Identifying sperm-egg receptor pairs essential for mammalian fertilization to select new targets for fertility treatment and contraception.

In humans and many other organisms, new life starts when a sperm and an egg recognise each other and permit the fusion of the membranes that surround them to form an embryo in a process we call fertilization. The way in which sperm and egg are able to recognise each other is very likely to involve a pair of recognition molecules: one displayed on the surface of the sperm and the other on the egg's surface that specifically bind to each other and finally enable the sperm and egg to fuse and create a genetically distinct embryo. Despite the fact that fertilization is a fundamental biological process our current molecular understanding is remarkably poor, particularly in mammals. This can be partly explained in humans due to the ethical issues involved, but there are also significant technical challenges that make working with sperm and eggs and the molecules on their surface in the laboratory very difficult. For example, eggs are a very rare cell type in mammals (humans usually release just a single egg each fertility cycle and mice less than ten) limiting the amount of biological material available. In addition, the interactions between cell surface recognition molecules are known to be extremely weak (often having half-lives of just fractions of a second), requiring the use of specialised approaches to detect them. The Cell Surface Signalling Laboratory at the Sanger Institute specialises in identifying these transient receptor interactions and we have developed a set of tools and techniques to circumvent these difficulties. We have recently shown, by identifying the first essential sperm-egg receptor pair (see Bianchi et al. Nature 2014 v508 p483), that applying these techniques can make important discoveries in this field. In this grant application, we propose to apply the same techniques in a systematic manner to identify additional sperm-egg recognition molecules but also begin to apply these findings to reveal new targets for fertility treatment and contraceptives.
Identifying the cell surface receptors that are important for fertilization could have significant implications for the development of novel contraceptives and fertility treatments. For example, identifying which receptors are required for fertilization and how they interact could provide the starting point to develop new drugs that specifically block the interaction and therefore be used as a contraceptive. Perhaps surprisingly, no new contraceptives have been developed for over 40 years. The rapidly expanding human population (currently over 7 billion and predicted to reach 10 billion by 2050) has raised concerns globally that the limited resources on the planet will not sustain such a continued expansion. A drug that blocks the interaction between sperm and egg could conceivably be taken by both men and women and be effective for just a short period of time (a few hours to several days). It is also likely that the receptor interaction will be used by other animal species and so could be used to design methods to effectively control animal populations in a humane way.
Finally, our previous work has shown that interactions between sperm and egg receptors can be essential for fertilization and so our planned research may reveal the identity of new infertility genes. Infertility is a growing problem, particularly in Western countries where the average age of couples having their first child has increased in recent years. By discovering new infertility genes, this research could open up the possibility of offering simple and inexpensive genetic screening tests to infertile couples that may guide their fertility treatment and save the expense and inconvenience of failed rounds of fertility treatment. In addition, by providing a molecular explanation for infertility, this may suggest methods of clinical intervention to help couples conceive.

Fertilization involves the cellular recognition and subsequent fusion of haploid gametes to create a diploid zygote. We know little about the molecular events involved in mammalian fertilization, including the identity of the sperm-egg receptor pairs involved. Largely, this is due to technical challenges due to the rarity of eggs and because the interactions between extracellular receptors are weak requiring specialised techniques to identify them. We have developed methods based on creating highly avid binding probes to detect weak extracellular interactions and have applied them to the problem of sperm-egg recognition and identified the first sperm-egg receptor pair that is essential for mammalian fertilization (Bianchi et al. Nature 2014 v508 p483). There is good evidence to suggest that there are additional important sperm-egg recognition molecules and this grant application outlines how we intend to apply the same approach to identify additional sperm-egg receptor pairs that could be useful for developing novel contraceptives and fertility treatments.
We will use a library of ~40 highly avid recombinant proteins that represents the secretome and cell surface receptor repertoire of the mouse spermatozoon and systematically test them for interactions with the oolemma and zona pellucida. For those sperm probes that exhibit positive staining with the egg, we will identify the molecular nature of the egg receptor using expression cloning (an approach we have successfully used in the past) and other receptor matching techniques that we have developed in the laboratory. The function of any identified interactions will be determined using transgenic mice.

The successful conclusion of this research would have a range of beneficiaries.
1) Academics: There would be a wide range of immediate academic beneficiaries of this work ranging from scientists who are specifically interested in the molecular mechanism of fertilization to those interested in other biological systems that involve membrane fusion such as the development and function of skeletal muscle, placenta and osteoclasts.
2) Commercial sector: The main impact that this research could have on society would be to identify a starting point for the development of novel contraceptives. No new contraceptives have been developed since the development of "the pill", which works by hormonal control, over 40 years ago. While this method is effective, many women are unable or recommended not to use it for medical or religious reasons. The identification of a new target/s required for fertilization could therefore benefit the pharmaceutical industry in the development of novel contraceptives and improve the lives of the recipients of these novel drugs. Naturally, because of the long development time and necessary safety testing for new drugs, these benefits would not be realised for 10 to 15 years.
3) Health workers, particularly those working in fertility clinics: The identification of receptor-ligand pairs involved in mammalian fertilization would potentially provide an explanation for couples who are unable to conceive naturally, and may lead to the development of novel fertility treatments.
4) Governments and wider society: If this research led to the development of new contraceptives then the ultimate beneficiaries of this research could include large sections of society. Related to this, a major issue facing the world is overpopulation. Improvements in healthcare systems have meant that people are now living much longer, so that today, there are over 7 billion humans on the planet and some forecasts predict that this will rise to 10 billion by the year 2050. Such a vast and rapidly increasing human population has already led governments to consider how they can maintain and improve future living standards within the confines of the planet's finite resources. Scientists will have to provide solutions to these problems and having new targets that could result in novel contraceptives would be an important starting point.
5) Veterinary science: Veterinary science may also benefit from this research since it is likely that contraceptives for other animal species could also be developed. This would be useful in controlling animal populations in an ethically agreeable manner. For example, based on our findings, one might be able to develop a contraceptive vaccine that could easily and irreversibly sterilise animals to control their populations within certain geographical areas where they are considered pests (such as badgers, elephants, deer, horses etc..) or for animal welfare (e.g. pets, zoo animals) without the need for surgical neutering. Again this may take up to 10 years to develop.
6) Scientific training: This grant would involve the employment of both a postdoctoral scientist and a research assistant. By joining the Cell Surface Signalling Laboratory at the Sanger Institute, they will be trained in a range of specialist biochemical techniques that are specifically designed to identify low affinity extracellular interactions. The product of this training would be an individual who would be ideally placed to apply these novel techniques to wider problems of gamete recognition to gain a more complete understanding of the molecular basis of fertilization.