Regulation and functions of male-derived shed microvesicles in Drosophila reproduction

Although males and females share a common goal when they mate, the production of offspring who carry copies of their genes, there is at the same time an important conflict between them. While on the one hand, males of many species can best maintain their gene pool by mating with many females and preventing other males from competing, females are often best served by sampling sperm from multiple males. Three years ago, we started to study the ways in which males have adapted to fight this battle of the sexes. We speculated that males had developed specialised ways of delivering signals to females that made their sperm work at their best and also stopped other males from mating with these females. We decided to work with the fruit fly, partly because it was already known that male flies had developed ingenious ways to fight the reproductive battle. They assemble a plug in the female uterus that blocks remating and also alter female behaviour so she rejects subsequent advances from other males. Flies are ideal for studies of this kind because they can be easily manipulated to identify the genes involved and find out how they work. About 70% of all genes known to be involved in human disease are also found in flies. Lots of the basic mechanisms in human biology were originally studied in flies or other simple organisms before being looked at in humans.

We are studying a male gland in flies called the accessory gland that seems to share many features of the human prostate and seminal vesicles, which generate most of the main components of semen. We have already shown that one type of cell in this gland secretes tiny membrane-bound packages that are passed into females on mating and fuse to sperm. These packages, called exosomes, are required to alter female behaviour after mating. Remarkably the human prostate also produces exosomes that fuse to sperm. Our work is starting to reveal how these exosomes are made and how they affect sperm, and we are beginning to see how defects in exosome production might be involved in aspects of prostate cancer, ideas that we are now following up with clinical colleagues.

Recently we discovered that another type of cell in the accessory gland secretes larger membrane-bound structures called shed microvesicles (SMVs) that are also passed to females in huge numbers during mating. These vesicles carry several key proteins that are already known to drive changes in the female after mating, like increased egg laying, sperm storage, mating plug formation and altered remating behaviour. SMVs coalesce to make a mating plug in one part of the female reproductive tract, while in other parts, they remain intact, but release one of their attached proteins, which then binds to sperm and allows it to be stored. These SMVs may also bind to female cells, raising the possibility that they could communicate to females in this way.

Several important fly molecules involved in this process are related to mammalian reproductive proteins. In fact, the discovery of SMVs in fly semen explains a mystery in both fly and mammalian reproduction - several proteins secreted into semen look like they should be attached to membranes, and not secreted. We will study how SMVs are made, work out what they do in females after mating and how these events are controlled by proteins made in the accessory gland. This work will not only help us to understand fundamental principles of reproduction, it may give us new ideas for reproductive therapies and contraception. SMVs are also important in blood clotting, inflammation and growth. We have evidence that the basic controls on these different processes are similar, opening up the possibility that our work will provide new insights into diseases where these processes go wrong. Our proposed work, looking into an important, but previously intractable problem, may therefore impact on several areas of biology relevant to human health.

When males mate, they must deliver sperm and a diverse range of seminal proteins to different parts of the female reproductive tract that fulfil several key reproductive functions. In studying this process in flies, we discovered that epithelial cells in the male accessory gland (AG) produce shed microvesicles (SMVs) that carry key seminal proteins into females. We have developed genetic tools to analyse the biogenesis and functions of these SMVs in the AG, which we will combine with available mutants, cell type-specific knockdown and overexpression to genetically analyse SMV biology in fly reproduction. Our key objectives are to:
1. genetically dissect the process of SMV biogenesis by screening a series of very strong candidate genes identified through microarray analysis;
2. identify the genes that target SMVs to different regions of the female reproductive tract and that then allow release of effector proteins from these vesicles, which promote sperm storage, suppress immune responses and alter female behaviour;
3. determine the functions of SMVs in reproduction by genetically blocking their secretion, using the tools developed in our first objective.
Several of the key molecules expressed by the AG that are vital for male reproduction have already been characterised genetically. However, our data suggest that these proteins interact with SMVs to perform their functions. Our proposed study should elucidate the roles of SMVs in these processes and determine how their interactions with seminal proteins change after mating. SMVs are not only implicated in reproduction, but also in blood coagulation, inflammation and diseases such as cancer, so our ability to study their function in a system that can be easily genetically manipulated may provide significant insights into several processes that rely on these poorly studied signalling structures.

Potential beneficiaries in academia are outlined in the previous section. Our work has wide-ranging implications, given the postulated roles of SMVs in animal biology, 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
Key findings that could emerge from our work are the identification of : i. molecular genetic mechanisms by which SMVs are formed in secreting cells; ii. molecular genetic mechanisms controlling SMV coagulation; iii. general molecular mechanisms by which SMVs release attached proteins (eg., SP, Acp36DE); iv. molecular genetic mechanisms promoting SMV attachment to cell membranes and potential crossing of epithelia. 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, blood coagulation and inflammation. Although SMVs are implicated in all these processes, we know remarkably little about several aspects of their basic biology, so at this point, it is really difficult to determine what the impact of our studies will be. But our link with Ian Sargent, a key figure in Oxford in the clinical exosome and SMV fields, will allow us to make contact with the appropriate group of clinicians to take any discoveries forward.
2. Pharmaceutical Industry and Biotechnology
As discussed above, the translation of our work into human systems may suggest new ways of intervening in processes like blood coagulation, which are believed to be initiated by SMV signals, although this is likely to be a relatively long-term goal. But if we learn how SMV secretion is controlled through our studies, and how these structures deliver their contents to the appropriate target site, this could suggest very novel ways to target hydrophobic or hydrophilic drugs to specific sites in the body. Again, at present the possibilities are tantalising because this is such an unexplored area. It is not possible at present to develop concrete ideas of how our work may impact in these areas, but our contacts with the Wood lab should allow us to exploit opportunities as they arise.
It is possible that SMVs could deliver novel compounds to females during insect reproduction. Although we think it will probably be too costly to develop this commercially, we will explore this possibility with colleagues in this area.
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 packages of active molecules to reprogramme female behaviour and optimise male fecundity, will be both surprising and fascinating to the public, particularly if there are clinical implications. And the fact that we can visualise SMVs 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 (replacement). 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.