Integrating systems biology and transgenic technologies to unlock the secrets of Sertoli cell development and function

The testes function to produce germ cells (sperm) and to make androgens (testosterone). These actions are essential for normal male fertility, male behaviour and for general adult male wellbeing. This project is designed to find out how the different component cell types in the testis interact to ensure that the testes develop and function normally. This information will be combined with previously published data to create a computer model of how the testis works and this model will be made available to allow scientists to test novel scenarios and hypotheses of testis function.
The testes are composed largely of an array of small tubules (the seminiferous tubules) in which the sperm develop supported by a group of cells called the Sertoli cells. The seminiferous tubules are surrounded by another cell type called the peritubular myoid cells which are thought to help sperm development. Androgens are secreted by the Leydig cells and these are found lying in between the seminiferous tubules. Early formation of the testis is known to be dependent upon the Sertoli cells. They develop first and then act to promote the subsequent differentiation of other cell types. What is much less clear, however, is how important the Sertoli cells are for later testis development and for overall function in the adult. In other words, we don't know if the Sertoli cells orchestrate overall testicular function or just act within the tubules to maintain sperm development. This is important because it is fundamental to our understanding of testis biology and normal development of the testis is essential for adult fertility and normal adult health.
For studies designed to examine the overall function of a particular cell type the most effective approach is to destroy that cell and see what happens to organ development or organ function in the adult. Until recently that was only possible using cell-specific toxins and these only existed for a very small number of cell types. Recent developments in mouse transgenics, however, now mean that almost any cell type can be targeted. The new techniques depend upon rodent insensitivity to the effects of diphtheria toxin which can be lethal in the human. In humans, diphtheria toxin binds to a receptor present on the cell surface (called the diphtheria toxin receptor or DTR) allowing part of the diphtheria toxin molecule to enter the cell and destroy it. Mice normally lack this DTR but using transgenics it is now possible to create mice that have the DTR on specific cell types. This then makes those cells sensitive to the toxic effects of diphtheria toxin. We have now made a line of mice that have the DTR on the Sertoli cells. Preliminary experiments have shown that when we inject diphtheria toxin into these mice it very quickly kills the Sertoli cells but does no other damage to the mouse. What we now propose to do is find out what happens to the other cell types in the testis when we kill some or all of the Sertoli cells. In addition, because we can choose when to inject the diphtheria toxin we can find out whether the function of the Sertoli cell changes as the animal develops. For example, will Sertoli cell death in the adult animal have the same effect on the peritubular myoid cells as cell death in the newborn?

Data from this work and from previous studies will be brought together within our modelling programme (Biolayout Express 3D, see www.biolayout.org) to create a computer model of how the cells and molecules within the testis interact to promote correct testis function. Together these computer and mouse models will allow us to determine the complex ways in which the Sertoli cell interacts with other cell types in the testis and how they act to promote testis growth and ensure fertility and wellbeing in the adult male.

Adult male fertility and wellbeing are dependent upon appropriate fetal and postnatal testis development. This proposal is designed to develop our fundamental understanding of testis development through a combination of cell ablation studies and generation of a computer modelling system. Early events in testis development are induced by Sertoli cell (SC) differentiation which leads to formation of the seminiferous tubules and development of the fetal Leydig cells (LC) and peritubular myoid cells (PTMC). We do not know, however, the extent to which the SC remain central to testis biology thereafter, beyond local maintenance of spermatogenesis. To identify the role of the SC in overall testis biology we have generated a mouse line that expresses the simian diphtheria toxin receptor (DTR) specifically in the Sertoli cells. Mice normally lack a functional DTR so that treatment of these animals with diphtheria toxin (DTX) will lead to specific ablation of the SC. This will allow us to determine what happens to the testis if SC are ablated at different stages of development. In preliminary experiments with neonatal mice we have shown that a single injection of DTX causes near total SC ablation with no other harmful effects on the mice. We now propose to use this model system to determine the role of the SC in (i) PTMC, LC and gonocyte survival, function and development in the fetus (ii) adult LC differentiation, development and function and (iii) PTMC function and differentiated status in the adult animal. In addition, through partial ablation of the SC, our studies will show how adaptive the remaining SC are during proliferation and following final differentiation. Data from these studies will be integrated into a comprehensive in silico model of testis development and function. This will enhance our understanding of the ongoing biological processes and will provide the opportunity to test hypotheses related to testis development, function and dysfunction.

Beneficiaries
Immediate beneficiaries will be research professionals. It is anticipated that this will expand over time to include livestock breeders, veterinarians and healthcare professionals and the ageing population as this research impacts on our understanding of testis development and its role in determining adult health and fertility. Through public engagement there will also be benefit to the general public.

How will they benefit
The basic nature of the proposed work means that it is unlikely to have immediate impact beyond research professionals but, in the longer term, the impact of this work will extend to those who are engaged in animal breeding, infertility and male reproductive health. Cattle fertility in the UK has been in declinefor a numbers of years which has a major impact on the economics of the industry (1). The underlying reasons for this subfertility are diverse but seriously deficient semen quality is seen in a significant number of young bulls indicating that male reproductive dysfunction is an important contributory factor (2). Human infertility is also an increasing problem in the developed world with male factors the single biggest cause. There is also good evidence that human reproductive health is declining and that the origins of this decline are during fetal testicular development. Increased understanding of the basics of male reproduction will, therefore, have an impact on the pregnant woman or animal as well as the professionals who advise or maintain them. In addition, there is growing evidence that testicular androgens contribute significantly to the well-being of middle-aged and ageing males. Low testosterone, for example, is linked to metabolic syndrome which increases the likelihood of developing cardiovascular disease or diabetes during adult life and dementia during ageing. The studies outlined here will impact directly on our understanding of the development and maintenance of the Leydig cells - the testicular cell type that produces androgen. In the longer term this is likely to increase the chances of ameliorative treatment for these conditions and, thereby, have impact on healthcare professionals and patients.
This project will provide benefit to the general public through a greater understanding of reproduction and reproductive health and the issues facing both the individual and wider society. Greater awareness of the health issues will, for example, encourage pregnant women to avoid the kinds of lifestyles and behaviours that may affect reproductive development.
Economic Impact: We don't expect data or models to arise from this project that would be commercially sensitive. However, we have previous experience of commercial licencing of mouse models arising from our work (3), and we will liaise closely with Edinburgh Research and Innovation to protect and exploit any IP arising from our work.
Training: The new staff will join vibrant research laboratories, which are actively developing and exploiting new methodologies. They will benefit from the research environment and they will develop experience and knowledge of techniques in transgenics, molecular genetics, immunohistochemistry, transcript measurement, stereology and systems modelling, all of which are in both academic and commercial demand. TF acts as convenor of an undergraduate course in systems biology at the University of Edinburgh and students will receive training in computer modelling using and populating our in silico model. Staff will also be expected to attend relevant courses run by the lead Institutes which are designed to improve skills such as time management and staff supervision and they will be encouraged to join the public engagement outreach of the group, and will receive formal training on media and public engagement.
(1) Royal M, et al 2000 Vet J 160, 53-60. (2) Revell SG et al 2007 http://www.bsas.org.uk/downloads/animalbytes/Dairycow_Fertility.pdf (3) Smith et al 2011, CardioRes 90,182