-species fertilization: the hamster egg receptor, Juno, binds the human sperm ligand, Izumo1. Phil. Trans. R. Soc. B 370 (2015)

  15. Inoue, N., Hagihara, Y., Wright, D., Suzuki, T. & Wada, I. Oocyte-triggered dimerization of sperm IZUMO1 promotes sperm–egg fusion in mice. Nat. Commun. 6, 8858 (2015)

    Article  ADS  CAS  Google Scholar 

  16. Inoue, N., Ikawa, M. & Okabe, M. Putative sperm fusion protein IZUMO and the role of N-glycosylation. Biochem. Biophys. Res. Commun. 377, 910–914 (2008)

    Article  CAS  Google Scholar 

  17. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  18. Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)

    Article  CAS  Google Scholar 

  19. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  PubMed  Google Scholar 

  20. Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protocols 3, 1171–1179 (2008)

    Article  CAS  Google Scholar 

  21. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  22. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  23. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–25 (2010)

    Article  CAS  Google Scholar 

  24. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  Google Scholar 

  25. DeLano, W. L. The PyMOL Molecular Graphics System. (2008)

  26. Dundas, J. et al. CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res. 34, W116–W118 (2006)

    Article  CAS  Google Scholar 

  27. Brautigam, C. A. Calculations and publication-quality illustrations for analytical ultracentrifugation data. Methods Enzymol. 562, 109–133 (2015)

    Article  CAS  Google Scholar 

  28. Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619 (2000)

    Article  ADS  CAS  Google Scholar 

  29. Brookes, E., Demeler, B., Rosano, C. & Rocco, M. The implementation of SOMO (SOlution MOdeller) in the UltraScan analytical ultracentrifugation data analysis suite: enhanced capabilities allow the reliable hydrodynamic modeling of virtually any kind of biomacromolecule. Eur. Biophys. J. 39, 423–435 (2010)

    Article  CAS  Google Scholar 

  30. Hashiguchi, T. et al. Crystal structure of measles virus hemagglutinin provides insight into effective vaccines. Proc. Natl Acad. Sci. USA 104, 19535–19540 (2007)

    Article  ADS  CAS  Google Scholar 

Download references


We thank the beamline staff members at the Photon Factory and SPring-8 for their assistance with data collection. We thank Y. Yamada and D. Liebschner for assistance with S-SAD data collection at PF-1A. This research is supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Japan Agency for Medical Research and Development (AMED). This work was supported by a Grant-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (U.O., S.U., N.I. and T.S.); CREST, JST (T.S.); the Takeda Science Foundation (U.O., N.I. and T.S.); the Mochida Memorial Foundation for Medical and Pharmaceutical Research (U.O.); and the Daiichi Sankyo Foundation of Life Science (U.O.).

Author information

Authors and Affiliations



H.I. expressed and purified recombinant proteins and performed size-exclusion chromatography and isothermal titration calorimetry experiments. H.I. and U.O. performed crystallization and structure determination. E.K and S.U performed AUC analyses. N.I. performed cellular assays. U.O and T.S. directed the research and wrote the paper with assistance from all other authors.

Corresponding authors

Correspondence to Umeharu Ohto or Toshiyuki Shimizu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks K. Melcher, M. Okabe and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Sequence alignments of IZUMO1 and JUNO.

Sequence alignments of IZUMO1 (top) and JUNO (bottom) from Homo sapiens, Macaca mulatta, Equus caballus, Bos tauras or Bos mutus, Oryctolagus cuniculus and Mus musculus are shown. Secondary structure elements are displayed above the sequences. The residues of the IZUMO1–JUNO interface are indicated by yellow highlighting. The sequence identity between human and mouse IZUMO1 and JUNO are 59% and 69%, respectively, for the extracellular regions used in this study. In contrast, the interface residues exhibit less conservation (35% and 53% identity for IZUMO1 and JUNO, respectively). The N-glycosylation sites are indicated by blue Y-shaped characters. Alignments were performed using Clustal Omega software (EMBL-European Bioinformatics Institute). Residues are coloured to indicate the degree of similarity: red residues are those with the highest similarity, followed by green, blue, and black (lowest similarity).

Extended Data Figure 2 Sequence alignment of JUNO and folate receptor (FR).

Sequence alignment of JUNO from Homo sapiens, Equus caballus and Mus musculus and FRα, FRβ, and FRγ from Homo sapiens. Secondary structure elements for JUNO and FR are displayed above and below the sequences, respectively. The residues involved in the folate binding in FRs are indicated by yellow highlighting. Alignments were performed using Clustal Omega software (EMBL – European Bioinformatics Institute). Residues are coloured to indicate the degree of similarity: red residues are those with the highest similarity, followed by green, blue, and black (lowest similarity).

Extended Data Figure 3 Anomalous difference Fourier maps from S-SAD data.

The anomalous difference Fourier maps from the data collected at 2.7 Å wavelength contoured with green at the 3σ level are superposed onto the refined model of the IZUMO1–JUNO complex. The disulfide bonds are shown in stick representations and indicated by green (IZUMO1) and cyan (JUNO) labels.

Extended Data Figure 4 Superpositions of IZUMO1 and JUNO structures.

a, Superposition of the six IZUMO1 molecules (chains A–F) in the asymmetric unit. b, Superposition of the two (form 1; chains A and B) and four (form 2; chains A–D) JUNO molecules in the asymmetric unit. c, Superposition of IZUMO1 (chain A) and JUNO (chain A) structures onto the IZUMO1–JUNO complex structure (form 1, form 2, and form 3). IZUMO1 (chain A) and JUNO (chain A) are coloured grey. The IZUMO1–JUNO complexes in the form 1, form 2, and form 3 (chain A–B and chain C–D) crystals are coloured green, cyan, orange, and magenta, respectively. The r.m.s.d. values for each pair of molecules are shown schematically.

Extended Data Figure 5 Cell surface expression of IZUMO1 mutants in COS-7 cells (related to Fig. 4).

Surface localization of wild-type IZUMO1 or its mutant in COS-7 cells. IZUMO1 proteins on the cell surface (green) and nuclei (blue) were stained. Scale bars, 20 μm.

Extended Data Figure 6 Dimerization of IZUMO1 and IZUMO1–JUNO complex analysed by SV–AUC and size-exclusion chromatography.

a, b, The oligomerization states of IZUMO1 (a) and IZUMO1–JUNO complex (b) were analysed by SV–AUC at various concentrations. The normalized c(s) distributions were plotted against the sedimentation coefficients, s20,w (S). The observed sedimentation coefficient of the IZUMO1 dimer (3.2 S) at a high concentration (100 μM) in a was smaller than the expected value for IZUMO1 dimer (3.7 S), owing to the fast monomer–dimer interconversion kinetics. c, The oligomerization of IZUMO1 was analysed by gel-filtration chromatography. In each experiment, 20, 100 or 740 μM IZUMO1 (total volume of 50 μl) was injected into a Superdex 200 Increase 5/150 GL gel-filtration column (running buffer; 10 mM Tris-HCl pH 7.5 and 150 mM NaCl).

Extended Data Figure 7 Effect of reducing agent and acidic pH on the IZUMO1–JUNO interaction.

a, DTT-induced aggregation of IZUMO1. Each sample was incubated at room temperature for 2 h in the presence of 1 mM DTT, and then injected into a Superdex 200 Increase 5/150 GL gel-filtration column (running buffer; 10 mM Tris-HCl pH 7.5 and 150 mM NaCl, 1 mM DTT). Eluents were analysed by SDS–PAGE. For gel source data, see Supplementary Fig. 1. b, The interaction between IZUMO1 and JUNO at acidic pH. The IZUMO1–JUNO interaction at acidic pH was analysed by size-exclusion chromatography (SEC; top) and isothermal titration calorimetry (bottom) as in Fig. 1. In SEC analysis, each sample was injected into a Superdex 200 Increase 5/150 GL gel-filtration column (running buffer; 10 mM MES-NaOH pH 5.5 and 150 mM NaCl). Eluents were analysed by SDS–PAGE. c, The SEC chromatograms at a neutral pH of 7.5 is shown as a control experiment.

Extended Data Table 1 Isothermal titration calorimetry results
Extended Data Table 2 Crystallization and cryoprotectant conditions
Extended Data Table 3 Data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains the uncropped blots with size marker indications. (PDF 655 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ohto, U., Ishida, H., Krayukhina, E. et al. Structure of IZUMO1–JUNO reveals sperm–oocyte recognition during mammalian fertilization. Nature 534, 566–569 (2016).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing