Knowledge of the sperm-specific protein that is required for the attachment of sperm to eggs during fertilization in mammals has led to the identification of the protein's receptor on the egg's plasma membrane. See Article p.483
Biology is full of surprises. Such is the case with the findings of a paper by Bianchi et al.1 reported on page 483 of this issue. The authors report the end of a decade-long search for a partner for the sperm protein Izumo1, which is responsible for sperm–egg adhesion during fertilization. The elusive mate is a member of the folate-receptor protein family, and is located on the plasma membrane of unfertilized eggs. Because of its essential role in fertilization, the researchers propose that the folate receptor, currently known as Folr4, should be renamed Juno, after the Roman goddess of fertility and marriage.
Fertilization defines the process by which a sperm and an egg combine to form a single-celled zygote that, in time, gives rise to a new individual. In 2005, researchers reported2 that a protein that spans the sperm's plasma membrane, called Izumo1 (named after a Japanese marriage shrine), is essential for fertilization in mammals. Male mice that lack Izumo1 are infertile — their sperm seem normal, but are unable to fuse with eggs. Subsequently, it was found that Izumo1 is part of a multiprotein family whose members form large complexes on sperm that may be essential for sperm–egg fusion3. However, the identity of the receptor to which Izumo1 binds remained a mystery, owing largely to inherent difficulties in working with small numbers of mammalian eggs, as well as to the transient nature of extracellular interactions between proteins tethered to the membrane.
Bianchi et al. identified the mammalian egg receptor for Izumo1 using a method previously developed in their laboratory for detecting weak interactions between membrane-tethered proteins4. The authors used a region of the Izumo1 protein — the part that extends into the extracellular space — as a binding probe, and found that a member of the folate-receptor family, Folr4, is the sole receptor for Izumo1 on unfertilized eggs. Furthermore, they confirmed previous suggestions that, unlike other Folr proteins, Folr4 is unable to bind folic acid5,6 (a form of folate). Bianchi and colleagues found that Folr4, which they rename Juno, is membrane-tethered and is present on the eggs of mice, opossums, pigs and humans.
The researchers examined three Izumo and three folate-receptor family members and found that only Izumo1 and Juno could interact with one another. Exposure of eggs to an antibody that blocks Juno prevented both the binding of Izumo1 to Juno and fertilization in vitro. Furthermore, female mice that lacked Juno were infertile, and eggs recovered from these mice could not be fertilized by sperm in vitro. In this situation, the authors found that sperm were unable to adhere to and fuse with the egg's membrane, but instead accumulated in the perivitelline space that lies between the egg's membrane and its thick extracellular coat, the zona pellucida. Even when the researchers removed the zona pellucida, exposing the plasma membrane, eggs that lacked Juno could not be fertilized by sperm in vitro and fewer sperm bound to the egg's membrane than to the membranes of control eggs. Collectively, these results strongly suggest that the formation of an Izumo1–Juno adhesion complex is essential for fertilization in mammals (Fig. 1a).
These observations beg the question: is the interaction between Izumo1 and Juno the only requirement for sperm–egg fusion? Some recent evidence7 suggests that, although Izumo1 induces surface interactions such as adhesion between cell membranes, it does not promote membrane fusion. Indeed, when Bianchi and co-workers induced expression of Juno or Izumo1 in non-fusing cell types, they found no evidence for cell fusion. They concluded that the Izumo1–Juno interaction is a necessary event for adhesion between sperm and eggs, but that cell fusion requires other membrane proteins — perhaps similar to those required for fusion in other cell types, such as EFF-1 (ref. 8) and myomaker9. The authors go on to propose that local clustering of Juno on the egg's membrane, possibly organized by another membrane-spanning protein such as Cd9 (ref. 10), may occur to increase the strength of sperm binding.
Bianchi et al. got another surprise when they looked for Juno on the fertilized egg's membrane. Thirty to forty minutes after sperm–egg fusion, Juno was barely detectable, having made its exit in extracellular vesicles. The authors suggest that loss of Juno after fertilization may account for the fact that fusion is limited (by an unknown mechanism) to one sperm per egg, a phenomenon known as the block to polyspermy11 (Fig. 1b). In keeping with this suggestion, Juno did not exit the egg's membrane when sperm were injected directly into eggs (known as intracytoplasmic sperm injection), or when eggs were activated with ethanol. Neither of these treatments results in a block to polyspermy12 and this could be due to retention of Juno on the fertilized egg's membrane, permitting binding and fusion of additional sperm.
As with other advances in biology, this discovery raises questions and creates possibilities. For example, whether cases of infertility in women can be ascribed specifically to mutations in the gene encoding Juno remains to be evaluated. However, Izumo1 has proved to be a suitable candidate for the development of a contraceptive vaccine13, and it is likely that Juno and Izumo1–Juno complexes will provide additional targets for contraception. Detailed knowledge of the three-dimensional structure of the Izumo1–Juno complex could certainly facilitate the design of small molecules that specifically inhibit formation of the adhesion complex and, consequently, prevent fertilization.
Another logical next step is to identify proteins that interact with Izumo1–Juno complexes and stimulate sperm–egg fusion. Further application of the techniques described by Bianchi et al. could aid the identification of such proteins. In a similar vein, the mechanism by which Juno is ejected from the egg membrane after sperm–egg fusion is not yet understood. Removal of Juno in extracellular vesicles may depend on the cortical reaction14, a part of the process of preventing polyspermy that involves fusion of vesicles known as cortical granules with the egg's membrane after fertilization. Much remains to be done, but the identification of an egg-based partner for Izumo1 represents a significant step forwards for our understanding of the fertilization process.
About this article
Molecular and Cellular Mechanisms of Sperm-Oocyte Interactions Opinions Relative to in Vitro Fertilization (IVF)
International Journal of Molecular Sciences (2014)