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Juno is the egg Izumo receptor and is essential for mammalian fertilization

Nature volume 508, pages 483–487 (2014)Cite this article

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Abstract

Fertilization occurs when sperm and egg recognize each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm–egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell-surface protein, but its receptor on the egg has not been described. Here we identify folate receptor 4 (Folr4) as the receptor for Izumo1 on the mouse egg, and propose to rename it Juno. We show that the Izumo1–Juno interaction is conserved within several mammalian species, including humans. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilization suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives.

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Figure 1: Juno is the GPI-anchored oocyte surface receptor for Izumo1.
Figure 2: The Izumo1–Juno interaction is direct, transient and conserved across mammals.
Figure 3: Juno is essential for female fertility.
Figure 4: Juno is rapidly shed from the oolemma of normally fertilized eggs, but not ICSI-fertilized or parthenogenetically activated eggs.

References

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

    Article  CAS  PubMed  Google Scholar 

  2. Gardner, A. J. & Evans, J. P. Mammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm. Reprod. Fertil. Dev. 18, 53–61 (2006)

    Article  CAS  PubMed  Google Scholar 

  3. Ikawa, M., Inoue, N., Benham, A. M. & Okabe, M. Fertilization: a sperm’s journey to and interaction with the oocyte. J. Clin. Invest. 120, 984–994 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 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  PubMed  Google Scholar 

  5. Le Naour, F., Rubinstein, E., Jasmin, C., Prenant, M. & Boucheix, C. Severely reduced female fertility in CD9-deficient mice. Science 287, 319–321 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  6. Miyado, K. et al. Requirement of CD9 on the egg plasma membrane for fertilization. Science 287, 321–324 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  7. Kaji, K. et al. The gamete fusion process is defective in eggs of Cd9-deficient mice. Nature Genet. 24, 279–282 (2000)

    Article  CAS  PubMed  Google Scholar 

  8. Satouh, Y., Inoue, N., Ikawa, M. & Okabe, M. Visualization of the moment of mouse sperm-egg fusion and dynamic localization of IZUMO1. J. Cell Sci. 125, 4985–4990 (2012)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  10. Coonrod, S. A. et al. Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion. Dev. Biol. 207, 334–349 (1999)

    Article  CAS  PubMed  Google Scholar 

  11. Alfieri, J. A. et al. Infertility in female mice with an oocyte-specific knockout of GPI-anchored proteins. J. Cell Sci. 116, 2149–2155 (2003)

    Article  CAS  PubMed  Google Scholar 

  12. Wright, G. J. Signal initiation in biological systems: the properties and detection of transient extracellular protein interactions. Mol. Biosyst. 5, 1405–1412 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yamaguchi, T. et al. Control of immune responses by antigen-specific regulatory T cells expressing the folate receptor. Immunity 27, 145–159 (2007)

    Article  CAS  PubMed  Google Scholar 

  14. Teng, M. W. et al. Multiple antitumor mechanisms downstream of prophylactic regulatory T-cell depletion. Cancer Res. 70, 2665–2674 (2010)

    Article  CAS  PubMed  Google Scholar 

  15. Liang, S. C., Moskalenko, M., Van Roey, M. & Jooss, K. Depletion of regulatory T cells by targeting folate receptor 4 enhances the potency of a GM-CSF-secreting tumor cell immunotherapy. Clin. Immunol. 148, 287–298 (2013)

    Article  CAS  PubMed  Google Scholar 

  16. Kunisawa, J., Hashimoto, E., Ishikawa, I. & Kiyono, H. A pivotal role of vitamin B9 in the maintenance of regulatory T cells in vitro and in vivo. PLoS ONE 7, e32094 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kinoshita, M. et al. Dietary folic acid promotes survival of Foxp3+ regulatory T cells in the colon. J. Immunol. 189, 2869–2878 (2012)

    Article  CAS  PubMed  Google Scholar 

  18. Salbaum, J. M., Kruger, C. & Kappen, C. Mutation at the folate receptor 4 locus modulates gene expression profiles in the mouse uterus in response to periconceptional folate supplementation. Biochim. Biophys. Acta 1832, 1653–1661 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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  PubMed  PubMed Central  Google Scholar 

  21. Jia, Z. et al. A novel splice variant of FR4 predominantly expressed in CD4+CD25+ regulatory T cells. Immunol. Invest. 38, 718–729 (2009)

    Article  CAS  PubMed  Google Scholar 

  22. 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  PubMed  PubMed Central  Google Scholar 

  23. Bushell, K. M., Sollner, C., Schuster-Boeckler, B., Bateman, A. & Wright, G. J. Large-scale screening for novel low-affinity extracellular protein interactions. Genome Res. 18, 622–630 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Jaffe, L. A. Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature 261, 68–71 (1976)

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Sabharanjak, S. & Mayor, S. Folate receptor endocytosis and trafficking. Adv. Drug Deliv. Rev. 56, 1099–1109 (2004)

    Article  CAS  PubMed  Google Scholar 

  26. Jackowski, S. & Dumont, J. N. Surface alterations of the mouse zona pellucida and ovum following in vivo fertilization: correlation with the cell cycle. Biol. Reprod. 20, 150–161 (1979)

    Article  CAS  PubMed  Google Scholar 

  27. Podbilewicz, B. et al. The C. elegans developmental fusogen EFF-1 mediates homotypic fusion in heterologous cells and in vivo. Dev. Cell 11, 471–481 (2006)

    Article  CAS  PubMed  Google Scholar 

  28. Mi, S. et al. Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 403, 785–789 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  29. Abmayr, S. M. & Pavlath, G. K. Myoblast fusion: lessons from flies and mice. Development 139, 641–656 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Powell, G. T. & Wright, G. J. Jamb and Jamc are essential for vertebrate myocyte fusion. PLoS Biol. 9, e1001216 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Millay, D. P. et al. Myomaker is a membrane activator of myoblast fusion and muscle formation. Nature 499, 301–305 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hemler, M. E. Tetraspanin functions and associated microdomains. Nature Rev. Mol. Cell Biol. 6, 801–811 (2005)

    Article  CAS  Google Scholar 

  33. Jégou, A. et al. CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization. Proc. Natl Acad. Sci. USA 108, 10946–10951 (2011)

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  34. Runge, K. E. et al. Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution. Dev. Biol. 304, 317–325 (2007)

    Article  CAS  PubMed  Google Scholar 

  35. Żyłkiewicz, E., Nowakowska, J. & Maleszewski, M. Decrease in CD9 content and reorganization of microvilli may contribute to the oolemma block to sperm penetration during fertilization of mouse oocyte. Zygote 18, 195–201 (2010)

    Article  PubMed  CAS  Google Scholar 

  36. Wolf, D. P. The block to sperm penetration in zona-free mouse eggs. Dev. Biol. 64, 1–10 (1978)

    Article  CAS  PubMed  Google Scholar 

  37. Gardner, A. J., Williams, C. J. & Evans, J. P. Establishment of the mammalian membrane block to polyspermy: evidence for calcium-dependent and -independent regulation. Reproduction 133, 383–393 (2007)

    Article  CAS  PubMed  Google Scholar 

  38. Horvath, P. M., Kellom, T., Caulfield, J. & Boldt, J. Mechanistic studies of the plasma membrane block to polyspermy in mouse eggs. Mol. Reprod. Dev. 34, 65–72 (1993)

    Article  CAS  PubMed  Google Scholar 

  39. Wortzman-Show, G. B., Kurokawa, M., Fissore, R. A. & Evans, J. P. Calcium and sperm components in the establishment of the membrane block to polyspermy: studies of ICSI and activation with sperm factor. Mol. Hum. Reprod. 13, 557–565 (2007)

    Article  CAS  PubMed  Google Scholar 

  40. Skarnes, W. C. et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337–342 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Nakanishi, T. et al. Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein. FEBS Lett. 449, 277–283 (1999)

    Article  CAS  PubMed  Google Scholar 

  42. Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo: A Laboratory Manual 3rd edn (Cold Spring Harbor Laboratory Press, 2003)

    Google Scholar 

  43. Kimura, Y. & Yanagimachi, R. Intracytoplasmic sperm injection in the mouse. Biol. Reprod. 52, 709–720 (1995)

    Article  CAS  PubMed  Google Scholar 

  44. Sun, Y., Gallagher-Jones, M., Barker, C. & Wright, G. J. A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions. Anal. Biochem. 424, 45–53 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Martin, S. et al. Construction of a large extracellular protein interaction network and its resolution by spatiotemporal expression profiling. Mol. Cell. Proteomics 9, 2654–2665 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Crosnier, C. et al. A library of functional recombinant cell-surface and secreted P. falciparum merozoite proteins. Mol. Cell. Proteomics 12, 3976–3986 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Durocher, Y., Perret, S. & Kamen, A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 30, e9 (2002)

    Article  PubMed  PubMed Central  Google Scholar 

  48. Golemis, E. A. & Adams, P. D. Protein-Protein Interactions: A Molecular Cloning Manual. (Cold Spring Harbor Laboratory Press, 2001)

    Google Scholar 

  49. Kerr, J. S. & Wright, G. J. Avidity-based extracellular interaction screening (AVEXIS) for the scalable detection of low-affinity extracellular receptor-ligand interactions. J. Vis. Exp. 61, e3881 (2012)

    Google Scholar 

  50. Bartholdson, S. J. et al. Semaphorin-7A is an erythrocyte receptor for P. falciparum merozoite-specific TRAP homolog, MTRAP. PLoS Pathog. 8, e1003031 (2012)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

This work was supported by the Wellcome Trust grant number 098051. Juno-deficient mice were generated by the Sanger Institute Mouse Genetics Project. We thank W. Skarnes and J. Bussell for advice on transgenic mice; J. Kerr for construct design; A. Bradley and L. Jovine for helpful comments on the manuscript; and M. Okabe and N. Inoue for the OBF13 antibody.

Author information

Authors and Affiliations

  1. Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK,

    Enrica Bianchi & Gavin J. Wright

  2. Mouse Production Team, Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK,

    Brendan Doe

  3. Electron and Advanced Light Microscopy Suite, Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK,

    David Goulding

Authors
  1. Enrica Bianchi
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  2. Brendan Doe
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  3. David Goulding
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  4. Gavin J. Wright
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Contributions

E.B. and G.J.W. conceived the project, designed and analysed the experiments, and wrote the manuscript. E.B. performed all experiments with technical help from B.D. (ICSI) and D.G. (electron microscopy).

Corresponding author

Correspondence to Gavin J. Wright.

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Extended data figures and tables

Extended Data Figure 1 Expression of a soluble, biochemically active, highly avid recombinant mouse Izumo1 ectodomain.

a, Schematic diagram of the mouse Izumo1 cell-surface protein as it would be displayed in the membrane. Izumo1 is a type I membrane protein containing an Ig-like domain predicted to contain a disulphide bond (S–S) and a single N-linked glycosylation site (filled lollipop). b, Diagram representing the plasmid encoding the highly avid, pentamerized Izumo1 ectodomain. The expression plasmid contains a cytomegalovirus (CMV) promoter driving the transcription of the entire ectodomain of mouse Izumo1 followed by the rat CD4 domains 3 and 4 tag, a region from the cartilage oligomeric matrix protein (COMP) that forms pentamers, a beta-lactamase (β-LAC) enzymatic tag, a 3×FLAG-tag for immunological detection and a C-terminal 6-HIS tag for purification. c, Immunoreactivity of the recombinant soluble mouse Izumo1 protein expressed as a biotinylated bait with the fertilization-blocking anti-Izumo1 monoclonal antibody OBF13, as assessed by ELISA. Untreated recombinant Izumo1 was highly immunoreactive, but this immunoreactivity was significantly reduced after heat treatment at 90 °C for 10 min, demonstrating the presence of a conformational heat-labile epitope. Bar graphs show mean ± s.e.m., n = 3.

Extended Data Figure 2 A schematic illustrating the expression cloning approach that identified Juno/Folr4 as an Izumo1 binding partner.

a, A normalized mouse oocyte cDNA expression library was purchased as a glycerol stock from Express Genomics (MGC-257). The library was subsequently plated over 360 100-mm-diameter plates at a density of ∼100 colonies per plate. The plasmids were purified as 360 pools from the colonies on each plate, and transfected into adherent HEK293T cells which were grown in 48-well microtitre plates and then tested for their ability to bind the recombinant Izumo1 probe using an anti-Flag-Cy3 secondary antibody followed by direct visual inspection. An example positive pool (pool 64) is shown—the arrowhead indicates a positively-stained cell. b, Pool 64 was retransformed and plasmids purified from 96 individual colonies. Further pools containing 12 colonies each were again transfected into HEK293 cells and assessed for Izumo1 binding; here, Row B contained a positive clone. c, Izumo1 staining of HEK293 cells transfected with individual plasmids from Row B identified clone B2 as encoding an Izumo binding partner which was then revealed as Folr4/Juno by sequencing and BLAST searching. Three individual plasmids were independently identified using this approach and all three contained an identical Folr4/Juno sequence.

Extended Data Figure 3 Juno/Folr4 does not bind folate, consistent with differences in amino acids known to be involved in folate binding.

a, Soluble, biotinylated recombinant proteins corresponding to the entire ectodomains of mouse Folr1, Folr2 and Juno or human Juno were captured on a streptavidin-coated plate and washed. Folic acid binding was quantified relative to a CD200R negative control by adding a folic acid–HRP conjugate followed by an HRP substrate. Folic acid bound Folr1 and Folr2, but not mouse or human Juno. b, A multiple sequence alignment of human and mouse Folr1, Folr2 and Juno highlighting residues which are critical for folic acid binding. Sequences were aligned and structural features annotated: the signal peptide is in green, the GPI-anchor cleavage sites are underlined in bold for Folr1 and Folr2, and the best-scoring prediction is underlined pink for Juno. Residues located in the folic acid binding pocket and which affect folic acid binding are marked in red and differences highlighted in blue. Only 9 out of 15 residues involved in folic acid binding are conserved in human Juno relative to human Folr1, and 6 out of 15 are conserved in mouse Juno. Bar graphs represent mean ± s.e.m., n = 3. *One way ANOVA, P < 0.05 (Tukey’s test).

Extended Data Figure 4 Mouse Izumo1 and Juno interact with a very low equilibrium binding constant.

Serial twofold dilutions of purified and gel-filtrated JunoCD4d3+4–His were injected (solid bar, inset) through flow cells containing 450 RU CD4d3+4–bio (used as a reference) and 1,150 RU (approximate molar equivalent) of biotinylated Izumo1CD4d3+4 captured on a streptavidin-coated sensor chip. Binding after reference subtraction was quantified once equilibrium had been reached (see inset) and plotted as a binding curve (main figure). An equilibrium dissociation constant (KD) was calculated using nonlinear regression fitting of a simple Langmuir binding isotherm to the data (solid line). The KD of 12.3 ± 0.2 µM was in excellent agreement with independently obtained kinetic data (KDcalc = kd/ka = 1.387 s−1/1.03 × 105 M−1 s−1 ≈ 13.5 µM).

Extended Data Figure 5 The N-terminal Izumo domain of Izumo1 contains both the anti-Izumo1 fertilization-blocking OBF13 monoclonal antibody epitope and the Juno binding site.

a, Schematic representation of the Izumo1 protein. b, The entire ectodomain, the N-terminal Izumo domain and Ig-like domain of Izumo1 were expressed as soluble biotinylated proteins at the expected size as shown by western blotting under non-reducing conditions using streptavidin–HRP (left panel). Probing the blotted proteins with the anti-Izumo1 fertilization-blocking monoclonal antibody OBF13 (right panel) demonstrated that the OBF13 epitope was located in the N-terminal Izumo domain. c, The binding site for Juno was localized to the N-terminal Izumo domain by using the AVEXIS assay. AVEXIS is an approach designed to detect direct low-affinity extracellular interaction between recombinant proteins expressed as either monomeric biotinylated ‘bait’ proteins or pentameric enzyme-tagged ‘prey’ proteins. The biotinylated bait proteins were captured on a streptavidin-coated microtitre plate and probed for interactions with the Juno prey. Both the entire ectodomain and Izumo domain alone bound the Juno prey relative to the Ig-like domain or CD4 negative control. The CD200 (prey)–CD200R (bait) interactions was used as a positive control. Bar charts show mean ± s.e.m., n = 3.

Extended Data Figure 6 An anti-Juno/Folr4 antibody potently blocked fertilization in IVF assays.

a, Titrating the dose of an anti-Juno monoclonal antibody in IVF assays showed that antibody concentrations of 0.1 µg ml−1 or above potently prevented fertilization. The numbers in parentheses report the total number of scored eggs from three independent experiments and fertilization was quantified by observing the presence of pronuclei six hours after addition of sperm. b, Images of the eggs at the end of the IVF assay performed in the presence of 10 µg ml−1 of either the isotype-matched control antibody (left image) or anti-Juno (right image). Pronuclei (arrows) and second polar body extrusion were clearly visible in the fertilized control eggs, but not in those incubated with the anti-Juno antibody. Scale bar, 20 μm.

Extended Data Figure 7 Juno-deficient mice: allele architecture, lack of detectable Juno on Juno−/− oocytes and fertility rescue with a genetic revertant.

a, Juno-deficient mice were generated using embryonic stem cells targeted at the Juno/Folr4 locus using a gene trapping cassette (Folr4tm1a(KOMP)Wtsi) containing a strong splice acceptor site (SA) followed by an internal ribosome entry site (IRES), lacZ reporter and a polyadenylation site (pA) between Juno exons III and IV. Splicing from exon III of Juno to the transgene disrupts the Juno gene. FRT and loxP indicate recombinase recognition sites. Juno exons are indicated as blue boxes. Juno-F and Juno-R primers amplify a 440-bp PCR product from wild-type genomic DNA, and Juno-F and Cas-R primers amplify a 128-bp PCR product from the tm1a knockout (tm1a) or heterozygous mice. b, Eggs were collected from wild-type and Junotm1a/tm1a mice and stained with an anti-Juno monoclonal antibody (green). Whereas Juno was highly expressed on eggs from wild-type mice, no Juno could be detected on eggs homozygous for the Juno tm1a allele. Scale bar, 50 μm. c, A second colony of Juno-deficient mice were created using an independent embryonic stem cell clone targeted with the same gene trapping allele (Folr4tm2a(KOMP)Wtsi). The homozygous Junotm2a/tm2a mutant females were also infertile, indicating that the infertility phenotype of Junotm1a/tm1a females was not due to closely linked secondary mutations. To confirm this, the Junotm2a allele was reverted essentially to a wild-type ‘floxed’ Junotm2c allele by crossing heterozygous Junotm2a/+ mice to mice that constitutively and ubiquitously express the FLPe-recombinase from the Rosa26 locus (Rosa26Fki mice). Homozygous Junotm2c/tm2c (indicated by −/−) female mice were obtained by breeding Junotm2c/+ heterozygotes and genotyped by PCR using the Juno-F and Juno-R primers; a representative PCR genotyping experiment is shown. d, e, Eggs from reverted Junotm2c/tm2c mice are fertile. d, Image of a reverted Junotm2c/tm2c egg fertilized by IVF relative to wild-type control. Successful fertilization is scored by the clear presence of pronuclei as detected by DNA staining (DAPI, blue). e, Quantification of the number of eggs fertilized by IVF from the reverted Junotm2c/tm2c eggs relative to wild-type controls (n = 40 eggs from wild-type and n = 20 from Junotm2c/tm2c, collected from two mice).

Extended Data Figure 8 The Izumo1–Juno interaction is not sufficient for cell fusion but is required for efficient sperm–egg adhesion.

a, HEK293T cells transfected with plasmids encoding either Juno or full-length, GFP-tagged Izumo1 were mixed and cultured for 24 h before analysis by confocal microscopy. Izumo1 was detected by GFP fluorescence (green) and Juno using an anti-Juno antibody (red) and nuclei using DAPI (blue). No fused cells were observed, but both Izumo1 and Juno were enriched at sites of cellular contact. Scale bar, 10 μm. b, Sperm were collected from (B6;B6C3-Tg(Acro3-EGFP)01Osb) acrosome reporter mice and capacitated before mixing with zona-free eggs at a 1:70 egg:sperm ratio. Acrosome-reacted (GFP-negative) sperm bound less efficiently to Juno-deficient (−/−) than wild-type (+/+) control eggs. Bars represent mean ± s.e.m., the number of eggs is indicated in parentheses.

Extended Data Figure 9 Juno staining is essentially undetectable on fertilized anaphase II zona-free eggs.

Wild-type eggs were removed from IVF assays at different time points after addition of sperm, and Juno detected by confocal microscopy using an anti-Juno antibody (green); fertilized eggs were staged by the morphology of the chromosomes stained by DAPI (blue). Juno is highly expressed on the surface of metaphase II eggs (top panel), but is barely detectable by anaphase II (middle panel, approximately 45 min after fertilization), and late telophase II (bottom panel, approximately 60 min after fertilization). Scale bar, 20 μm. Note: DAPI images are taken from a different focal plane to clearly show chromosomes.

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Bianchi, E., Doe, B., Goulding, D. et al. Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature 508, 483–487 (2014). https://doi.org/10.1038/nature13203

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