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

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.

Figure 1. Juno is the GPI-anchored oocyte surface receptor for Izumo1

(a) An avid recombinant Izumo1 protein but not a control bound the oolemma. (b) Izumo1 bound HEK293 cells transfected with a Juno cDNA (clone B2) but not untransfected controls. (c) Juno is highly expressed on the oolemma of unfertilised eggs. (d) Preincubation of eggs with an anti-Juno but not an anti-Cd55 control antibody blocked binding of recombinant Izumo1 protein. (e, f) Cell surface Juno was lost after treatment with PIPLC on Juno-transfected HEK293 cells (e) or eggs (f). Images are single optical sections of unfertilised mouse metaphase II eggs; scale bars are 20 μm in a, c, d, f, and 10 μm in b.

Figure 2. The Juno-Izumo1 interaction is direct, transient and conserved across mammals

(a) Biophysical analysis of the Juno-Izumo1 interaction using SPR. Serial dilutions of purified, soluble Juno were injected over immobilised Izumo1, and kinetic parameters derived from a 1:1 Langmuir binding model (red lines). (b) Binding specificity within the paralogues of the mouse Izumo and Folr families: only Juno (Folr4) bound Izumo1. (c) The Juno-Izumo1 interaction is conserved across mammals. AVEXIS was used for binding analysis in (b) and (c) using recombinant Folr/Juno proteins as preys and Izumo proteins as baits; control bait in (c) was Cd4. All bar charts show mean ± SEM; n =3.

Figure 3. Juno is essential for female fertility

(a) An anti-Juno monoclonal antibody potently blocked in-vitro fertilisation; anti-Izumo1 is shown for comparison. (b) Female Juno^−/− mice are infertile. Continuous mating of Juno^−/− female mice to proven wild-type males for three months did not result in any pups. (c) Greater numbers of sperm were observed in the perivitelline space (PVS) of eggs from superovulated Juno-deficient (−/−) mice relative to wild-type (+/+). (d, e) Juno^−/− eggs do not fuse with wild-type sperm in vitro. Sperm were added to Juno-deficient (−/−) and wild-type (+/+) eggs and fused sperm quantified after two hours (d), or pronuclei after six hours (e). All bar charts show mean ± SEM; numbers in parentheses are total number of eggs (a, c, d, e) or number of mating pairs (b).

Figure 4. Normally-fertilised, but not ICSI-fertilised or parthenogenetically-activated eggs, rapidly shed Juno from the oolemma

(a) Cell surface Juno rapidly becomes undetectable after fertilisation. Juno (green) is expressed on ovulated metaphase II eggs but is barely evident at telophase II and undetectable on pronuclear-stage zygotes. Arrow and asterisk indicate sites of first and second polar body extrusion respectively; chromosomes are not within the plane of focus. (b) Immunogold electron microscopy localised Juno primarily to the oolemma in unfertilised, ovulated eggs but was redistributed to vesicles within the perivitelline space within one hour after fertilisation. (c) Eggs fertilised by ICSI or parthenogenetically-activated retain oolemmal Juno staining until at least the pronuclear stage, as shown. Nuclei/pronuclei were visualised with DAPI (blue). Images show representative eggs from three independent experiments. Scale bars represent 20 μm (a and c) and 1 μm (b).