08 July 2014
By Enrica Bianchi
The dictionary definition of fertilisation is plain and straightforward: “the union of male and female gametes, during sexual reproduction, to form a zygote”. Yes, we know how reproduction occurs but when it comes to the molecular mechanisms of fertilisation our understanding is surprisingly poor.
To date only one pair of binding proteins has been found to be essential for sperm-egg fusion in mammals: Izumo1 and Juno. Izumo1 is expressed exclusively by sperm cells and was first identified almost 10 years ago. It wasn’t until April this year that we published the identification of Juno – formerly known as Folate receptor 4 and renamed after the Roman goddess of marriage and fertility – as the sole Izumo1 receptor present on eggs.
Why did it take so long to identify Juno despite research in the reproduction field having a solid, decade-long history? The major limiting factors are, firstly, the small amount of biological material available and, secondly, the biochemical characteristics of membrane protein interactions.
The first difficulty arises because, once gametes have matured, they are destined to die unless they fuse to generate a new organism. So, the cells responsible for species propagation are actually capable of doing so for only a very short time of their life. And there are no cell lines that can successfully substitute sperm and eggs in the lab. Furthermore, every mammalian female has a limited reservoir of eggs that cannot be replenished.
The second difficulty arises from the interactions occurring among proteins displayed on the surface of cells, which are often highly transient, making them difficult to detect. Biochemically manipulating these proteins is even more challenging due to the presence of a greasy region that is required to tether these proteins within the plasma membrane, making them difficult to dissolve while preserving the rest of the protein structure.
Being acutely aware of these challenges, we selected an expression cloning approach, which is where you develop a library containing all the genes expressed in a particular cell, the egg in our case, and then you screen the library for the gene encoding for a particular protein as we did for the gene encoding the egg Izumo receptor. Expression cloning in mammalian cells was a true leap forward in immunology that led to the identification of many receptors including that for capsaicin.
To increase the chance of identifying the binding partner of Izumo1, we expressed the whole extracellular domain of Izumo1 as a multimeric form. This modification makes the receptor binding stronger and can be used to identify weaker interactions. Pentameric Izumo1 was therefore used as a probe to screen an egg complementary DNA library.
Eventually, after analysing 251 pools of clones we finally found the Izumo1 binding partner: Juno.
Our approach proved to be successful and overcame the two limits outlined above by using a mammalian cell line to screen the genes expressed in the egg and using a protein created in the laboratory as a probe instead of the protein extracted from sperm cells.
The emerging model of fertilisation is that the proteins expressed on the surface of gametes are organised in complexes that interact with each other. Therefore it is likely that molecules with different roles are involved: some (like Juno and Izumo1) are directly responsible for sperm-egg recognition while others assemble and maintain the structures necessary for the interaction. The discovery of the first binding pair essential for fertilisation provides a starting point for further investigation, and hopefully will help in elucidating the roles of other proteins.
Notably, we still don’t know the identity of the molecule that mediates membrane fusion (the “fusogen”) or the role played by CD9 and CD81, two egg proteins that are necessary for fusion but do not bind Izumo1 directly.
To gain a comprehensive understanding of molecular mechanisms beyond the simple dictionary definition of fertilisation, we certainly need more scientific investigations, and combining methods applied in different fields could provide a valid way of getting there.
Enrica Bianchi is a postdoctoral fellow at the Wellcome Trust Sanger Institute. In 2011 she joined Dr Gavin Wright’s group to study the molecular basis of mammalian sperm-egg recognition.
- Inoue, N et al (2005). The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature. doi:10.1038/nature03362
- Bianchi, E et al (2014). Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature. doi:10.1038/nature13203
- Aruffo, A et al (1987). Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proceedings of the National Academy of Sciences of the United States of America. PMCID: PMC299587
- Caterina, M et al (1997). The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature. doi:10.1038/39807
- Bushell, K et al (2008). Large-scale screening for novel low-affinity extracellular protein interactions. Genome Research. doi: 10.1101/gr.7187808
- Bianchi, E et al (2014). Izumo meets Juno: Preventing polyspermy in fertilization Cell Cycle. doi:10.4161/cc.29461