Exosome signalling and cellular reprogramming in the Drosophila reproductive system

The critical event that takes place in the earliest stages of reproduction is the fertilisation of the egg by a single sperm within the female reproductive tract. However, for this event to happen, a number of hurdles have to be negotiated. Sperm need to be activated and mobilised after intercourse. The female also mounts an immune response to the foreign material in the ejaculate and molecules in the semen must block this. And finally, some components of semen in insects affect female behaviour to increase the number of offspring that a male can produce, and there is some evidence that this could also take place in mammals. In species as diverse as humans and fruit flies, males contain reproductive glands, like the prostate in men, which make the constituents of semen involved in these different processes. Surprisingly, we know little about the actual molecules that are responsible for these signals that pass between males and females when they mate, even though a better understanding might give us important new insights relevant to in vitro fertilisation (IVF) or contraception.

Recently, it has been shown that human prostate cells release into semen small membrane-bound structures called exosomes that at least in a petri dish, can fuse to sperm and make them more mobile. In an independent study, we found that an organ in the fruit fly called the accessory gland, which secretes most of the fluid in fly semen, also makes exosomes that fuse with sperm inside the female reproductive tract after mating. These exosomes also seem to be important in affecting the female's behaviour, so that she becomes unreceptive to other males, who want to mate with her.

Fortunately studies over the last 30 years in flies have revealed amazing similarities between flies and humans. About 70% of all the genes known to be involved in human disease are also found in flies and lots of the basic mechanisms by which human cells work were originally studied in flies or other simple organisms before being looked at in humans. The parallels between flies and humans suggest that if we find out how exosomes affect reproduction in flies, it is likely to give us important clues about how exosomes work in humans and other animals. The advantage we have in flies is that we can use a remarkable range of experimental tricks to mark the exosomes produced by the accessory gland in living flies, selectively block exosome secretion in this gland and remove individual components from the exosomes to test their function. As far as we are aware, this is a completely new approach and there are no other animals in which similar studies can currently be undertaken.

We will try to work out whether there are different types of exosome, what they do in the female fly, how they are targeted to certain cells and which molecules within the exosome affect what the target cell does. Our findings could suggest important new research angles that will then need to be studied in human or animal reproduction. For example, if we identify a key molecule that is needed for exosomes to work, it may be possible to block that equivalent molecule in humans as part of a male contraception strategy or enhance its activity if the molecules is defective in some cases of male infertility. There will probably be additional more indirect benefits from our studies. For example, exosomes have been implicated in diseases, like cancer, where they may drive some of the early stages of tumour spreading, the most lethal aspect of this disease. Exosomes are also being developed as carriers for drugs that could be introduced into patients, and get into inaccessible organs like the brain. Our system in flies really provides the first opportunity in a living animal to address some of the basic questions that scientists working in all these areas, some of whom we work with, wish to answer, so that they can work out the best ways to design their experiments and use exosomes in medicine.

Exosomes are thought to be key mediators of cell-cell signalling, but functional studies in vivo are limited. We have developed a broad range of in vivo genetic tools to study exosome production by secondary cells (SCs) and exosome function in fly reproduction. These tools include inducible SC-specific GAL4 drivers that allow us to modulate the function of genes of choice in adults, using publicly available fly RNAi lines, and dominant negative and activated constructs. Our key objectives are to:

1. establish a set of markers for different exosomes and identify their fusion specificities: We have already found that not all SC exosomes are identical. Using a range of Drosophila tetraspanin fusion molecules as well as more general exosome markers, we will characterise the different exosome subtypes and test whether they fuse with different cell types in females;
2. block production of SC exosomes to assess their cellular and physiological roles in reproduction: We have several GAL4-inducible tools that should allow us to block different stages of exosome production in SCs. We will test which tools are most effective, then determine how this affects fertility and female post-mating responses;
3. identify and characterise one or more molecules that alter cell function in females after exosome fusion: We have identified strong candidate proteins expressed in SCs that may be involved in exosome signalling, some of which we already know are carried in SC exosomes. We will test by SC-specific knockdown and in some cases, overexpression/activation, whether these molecules, and any new candidates emerging from parallel studies in the lab, mediate exosome function in females, and then determine the precise functions of the proteins that do play a role.

Overall, these studies will not only define the in vivo role of exosomes in reproduction, but are also likely to highlight fundamental functional mechanisms relevant to the exosome field in general.

Potential beneficiaries in academia are outlined in the previous section. Our work has wide-ranging implications, given the postulated roles of exosomes in health and disease, and their potential uses for biodelivery of drugs. We have indicated in the previous section how our work could be developed in academia within these areas.
Other areas of potential impact are:
1. Clinical Medicine
Three key findings that could emerge from our work are: i. the identification of different types of exosome; ii. the mechanisms by which exosomes fuse to their target cells in vivo and iii. cargo molecules that play a role in exosome function. As discussed in the previous section, the knowledge gained from these studies could ultimately impact on the clinic in several areas, such as reproductive medicine, stem cell therapies and cancer biology. For example, cancer cells are thought to increase their exosome production compared to normal cells and potentially alter the exosome contents. We are already setting up to test whether molecules like c-Src are selectively packaged in exosomes as they are in flies, through our pump-priming funds from CRUK. If this is the case, it will be important to test whether Src inhibitors in cancer patients reduce circulating exosome production, or whether these inhibitors affect cell-cell transfer of exosomes. We can take the same approach with inhibitors of other signalling pathways we are testing or using RNAi knockdown in the cells producing exosomes. Success in any of these areas is likely to inform clinicians about possible combinations of drug therapies that might block exosome function in addition to more classical tumour properties, and it might also suggest new molecules to detect in diagnostic screens for exosomes.
2. Pharmaceutical Industry
As discussed above, the translation of our work into human systems may suggest rational combinatorial drug treatments to suppress active cancer cell exosomes in addition to other tumour cell properties. If such approaches involve drugs under development, such as the new Src inhibitors, we will develop these studies with the pharmaceutical industry with whom our clinical collaborators have links. There is also of course the possibility that we may identify a new drug target for modulating exosome function that could be developed with a pharmaceutical company. If our work identifies good markers for all exosomes or subpopulations of exosomes, it may be possible to find parallels in human cells and set up genetic and/or small molecule screens to identify inhibitors of secretion. And finally if we develop ideas concerning approaches by which exosomes might be secreted, loaded or targeted in biodelivery, this may help in the development of this technology, given that the basic biology of exosomes is still relatively poorly understood.
3. General Public and Schools
We think it is important to disseminate our work to a wider public for two main reasons. First, our studies are frequently targeted at fundamental problems that are ultimately relevant to human health. Our experience is that this generates significant interest in the media. We think the main theme of our work, that males release tiny packages of active molecules to reprogramme cells in females after mating, will be both surprising and fascinating to the public, particularly if there are clinical implications. And the fact that we can visualise the exosomes in action should spark the imagination of an audience that knows very little about these structures at present. Potentially it might even have controversial implications concerning the biological interactions between men and women. Second, our work has advantages in the context of the 3Rs. We strongly believe that aspects of physiological research must be pursued in vivo, and our work exemplifies how simpler organisms can avoid some of the potential ethical issues, while answering questions of fundamental importance to human and other animal health.