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

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

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.

Technical Summary

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.

Planned Impact

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
 
Description Achievement 1) We have identified the roles played by the Sertoli cells in regulating development of the adult testis and in maintaining adult testis function. As part of this project (and in collaboration with Prof S Nef) we developed two transgenic models that allow controlled ablation of Sertoli cells in fetal or post-natal life. These models have been used to show, for the first time, that during development Sertoli cells are required: (1) to maintain the testicular peritubular myoid cells in prepubertal life; (2) to maintain the adult Leydig cell progenitor (precursor) population in the postnatal testis; (3) for development of normal adult Leydig cell numbers and 4) for development of the testicular vasculature. Results also show that in the adult the Sertoli cells are required both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function (as well as their known role in supporting spermatogenesis). Data has been published in Development, PloS ONE and Endocrinology.
Achievement 2) The transgenic lines allow partial (rather than complete) ablation of the Sertoli cell population which has allowed us to model the effects of altering the number of Sertoli cells that develop (eg in the fetus or neonate) or the effects of loss of Sertoli cells in the adult (as happens during ageing). Results show that at all ages the size of the Sertoli cell population is highly predictive of resulting testicular cell composition. A reduction in Sertoli cell number/proliferation at any age will lead, therefore, to a proportional decrease in germ cell and Leydig cell number with likely consequential effects on fertility and health. Data has been published in Endocrinology.
Achievement 3) We have used the mouse cell ablation models, along with techniques that allow measurement of all genes expressed in a tissue (RNAseq), to identify all genes that are expressed only in the Sertoli cells. This will allow us to start investigating the potential mechanisms by which Sertoli cells regulate testis function. In addition, by using these results to interrogate previously unpublished array data from mice lacking specific reproductive hormones (FSH and androgens), we have shown how these hormones control Sertoli cell gene expression. Data has been published in BMC Genomics
Achievement 4) In a collaboration between this project and BBSRC grants BB/JO1446X/1 and BB/I001107/1 (Tom Freeman, PI), we have developed of a new modelling framework that includes: i) a biologist-friendly pathway modelling language (mEPN), ii) example pathway models that illustrate the power of this graphical notation scheme to depict a diverse range of pathway systems and iii) a simple but sophisticated method that supports model parameterisation according to accessible biological information. Published in PLoS Biology.
Overall, the wide-ranging success of the research carried out in this project has fundamentally changed our understanding of testis biology, with significant implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.
Exploitation Route Data from this project is having a major impact on our understanding of testis biology. This is being re-enforced by academic reviews generated by the PIs during the course of the project and afterwards, and the availability of the modelling pathways developed through our publication in PLoS Biology. From an impact perspective, the most important finding from this study is that altered Sertoli cell number affects Leydig cell number (thereby reducing testosterone and increasing chances of ill health). This is relevant to both human development and ageing and should be of interest to a number of sectors. With respect to ageing this is being taken forward by Prof Smith who is funded by the MRC to investigate the role of androgens in lifelong male health. With respect to development Prof O'Shaughnessy is funded by the MRC to examine maternal lifestyle/exposure and human fetal development. It is intended that these data will be disseminated through our clinical and academic colleagues to agencies and bodies which influence policy related to human and animal exposure. Clinically, we are disseminating this data to veterinary and medical colleagues.
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The Research group have been invited to several international conferences to speak on the results of this project and interest in the research community is high. Our most recent data for example (published in Endocrinology) relates to the role of the Sertoli cell in development and ageing and shows that loss of Sertoli cells is probably central to Leydig cell decline and loss of testosterone during this process. Reduced testosterone is known to lead to many of the problems of ageing in men and so development of strategies to prevent Sertoli cell death during this process is likely to be of interest to several sectors.
First Year Of Impact 2017
Sector Digital/Communication/Information Technologies (including Software),Healthcare,Manufacturing, including Industrial Biotechology
 
Description MRC DTP
Amount £70,000 (GBP)
Organisation MRC Doctoral Training Program 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 05/2018
 
Description Response mode research grant
Amount £162,994 (GBP)
Funding ID MR/L010011/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2014 
End 01/2017
 
Title Development and roll out of an in silico modelling system, 
Description There is a need for formalised diagrams that both summarise current biological pathway knowledge and support modelling approaches that explain and predict their behaviour. Here, we present a new, freely available modelling framework that includes a biologist-friendly pathway modelling language (mEPN), a simple but sophisticated method to support model parameterisation using available biological information; a stochastic flow algorithm that simulates the dynamics of pathway activity; and a 3-D visualisation engine that aids understanding of the complexities of a system's dynamics. We present example pathway models that illustrate of the power of approach to depict a diverse range of systems. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact Only just published. No impacts as yet 
URL https://www.ncbi.nlm.nih.gov/pubmed/27509052
 
Title Testicular cell ablation using diphtheria toxin 
Description Generation of transgenic animals which allow ablation of specific cell types in the testis 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact Use of this technique has resulted in generation of nine reviewed publications 
URL https://www.ncbi.nlm.nih.gov/pubmed/29453574
 
Title Transgenic mouse expressing DTR on the Sertoli cells 
Description A mouse model has been generated which expresses the diphtheria toxin receptor only on the Sertoli cells. This allows controlled ablation of the Sertoli cells which then provides information about the normal function of these cells 
Type Of Material Biological samples 
Provided To Others? No  
Impact Two publications to date: Rebourcet D, O'Shaughnessy PJ, Monteiro A, Milne L, Cruickshanks L, Jeffrey N, Guillou F, Freeman TC, Mitchell RT, Smith LB. Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis. PLoS One. 2014 Aug 21;9(8):e105687 Rebourcet D, O'Shaughnessy PJ, Pitetti JL, Monteiro A, O'Hara L, Milne L, Tsai YT, Cruickshanks L, Riethmacher D, Guillou F, Mitchell RT, van't Hof R, Freeman TC, Nef S, Smith LB. Sertoli cells control peritubular myoid cell fate and support adult Leydig cell development in the prepubertal testis. Development. 2014 May;141(10):2139-49. 
 
Title Testis RNAseq 
Description Global transcript study by RNAseq which has identified all transcripts present in adult mouse testis and has all identified germ cell-specific and Sertoli cell-specific transcripts in the adult mouse testis 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact We have been able to identify Sertoli cell genes regulated by follicle-stimulating hormone and by androgen 
 
Description Lee Smith Edinburgh 
Organisation University of Edinburgh
Department MRC Centre for Reproductive Health
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided expert knowledge and techniques such as stereology to study of testicular development and function in transgenic animals
Collaborator Contribution The collaborators have provided samples from transgenic animals for study and have provided data which contributes to the overall study
Impact Currently there are two full papers published (details given under publications). A website is under construction.
Start Year 2008
 
Description American testis workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presented plenary talk related to the data from this award
Year(s) Of Engagement Activity 2017
URL https://andrologysociety.org/meetings/testis-workshop/past-workshops/searchable-program-schedule.asp...
 
Description Androgens conference (London) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk to present current data. Led to discussion/organisation of future projects
Year(s) Of Engagement Activity 2014
URL http://www.precisionmedicines.com/androgens-2014.html
 
Description European congress of Andrology (Barcelona) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited presentation to discuss ongoing studies. Impacts as yet unknown
Year(s) Of Engagement Activity 2014
URL http://onlinelibrary.wiley.com/doi/10.1111/andr.2014.2.issue-S2/issuetoc
 
Description Klotz symposium on reproductive development (Paris) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was a scientific talk at an annual symposium held in Paris; this year the topic was Human reproduction. The intended purpose was to inform other scientists as well as clinicians. The proceedings appeared in press, impacts are not known
Year(s) Of Engagement Activity 2014
URL http://www.em-consulte.com/en/article/895896
 
Description World congress of Reproductive Biology (Edinburgh) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited presentation to describe cell ablation studies in the testis. Potential collaborative studies were identified.
Year(s) Of Engagement Activity 2014
URL http://www.wcrb2014.org/