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

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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.

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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.

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Authors and Affiliations

Authors

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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Competing interests

The authors declare no competing financial interests.

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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