Functional dissection of Tmem95: a sperm cell surface protein essential for mammalian fertilization

In humans, new life starts at fertilization when a sperm and an egg recognise each other and their surrounding membranes fuse to form a new embryo. Sperm and egg interact using cell surface recognition molecules which bind specifically and enable them to fuse together. Although fertilization is a fundamental biological process, our understanding of the molecules involved is remarkably poor, especially in mammals; for example, we do not know the molecule/s that are responsible for fusing the sperm and egg together. These knowledge gaps can be partly explained in humans due to the ethical issues surrounding fertilization, and the difficulties in studying cell surface molecules. For example, eggs are a very rare cell type, with humans usually releasing just a single egg in each fertility cycle limiting the amount of biological material available. In addition, the physical interactions between cell surface molecules are known to be very weak (having half-lives of just seconds), requiring the use of specialised methods to detect them. The Cell Surface Signalling Laboratory at the Sanger Institute specializes in identifying these fleeting interactions and we have developed a set of techniques to circumvent these difficulties. We have previously used these techniques to identify the first essential sperm-egg receptor pair (see Bianchi et al. Nature 2014 v508 p483), which made an important contribution to the molecular understanding of fertilization.

We are excited about a new sperm surface protein prosaically named Tmem95 (for TransMEMbrane protein number 95) that is essential for the ability of sperm to fuse with eggs. Tmem95 was identified from research in the cattle industry where a case of idiopathic male infertility (sperm that is infertile despite looking and moving normally) was investigated. Researchers used the results of thousands of artificial inseminations performed in cows, and by careful record-keeping, identified the source to a mutation in the TMEM95 gene. Further research has shown that male mice which have an engineered mutation in their Tmem95 gene are also infertile because normal-looking sperm are unable to fuse with eggs in IVF assays. Humans also have a TMEM95 gene which looks very similar and we believe that it will have an important role in humans too; however, we know very little about this protein. This grant application is aimed at understanding more about the important role TMEM95 has in fertilization. Specifically, we will use our specialized technologies to determine the molecular identity the receptor it interacts with on the egg surface, describe how the protein is redistributed in the sperm during fertilization, establish if Tmem95 has a specific role in fusing membranes together, and investigate how it interacts with another essential sperm cell surface proteins called Izumo1.

These studies will both further our molecular understanding of a fundamental biological process, and provide a foundation to develop better contraceptives and fertility treatments both in humans and in the farming industry. The rapidly expanding human population (currently over 7 billion and predicted to reach 10 billion by 2050) has raised concerns that the limited resources on the planet will not sustain a continued expansion. Paradoxically, 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 pain of failed rounds of IVF. These studies could also improve reproduction technologies in the livestock industry and also in the development of contraceptive vaccines to control wild animal populations and sterilize companion animals in an ethically more acceptable way other than culling or neutering.

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 molecule/s involved in gamete membrane fusion in any animal. Recent research from the cattle industry supported by subsequent studies using gene-targeted mice has identified Tmem95 as a gene encoding a sperm cell surface protein that is essential for fusion with eggs. Our preliminary studies have shown that a highly avid soluble recombinant protein comprising the extracellular regions of Tmem95 specifically stains the egg membrane demonstrating it interacts with a receptor on the oolemma.

The main aim of this grant application will be to determine the molecular nature of this egg receptor using a range of assays including cell-based expression cloning and testing libraries of recombinant proteins that each address the particular technical challenges of identifying extracellular receptor-ligand interactions. We will also raise a high quality antibody to Tmem95 and determine the detailed subcellular localization of the protein in the sperm head throughout the acrosome reaction and during egg membrane fusion. Because Tmem95-deficient sperm are unable to fuse with zona-free eggs, Tmem95 is an excellent candidate for the elusive protein that mediates sperm-egg membrane fusion and so we will use a cellular fusion assay that we have developed to determine its fusogenic ability. We will also investigate how Tmem95 interacts with another essential sperm cell surface protein, Izumo1, to dissect their distinct functional roles during fertilization.

These studies will increase our molecular understanding of a fundamental biological process and provide a platform to permit the development of novel contraceptives and fertility treatments in both humans and livestock animals.

1) Commercial sector: The main impact that this research could have on society would be to identify a starting point for the development of better contraceptives. While the use of the contraceptive "pill" 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. These benefits would not be realised for 10 to 15 years due to lengthy safety and efficacy testing.

2) Livestock industry: The propagation of traits of economic value is the main purpose of selective breeding in livestock and fertility is an important economic trait per se. Advancements in our understanding of the molecular mechanism of fertilization could be translated in more efficient and widespread use of assisted reproduction technology (ART). For example, Tmem95 could be used to assess semen quality while the identification of its binding partner on the egg (objective 1) could inform female reproductive performance. Remarkably, artificial insemination has played a key role in the increase in milk production in the dairy industry. The swine and poultry industries have made similar improvements in efficiency and product consistency. Assisted reproductive technologies could benefit further by gamete selection based on molecular markers. Thanks to our collaboration with Professors Pilar Coy and Maria Jimenez-Movilla in Murcia we will be able to validate and assess the outcome of our proposed research in pig reproduction where polyspermy in vitro is a major barrier to ART.

3) Governments and wider society: If this research led to the development of new contraceptives then the 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 longer, so that today, there are over 7 billion humans on the planet which is predicted to 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. The identification of new essential proteins involved in mammalian fertilization would potentially provide an explanation for idiopathic infertility and provide infertile couples with a diagnosis, and may lead to the development of novel fertility treatments.

4) 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 one might be able to develop a contraceptive vaccine that could easily and irreversibly sterilise animals to control their populations where they are considered pests (e.g. badgers, deer, etc..) or for animal welfare (e.g. pets, zoo animals) without the need for surgical neutering.

5) Academics and scientific training: Scientists who are specifically interested in the molecular mechanism of fertilization would immediately benefit and those interested in other biological systems that involve cellular fusion such as the development and function of skeletal muscle, placenta and osteoclasts. This grant would involve the employment of a scientist who will be trained in a range of specialist biochemical techniques to identify low affinity extracellular interactions. Importantly, both researchers named on this grant (Drs Wright and Bianchi) have taught on an M.Phil reproduction course in the University of Murcia, Spain.