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

Lead Research Organisation: University of Oxford
Department Name: Physiology Anatomy and Genetics

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description This grant funded the investigation of lipid-containing cigar-shaped structures, which we had discovered while studying the biology of this gland. These structures, which we now call microcarriers, are released from the epithelial 'main cells' of the Drosophila male accessory gland; we had shown that they carry a key seminal protein involved in changing the behaviour of females after mating called Sex Peptide (SP). Since these structures appear to represent a new form of secretory vehicle, we set out to study how they are regulated and to determine their functions.
We have discovered that:
1. microcarriers contain a lipid-rich centre and carry SP on their surface. Following mating, these structures dissipate and release SP, which binds to the plasma membrane of sperm tails and is stored with sperm in the female sperm storage organs. We conclude that one role of microcarriers in the accessory gland is to act as a long term repository for key seminal proteins that are not fully soluble, and then to allow quick release of these proteins when they arrive in the female reproductive tract.
2. microcarrier formation is controlled by SP. SP null mutations and main cell-specific SP knockdown both lead to the production of highly enlarged, fused lipid globules that are no longer properly loaded with their cargos, which become diffusely distributed throughout the defective structures. Therefore SP has a new, previously unrecognised, function in males, the control of microcarrier formation, in addition to its well-established roles when transferred to females.
3. using inducible knockdown, we find that once defective microcarriers are formed in the absence of SP, they cannot recover their structure, even if SP is resupplied. In this latter scenario, males remain unable to induce long-term stimulation of ovulation in females, even though their seminal fluid contains SP. These experiments surprisingly suggest that SP's function in microcarrier biogenesis may explain some of the changes in mated females associated with SP loss in males.
4. a specific lipid metabolic enzyme is essential for normal microcarrier release from main cells, highlighting a very specific aspect of lipid metabolism that is involved in this process and highly upregulated in the main cells. Remarkably the enzyme involved is widely expressed in mammalian secretory cells and has roles in bile secretion, myelination and breast milk, and is frequently upregulated in cancer, suggesting that there may be functions for this new type of secretory vehicle in higher organisms too.
As of March 2018, we are assembling a first manuscript to describe the role of SP in microcarrier formation and function. This has taken more time than anticipated for two reasons: first, because the mechanisms we describe have not been previously reported and we have therefore needed to verify them by multiple approaches; second, because we have found that the classic SP mutant employed in many previous studies appears to carry a second mutation that affects male fertility, again requiring us to use additional approaches to confirm our findings. This latter observation is another significant finding of our studies.
Exploitation Route We have identified a new mechanism by which partially soluble molecules can be stored and then delivered to a specific location in response to as yet unidentified cues (ie. in the female reproductive tract). Going forward, we envisage the following:
1. characterisation in mammalian cells of the effects of overexpression of the novel lipid metabolic enzyme we have implicated in microcarrier formation, initially in cell culture, to test whether microcarriers can be formed by these cells;
2. analysis of microcarrier components in flies and humans by proteomics and lipidomics; determine what cargos are required to form normal microcarrier structures;
3. others, including our collaborators, could then test whether this system might be engineered for delivery of specific drugs, etc;
4. other groups or our collaborators could determine whether the microcarrier system might be employed in any pest control strategies, where they are used to deliver a pest control molecule to females in the wild.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Since 2016, we have presented aspects of our work in several engagement activities, particularly with schoolchildren. They are particularly interested and surprised to hear how using flies can impact our understanding of cancer and basic biological processes involved in human disease. Feedback is routinely positive and some of the individuals who come from schools that have previously very limited links to Oxford have applied here, a key objective of some of the events that we hold. We are now also involved in the UNIQ and UNIQ+ schemes in Oxford, where schoolchildren and undergraduate students from disadvantaged backgrounds come to Oxford to experience the academic opportunities here: schoolchildren stay for a week and receive a range of academic sessions, including a cell biology tutorial that I participate in, and undergraduates work in my lab on accessory gland projects inspired by our funded studies. These students often successfully apply to Oxford (and other world-class Universities) subsequently.
First Year Of Impact 2016
Sector Education
Impact Types Cultural,Societal

 
Description MRC DPhil studentship
Amount £110,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description Regulation of exosome heterogeneity and function
Amount £621,117 (GBP)
Funding ID BB/R004862/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2021
 
Description Responsive mode award
Amount £518,971 (GBP)
Funding ID BB/N016300/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2016 
End 09/2019
 
Description Wellcome Trust Chromosome and Developmental Biology Programme DPhil studentship
Amount £120,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 09/2017
 
Title Genetic model of dense-core granule secretion and its regulation 
Description We have developed the Drosophila secondary cell as a new genetic model for dense-core granule (DCG) biogenesis. We have developed new tools to mark DCGs in living cells, eg., GFP-GPI, and used this system to show that BMPs, loaded in DCGs, control secretion in an autocrine fashion. BMPs are implicated in insulin secretory regulation in beta cells and we postulate that similar mechanisms may be involved. Relevant to 3Rs. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2016 
Provided To Others? Yes  
Impact Redhai, S., Hellberg, J.E.E.U., Wainwright, M., Perera, S.W., Castellanos, F., Kroeger, B., Gandy, C., Leiblich, A., Corrigan, L., Hilton, T., Patel, B., Fan, S.-J., Hamdy, F., Goberdhan, D.C.I., Wilson, C. (2016) Regulation of dense-core granule replenishment by autocrine BMP signalling in Drosophila secondary cells. PLoS Genet. 12, e1006366. £518,971 from the BBSRC for a project entitled "Linking reproductive behaviour and dense core granule biogenesis in secondary cells of the Drosophila male reproductive system" Wilson, C. PI. (August 2016 - August 2019). - BB/N016300/1 Several invitations and platform presentations at meetings: 2016 & 2017 US Drosophila Conference. 
 
Title New model for insulin signalling-/diet-linked, age-related degenerative disease in Drosophila 
Description We identified a mutant in tumour suppressor gene Pten, which has an age-related muscle degeneration phenotype and showed that this defect was mTORC1-dependent and reduced mitochondrial integrity. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact Mensah LB, Davison C, Fan SJ, Morris JF, Goberdhan DC, Wilson C. (2015). Fine-Tuning of PI3K/AKT Signalling by the Tumour Suppressor PTEN Is Required for Maintenance of Flight Muscle Function and Mitochondrial Integrity in Ageing Adult Drosophila melanogaster. PLoS One 10:e0143818. 
URL http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143818
 
Description Cell-cell communication via lncRNA transfer 
Organisation University of Toronto
Department Donnelly Centre for Cellular and Biomolecular Research
Country Canada 
Sector Academic/University 
PI Contribution Expertise in analysing exosome- and microcarrier-mediated signalling between cells.
Collaborator Contribution Expertise in lncRNA expression and function in Drosophila.
Impact Canadian Institutes of Health Research grant 2019 - 2024. We will provide training, tools, experimental input and expertise as collaborators in analysing accessory gland cell biology.
Start Year 2019
 
Description ChemiBio Hub meeting - University of Oxford/industry conversation - 31.7.2015 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation poster presentation
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Discussions with pharmaceutical company representative at meeting led to subsequent visit to the labs of my collaborator and me and discussion of possible collaborative overlaps.

Discussion concerning collaboration and possible translation of our work in progress.
Year(s) Of Engagement Activity 2015
 
Description Medicine & Biomedical Sciences Taster Day, 28 May, 2016 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact We ran a whole day event at St. Hugh's College and our Department for students from schools that rarely send students to Oxford, including introductory talks, a lecture, mock interview, Q&A and then in the afternoon, research demonstrations related to our ongoing research on Drosophila, exosomes, etc.
Year(s) Of Engagement Activity 2016
 
Description Open Days in Oxford (St Hugh's College) for schoolchildren 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I meet small numbers of schoolchildren, typically three times per year to discuss studying at Oxford in medicine and biomedical sciences, and highlighting the importance of research in the Oxford academic environment.

I have had school work placements in my lab linked to these sessions. We have also has applications to Oxford from students studying at schools that do not have a history of student applications to Oxford, which I regard as a really important aspect of these Open Day sessions.
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
 
Description Press coverage of published work 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact A press release relating to Fan et al. (2015) Oncogene Oct 5. doi: 10.1038/onc.2015.363 was covered in several national newspapers and science websites - Daily Telegraph, Sun, Daily Mirror - http://www.mirror.co.uk/lifestyle/health/cancer-tumours-could-starved-death-6578860, medicaldaily, financialexpress, GoodNewsNetwork, medicalxpress
Year(s) Of Engagement Activity 2015
URL http://www.dpag.ox.ac.uk/news/identifying-cancer2019s-food-sensors-may-help-to-halt-tumour-growth
 
Description School Visit (Kent) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Presentation and Q&A session to post-GSCE schoolchildren from a region around St. Olave's Grammar School in Orpington, Kent. Discussing medicine and biomedicine as a degree and career option, admissions to Oxford, etc. After the Q&A session, I answered individual questions from students, two of whom indicated that their views on the most appropriate career choices had changed as a result of the meeting. I also received e-mails of thanks from the co-ordinating teacher at the meeting and some parents.
Year(s) Of Engagement Activity 2018,2019
 
Description School visit (Canterbury) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact About 50 people attended a careers talk and research discussion in biomedicine and medicine talk, which was followed by questions. Also had supper with a few students with specific interests in these areas.

School arranged visit to Oxford to discuss opportunities for students to study or undertake projects here.
Year(s) Of Engagement Activity 2011,2014
 
Description Super Genes Day - 300 Year 12/13 students - demonstration in Natural History Museum, Oxford, March 18, 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact We ran a 1.5 hour demonstration in the Natural History Museum on Super Genes Day, when 300 Year 12/13 students from schools nationally visit for a full day. We were showing students how flies can be used to study the genetics of biological processes and discussing the development of the fly as a model for prostate biology and cancer, which has been developed in my lab. Interactive displays looking at different fly genotypes, high-resolution imaging data and discussing exosome biology. Spoke to studnets and teachers, some of whom asked for advice on using Drosophila in practicals at school.
Year(s) Of Engagement Activity 2016
 
Description UNIQ Alumni Evening, 26 July 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I was involved in a event during a UNIQ event over the summer: students from schools that rarely, if ever, send students to Oxford stay in Oxford for a week and are involved in lectures, practical , tutorials and other events to introduce them to what is available here. I chaired an evening where we had a Q&A session for the students and also discussed our academic backgrounds, our research careers and our current research interests. The students were very engaged and many applied to Oxford.
Year(s) Of Engagement Activity 2016
 
Description UNIQ Summer School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Education week giving students from disadvantaged backgrounds the opportunity to experience University and Oxford environment and to encourage them to aspire to applying to the top national Universities. I am involved in a cell biology tutorial session with a group of ~ 6 students, where they can personally interact with academics and i can discuss our work, as well as basic aspects of cell biology. This session is highlighted by most students as the most useful event of the week. We are aware that many student on this course decide to apply to Oxford and they have a success rate that is typically higher than other students.
Year(s) Of Engagement Activity 2019