Functional significance and regulation of the reproductive 'transferome'

Even very simple creatures need to co-ordinate their activities. For example, the sea anemone has a simple net of connected neurons with which it can control the movements within its body wall. As organisms increase in complexity, there are ever more examples where coordinated control of bodily processes is required. We often know a lot about how individual components in these systems might function. However, we have a serious lack of knowledge about how groups of gene products are controlled in the highly precise and flexible way they often are. This is systems biology and is recognized as an increasingly important way in which to understand the complex world around us.

Our focus is on a group of vitally important semen proteins transferred along with sperm - the 'transferome'. It has been realized for many decades seminal fluid proteins are far more than a simple sperm buffer. In fact they can cause profoundly important effects on female behaviour and physiology. These effects have been best studied in the fruitfly, which we use as the model system here. However, similar effects are also seen across a huge variety of animal taxa. It has been reported recently that the transfer of seminal fluid proteins by human males causes changes in the expression of immune genes in the female cervix. This is thought to prepare the womb for implantation as well as protecting against sexually transmitted infections.

In the fruitfly there are about 130 semen proteins making up the transferome. They are made mostly in the male accessory glands (the fly equivalent of the human male prostate) the ejaculatory ducts and a few in the testes. They result in a huge variety of vitally important effects: they cause females to lay more eggs, to eat more (and of different types of foods), to be less sexually receptive to males, to switch on immune genes, to retain more sperm in storage, to show altered patterns of water balance and to sleep less!

We also know from our own work that males can respond in a highly sophisticated and individually flexible manner to their social and sexual environment. When males are exposed to rivals they mate for longer when they meet a female and transfer more transferome components during those longer matings. This results in a higher number of offspring. Furthermore, there is recent evidence to show that the composition of the transferome can change in response to social context.

Despite the importance of the transferome to both males and females and its high degree of flexibility, we know next to nothing about how it is controlled. However, we have gathered strong background data and have excellent experimental tools in the fruitfly to tackle this omission, giving us a unique and unparalleled opportunity to investigate for the first time the control of this complex and important system.

We hypothesise that an effective way to regulate 130 individual components of the transferome is to manage them in 'sets' controlled by the same activator / inactivator. This facilitates the quick and co-ordinated release of groups of substances as soon as they are required, rather than trying to make them all individually from scratch.

We predict that this level of control is achieved in practice by transcription factors that turn on the expression of genes and by different types of small RNAs that then bind to, repress and 'manage' gene sets. Our investigations provide strong evidence that is consistent with these predictions. What we propose here are important tests of these ideas by experimentally altering directly these different types of gene regulation and testing the effects on the control and function of the transferome. This will elucidate how it is that the transferome can be regulated with robustness, precision and flexibility.

The reproductive transferome comprises the extracellular non-sperm molecules transferred during mating. These cause a remarkable remodelling of female behaviour, physiology and gene expression. Recent work shows that transferome quantity and composition can be modified according to the socio-sexual context. Such responses are highly precise, robust and flexible and result in significant fitness effects.

However, despite significant research into the functions and fitness effects of transferome components, we know very little about how the genes that encode them are regulated.

The aim is to address this omission by providing the first system-wide and hypothesis-driven investigation into the regulation of the reproductive transferome.

The work will elucidate in general terms how evolutionary robustness and flexibility can be conferred by specific regulatory features.

Our proof-of-principle research predicts that transferome genes are regulated in a highly structured way by transcription factors, microRNAs and other small RNA regulatory elements. Sets of genes show evidence of shared regulation at the 5', 3' or both gene regions, and differences in the number / type of shared regulatory sequences. The data suggest novel regulatory 'hubs' that control distinct sets of transcripts.

We will manipulate global regulators - transcription factors, microRNA (Drosha, Dicer-1) and small interfering RNA (Dicer-2) biogenesis components - and test the effect on the regulation of transferome gene expression by using simultaneous mRNA and small RNA sequencing on the same biological samples. We will also test the phenotypic effects of these manipulations on the ability of the transferome to respond effectively to the social environment.

These experiments will show whether the diversity of regulatory mechanisms confers stability and flexibility on the composition of the transferome - giving general insight into the control of fundamentally important gene network.

1. WHO WILL BENEFIT FROM THIS RESEARCH

We anticipate 4 main areas for potential impact:
1. Regulation of biological systems. We perceive significant general interest in terms of systems biology. The observed level of regulatory control that is evident in the reproductive transferome in response to the environment predicts that it exhibits robust, sophisticated, tight yet flexible regulation. There is intense interest in understanding how biological systems can exhibit these characteristics. We hypothesise that efficient regulation can be achieved through management of transcription factor generated mRNAs by post-transcriptional regulation by small RNAs. Our bioinformatic analyses conducted for this proposal support this scenario and also give evidence for putative miRNA regulatory hubs to control distinct gene sets, facilitating the modulation of the absolute and relative level of transferome components. We therefore have an unparalleled opportunity to reveal the mechanisms that confer both stability and the ability to make precise adjustments to ejaculate composition, and finally, to understand the regulation of an entire gene network.
2. Sexual interactions. The interplay between males and females is a complex and powerful evolutionary force. Such interactions can be co-operative or conflicting. There is intuitive appeal in the study of reproductive adaptations that can be good for males and bad for females, and vice versa. This is a topic that catches the public interest.
3. Husbandry practices for insect control: There is an applied context of this research in terms of the husbandry of insect pests that are mass reared for control. This is being explored through the applied partnerships described above, and also in a new NERC quota studentship (supervisor TC).
4. Communication between signals and receivers: The way in which signals are generated and received is a topic of broad importance and also of public interest.

The main beneficiaries are:
1. Academia: we aim to maximise impact of the research through open access papers, reviews, commentaries, lab research web sites etc. For this project, we plan a research blog to which the research team would contribute in turn.
2. Private sector: we are investigating the potential for conflicts of interest to impact upon husbandry and rearing regimes of insects used for pest control. We have already developed in the private sector the application of knowledge to fruitfly pests in two NERC-funded CASE studentships together with Oxford insect technologies (Oxitec).
3. General public, schools: We have recently devised new exhibition materials for open days and visit days at UEA. We aim to develop teaching resources for use in the Teacher Scientist Network (TSN) scheme. Here we will build models of the different level of gene regulation and see which of them results in greatest stability and co-ordination. Together with our engagement Director in the School, we are tailoring these resources to the National Science Curriculum, for taking into schools via the TSN.

2. HOW WILL THEY BENEFIT FROM THIS RESEARCH
The beneficiaries named above will benefit through:

1. Increased economic competitiveness of UK plc through increased visibility of research outputs and increased engagement with the private sector.
2. Enhanced effectiveness in the transmission of research findings through the academic community.
3. Increased learning and awareness (through school visits) of the opportunities, relevance and range career choices available through academia.
4. An increased range of career options through training in media, communication, business and private sector practices, which could be employed in a range of employment sectors.