Sex Peptide-dependent microcarrier signalling in reproduction

Reproductive biology affects our day-to-day lives in multiple ways. Many patients require assisted reproductive technologies, like in vitro fertilisation, to allow them to have children. Animals used in agriculture or threatened by extinction can benefit from similar approaches for selective breeding or to increase the chances of successful reproduction. Increasingly, pest control strategies are targeting reproduction to reduce numbers of insects that cause disease or damage crops.

Although sperm from the male are absolutely essential to fertilise the egg, seminal fluid also has a number of important roles, activating sperm function in females, affecting female reproductive organs, like the uterus, to enhance the chances of a successful pregnancy, and in some animals, like the fruit fly, reprogramming the female's brain, so that she rejects other males who try to mate with her. It is clear that understanding how seminal fluid can produce all these effects is likely to inform development of improved reproductive technologies. However, the processes involved are complex and studies in simple animals like the fly are needed to pick apart this complexity.

Flies look very different from humans, but they have equivalents of about 70% of the genes found within us. Perhaps surprisingly, they have frequently been used to unravel the fundamental mechanisms, which control many important biological processes, such as memory, sleep and development, and to study the genetic defects that underlie several major human diseases, such as cancer, diabetes and Alzheimer's.

Pioneering studies have shown that a small fly protein in seminal fluid called Sex Peptide plays a pivotal role in sending signals to females during mating. It is able to dramatically increase the female's ability to produce progeny, using stored sperm from the mating, for a period of more than a week. Although there is no human equivalent of Sex Peptide, several molecules that control Sex Peptide's activity in females belong to families of proteins that are also found in human seminal fluid. Furthermore, we have recently shown that Sex Peptide is stored and delivered to females on specialised structures called microcarriers. The structure of microcarriers is controlled by Sex Peptide, but also by a set of enzymes that are very highly evolutionarily conserved in the animal kingdom. Understanding how Sex Peptide and microcarriers co-operate together to enhance fertility is therefore likely to provide clues concerning the general mechanisms by which seminal fluid proteins carry out their reproductive functions in other animals, including humans.

We will now use the powerful techniques available to us in flies to work out how Sex Peptide controls microcarrier structure, how critical it is for Sex Peptide to be loaded on microcarriers for it to be properly delivered to females during mating, and what other proteins are involved in microcarrier-dependent, male-to-female signalling. Our experiments will identify new seminal proteins that are loaded on to microcarriers, and reveal how they are loaded and how they are dispersed when they arrive in females. They will also show how defects in these different molecules and processes affect different female responses to seminal fluid, such as increased egg laying, sperm storage and rejection of other males.

Our findings will not only allow us to build up a detailed picture explaining how different seminal proteins affect the composition of seminal fluid and fertility in flies, but they will also provide important clues about the ways in which similar molecules perform their roles in seminal fluid of other animals and humans. This could enhance our understanding of male infertility in our own species and in other mammals, and suggest new approaches for optimising fertility in assisted reproductive technologies. It may also indicate new ways of blocking fertility in insects in the design of pest control technologies.

We have recently developed a set of tools in Drosophila to identify and then characterise microcarriers and their association with Sex Peptide. These now allow us to study this interplay and the roles of other interacting proteins in seminal fluid signalling and the induction of female LTR. We will use a range of assays to:

a. Assess the regulation of microcarrier production, structure and release from secretory cells;

b. Investigate the loading of Sfps on microcarriers, using GFP-fusions such as SP-GFP, and the subsequent dispersal of these cargos in females;

c. Identify novel microcarrier cargos through a proteomics approach;

d. Determine the effect of mating on different long-term post-mating responses in females, namely egg laying, sperm competition and reduced receptivity to remating,

and combine these assays, most of which are quantifiable, to assess the effects of knocking down specific gene transcripts expressed in the accessory gland, which we hypothesise play a role in SP-dependent microcarrier signalling. Using these approaches, we will:

i. Determine how the SP network genes regulate SP and microcarrier function, particularly the release and relocalisation of SP in the female reproductive tract;

ii. Test how SP and microcarriers co-operate together to regulate microcarrier structure and delivery of Sfps, and promote fertility;

iii. Identify other microcarrier regulators and cargos from proteomics screens and determine their roles in microcarrier biology and reproductive signalling.

These studies will provide the most comprehensive picture to date of how specific proteins and signalling structures function together in seminal fluid to enhance the fertility of females and suppress competition from other males. Since many proteins we study are evolutionarily conserved or members of conserved protein classes, our findings should be relevant to fertility in other animals and the technologies employed to modulate it.