Gene targeting and control of ovarian function

Lead Research Organisation: MRC Human Reproductive Sciences Unit

Abstract

In adult reproductive life only only a minute fraction of all the eggs in an ovary laid down in fetal life ever grow to maturity and are released at ovulation in mid cycle where the mature egg can be fertilised and pregnancy established. Each day, factors released from the egg activate the growth of the follicle, but almost all eggs and follicles die until no eggs are left in the ovary at the menopause. During each menstrual cycle gonadotrophic hormones secreted from the pituitary gland stimulate follicle growth towards mid cycle when all but one follicle is selected to continue growth to ovulation This process can be disrupted for instance by excess androgen secretion by the ovary in PCOS. We are investigating the factors within the developing follicle that makes the follicle sensitive to gonadotrophins, and stops follicles from dying, to identify novel targets to manipulate follicle survival to promote or inhibit fertility. When follicle death does not occur in an ordered manner, ovarian tumours may arise. We are developing model systems to analyse the course of ovarian tumour formation and investigating different methods of gene targeting to normal ovaries including ultrasound to provide potential novel treatments to regulate fertility.

Technical Summary

The program will investigate the regulation of follicle activation, growth and atresia, and relate this to the causes of ovarian tumour formation. A principal aim is to develop an alternative model to the multi-ovular rodent to analyse gene function in ovarian activity in a mono-ovular species equivalent to the human through targeted cell-specific time-controlled gene manipulation. Studies in the Dazlko mouse, where all oocytes are lost after birth, showed that adult ovaries lacking oocytes produce steroids and develop ovarian tumours, providing a new model for some types of ovarian tumours in women. Heterozygous Dazlko mice have a reduction of total follicle numbers but increased litter sizes providing a new model for the role of oocyte factors in regulating gonadotrophin sensitivity and resistance to atresia. Although Polycystic Ovaran Syndrome (PCOS) is one of the most common causes of fertility problems in women exacerbated by increase in body weight, and associated with increased androgens, the causes of this disease and the role of androgens in the ovary remains unclear. We are investigating the function of androgens in the ovary both in a sheep model of PCOS, and using transgenc mice in which the androgen receptor (AR) can be removed in specific cell types within the ovary while leaving ARs in other tissues unaffected. Our observations in the Dazl kO mouse that stromal/thecal cells remain functional in ovaries devoid of oocytes and produce androgens potentially associated with tumour development suggest that control of androgen metabolism and regulation of thecal cell lifespan is important. We hypothesise that stromal/thecal cells require to be removed from the ovary in an ordered manner, which normally occurs as follicles become atretic and die. In the absence of oocytes and normal follicle development in our Dazlko mice, this normal pathway of thecal cell demise is absent, there is lack of control of stromal cell function, and this results in tumour formation either through an action of androgens or converted estrogens. Following our identification of the BMP1B receptor as the prolificacy gene in Booroola sheep, we continue collaborative studies on the role of the TGF beta superfamily of proteins, particularly activins and BMPs, in ovarian and pituitary function. Our studies have highlighted the need to develop a new in vivo model to evaluate intra-ovarian factors in the regulation of follicle growth and development in a mono-ovular species rather than relying entirely on the multi-ovulating rodent models for the human. Using our expertise in sheep ovarian and pituitary biology, our ability to monitor and manipulate the endocrine environment in sheep, and our collaboration with Medical Physics to enhance ultrasound-imaging techniques, we are developing gene delivery vectors for cell specific gene targeting to increase or decrease specific gene expression. This will allow for the first time a comparison between multi- and mono-ovular species. Overall these studies may lead to new methods of regulating ovarian function and fertility, and potentially ovarian tumour formation.

Publications


10 25 50
Carlson AA (2006) Cortisol levels are positively associated with pup-feeding rates in male meerkats. in Proceedings. Biological sciences
Dickinson RE (2010) Involvement of the SLIT/ROBO pathway in follicle development in the fetal ovary. in Reproduction (Cambridge, England)
Fraser KH (2006) Acoustic speed and attenuation coefficient in sheep aorta measured at 5-9 MHz. in Ultrasound in medicine & biology
 
Description Member of the Home Office Animal Procedures Committee for 8 years, and was Chairman of the Primate sub-committee for 3 of these years, preparing reports for the APC and minister.
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guidance committee
Impact Prepared several reports and on committee obviously joined discussions re future of research with animals, particularly primates. These included supply of primates from oversees suppliers, report on supposed overbreeding of animals for research, interactions with the pharmaceutical indusrty
 
Description Weatherall Committee on use of primates in research
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
Impact Explained to the committee the reproductive biology of the primate and how there were several unique features that could not be replicated in mouse or rat models. This appeared in the final report.; Empahsised the importance of primates for some research programs in reproductive medicine in the MRC.
 
Description BBSRC Research Grant
Amount £679,367 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start  
 
Description MRC
Amount £808,144 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start  
 
Description MRC Research Grant
Amount £888,149 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start  
 
Description MRC Research Grant
Amount £329,460 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start  
 
Title ovarian tumor model 
Description We are developing a new mouse model of ovarian cancer development which may allow us to track the aetiology of the disease and design new tools for monitoring women. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Local talks with caner experts suggest that this will be a valuable tool - mouse model - when we have fully evaluated it. 
 
Description Development of contrast-enhanced ultrasonography using the sheep ovarian model of microvascular regulation 
Organisation European Commission (EC)
Country European Union (EU) 
Sector Public 
PI Contribution Reagents have been supplied as necessary at no cost. WE have developed the use of the sheep ovary and particularly the corpus luteum as a model for the microvasculature. Our extensive knowledge of the control of ovarian function in sheep and how to manipulate this, together with the surgical skills to allow proper visualisation of the ovaries, and the infrastructure of our sheep facility allowed this unique program to proceed.
Collaborator Contribution The collaboration has allowed us to develop novel methods of contrast-enhanced ultrasound to monitor changes in the microvasculature in real time, by developing new software and supplying the knowledge of the latest technology to achieve our aims. In addition the USA connection has provided rfree reagent for these studies and the latest methods for targeting genes using the contrast agents.development of new softwaredevelopment of new software
Impact We have seen for the first time the changes in microvasculature in an organ after physiological manipulations known to alter the function of the tissue. New methods have been developed to analyse these data with direct application to routine ultrasound. The collaborations with our cardiovascular and gynaecological colleagues is leading to novel potential applications. The potential to use contrast agents as gene delivery vehicles is being explored with our USA collaborator. A phase 1 clinical study is being planned to identify changes in uterine vasculature related to preeclampsia and other conditions related to poor pregnancy outcomes.
Start Year 2006
 
Description Development of contrast-enhanced ultrasonography using the sheep ovarian model of microvascular regulation 
Organisation University of Cyprus
Country Cyprus, Republic of 
Sector Academic/University 
PI Contribution Reagents have been supplied as necessary at no cost. WE have developed the use of the sheep ovary and particularly the corpus luteum as a model for the microvasculature. Our extensive knowledge of the control of ovarian function in sheep and how to manipulate this, together with the surgical skills to allow proper visualisation of the ovaries, and the infrastructure of our sheep facility allowed this unique program to proceed.
Collaborator Contribution The collaboration has allowed us to develop novel methods of contrast-enhanced ultrasound to monitor changes in the microvasculature in real time, by developing new software and supplying the knowledge of the latest technology to achieve our aims. In addition the USA connection has provided rfree reagent for these studies and the latest methods for targeting genes using the contrast agents.development of new softwaredevelopment of new software
Impact We have seen for the first time the changes in microvasculature in an organ after physiological manipulations known to alter the function of the tissue. New methods have been developed to analyse these data with direct application to routine ultrasound. The collaborations with our cardiovascular and gynaecological colleagues is leading to novel potential applications. The potential to use contrast agents as gene delivery vehicles is being explored with our USA collaborator. A phase 1 clinical study is being planned to identify changes in uterine vasculature related to preeclampsia and other conditions related to poor pregnancy outcomes.
Start Year 2006
 
Description Development of contrast-enhanced ultrasonography using the sheep ovarian model of microvascular regulation 
Organisation University of Edinburgh
Department Centre for Cardiovascular Science, Queens Medical Research Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Reagents have been supplied as necessary at no cost. WE have developed the use of the sheep ovary and particularly the corpus luteum as a model for the microvasculature. Our extensive knowledge of the control of ovarian function in sheep and how to manipulate this, together with the surgical skills to allow proper visualisation of the ovaries, and the infrastructure of our sheep facility allowed this unique program to proceed.
Collaborator Contribution The collaboration has allowed us to develop novel methods of contrast-enhanced ultrasound to monitor changes in the microvasculature in real time, by developing new software and supplying the knowledge of the latest technology to achieve our aims. In addition the USA connection has provided rfree reagent for these studies and the latest methods for targeting genes using the contrast agents.development of new softwaredevelopment of new software
Impact We have seen for the first time the changes in microvasculature in an organ after physiological manipulations known to alter the function of the tissue. New methods have been developed to analyse these data with direct application to routine ultrasound. The collaborations with our cardiovascular and gynaecological colleagues is leading to novel potential applications. The potential to use contrast agents as gene delivery vehicles is being explored with our USA collaborator. A phase 1 clinical study is being planned to identify changes in uterine vasculature related to preeclampsia and other conditions related to poor pregnancy outcomes.
Start Year 2006
 
Description Dissection of a novel molecular pathway involved in seasonal timing in a melatonin-target tissue using an experimental and systems-level approach 
Organisation University of Manchester
Department School of Medicine Manchester
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have trained the technician in the required methods and she is utilising our skills to analyse the results. I assist in experimental design and execution of the in vivo sheep studies.
Collaborator Contribution A technical post has been assigned to me to undertake collaborative studies on this project.
Impact We have identified a number of genes that are either up or down regulated by melatonin and are currently analysing the transcriptional regulation of these genes. The bioinformatics involved have been extremely helpful in our own research program and we have benefited from using the latest technology, both for arrays and the intepretation of the results.WE have now established that some putative transcription factors in the brain do not act in this way in the pituitary, adding further interest to the mechanisms involved.
Start Year 2007
 
Description Identification of the THOKA fecundity gene in sheep 
Organisation School of Medicine & Dentistry Aberdeen
Department Division of Applied Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Having established the phenotype of the homozygous sheep which have inactive ovaries as adults, we then screened for candidate genes and identified a single base mutation leading to a single amino acid change in the oocyte specific GDF9. We determined the phenotype effect within the ovary and established the physiological consequences of the mutation. Subsequently the Roslin team showed that this mutation was the only one to explain the effects on fertility. We are now exploring how the mutation increases fertility in the heterozygous sheep.
Collaborator Contribution The genetics for identifying the chromosomal gene location was provided by the Roslin Institute.The sheep are supplied by the Macaulay Institute for our studies at nominal costWe have undertaken a proteomic screen of ovaries from the Thoka homozygous sheep in comparison with late pregnancy fettle sheep ovaries and identified a number of proteins that are either increased or decreased related to oocyte activation and follicle development. This has been mutually beneficial giving insight into how environmental factors may influence ovarian programming in adult life.
Impact We have identified and published (Nicol et al 2009; Reproduction 138: 921-933) the mutation so that all sheep in the flock can be genotyped. Our consortium are finalising an agreement with Innovis in Aberystwyth to exploit this genotype for the sheep industry introducing into commercial flocks for production. We are currently examining the mechanisms whereby there is an increase in fertility and fecundity in heterozygous sheep, and whether we can reverse the infertility in homozygous sheep by intra-ovarian gene transfer using lentiviral vectors both in vivo and in vitro.
Start Year 2006
 
Description Identification of the THOKA fecundity gene in sheep 
Organisation Scottish Government
Department James Hutton Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Having established the phenotype of the homozygous sheep which have inactive ovaries as adults, we then screened for candidate genes and identified a single base mutation leading to a single amino acid change in the oocyte specific GDF9. We determined the phenotype effect within the ovary and established the physiological consequences of the mutation. Subsequently the Roslin team showed that this mutation was the only one to explain the effects on fertility. We are now exploring how the mutation increases fertility in the heterozygous sheep.
Collaborator Contribution The genetics for identifying the chromosomal gene location was provided by the Roslin Institute.The sheep are supplied by the Macaulay Institute for our studies at nominal costWe have undertaken a proteomic screen of ovaries from the Thoka homozygous sheep in comparison with late pregnancy fettle sheep ovaries and identified a number of proteins that are either increased or decreased related to oocyte activation and follicle development. This has been mutually beneficial giving insight into how environmental factors may influence ovarian programming in adult life.
Impact We have identified and published (Nicol et al 2009; Reproduction 138: 921-933) the mutation so that all sheep in the flock can be genotyped. Our consortium are finalising an agreement with Innovis in Aberystwyth to exploit this genotype for the sheep industry introducing into commercial flocks for production. We are currently examining the mechanisms whereby there is an increase in fertility and fecundity in heterozygous sheep, and whether we can reverse the infertility in homozygous sheep by intra-ovarian gene transfer using lentiviral vectors both in vivo and in vitro.
Start Year 2006
 
Description Identification of the THOKA fecundity gene in sheep 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Having established the phenotype of the homozygous sheep which have inactive ovaries as adults, we then screened for candidate genes and identified a single base mutation leading to a single amino acid change in the oocyte specific GDF9. We determined the phenotype effect within the ovary and established the physiological consequences of the mutation. Subsequently the Roslin team showed that this mutation was the only one to explain the effects on fertility. We are now exploring how the mutation increases fertility in the heterozygous sheep.
Collaborator Contribution The genetics for identifying the chromosomal gene location was provided by the Roslin Institute.The sheep are supplied by the Macaulay Institute for our studies at nominal costWe have undertaken a proteomic screen of ovaries from the Thoka homozygous sheep in comparison with late pregnancy fettle sheep ovaries and identified a number of proteins that are either increased or decreased related to oocyte activation and follicle development. This has been mutually beneficial giving insight into how environmental factors may influence ovarian programming in adult life.
Impact We have identified and published (Nicol et al 2009; Reproduction 138: 921-933) the mutation so that all sheep in the flock can be genotyped. Our consortium are finalising an agreement with Innovis in Aberystwyth to exploit this genotype for the sheep industry introducing into commercial flocks for production. We are currently examining the mechanisms whereby there is an increase in fertility and fecundity in heterozygous sheep, and whether we can reverse the infertility in homozygous sheep by intra-ovarian gene transfer using lentiviral vectors both in vivo and in vitro.
Start Year 2006
 
Description Polycystic Ovarian Syndrome: modelling development, phenotype and progression using a developmental paradigm 
Organisation University of Edinburgh
Department Centre for Reproductive Biology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have supplied extensive knowledge of sheep pituitary regulation of ovarian function to thse studies. In addition we have added information from our own studies on PCOS in rats to complement these studies. WE are continuing these studies with renewed funding for the MRC to explore which steroids are involved in programming the metabolic and ovarian disruption that occurs in PCOS.
Collaborator Contribution The collaboration has allowed us to develop a novel model of PCOS. The skills of my reproductive medicine clinical colleague has allowed us to perfect the use of direct injection of androgens in the sheep fetus, to explore programming effects in adult life.It has also led to further collaborations and development of a number of new initiatives.
Impact We have shown for the first time that alterations in levels of steroids directly in the fetus can program aspects of adult metabolic activity with potential consequences on ovarian and thus, reproductive function in the adult. The results are dissecting apart the different aspects of the complicated phenotype of PCOS in women. OU recent results in the rat model have shown that the effects on metabolism are related to both altered fettle androgen and oestrogen action with subtle variations in effect.
Start Year 2006
 
Description Temporal analysis of the dynamic regulation of pituitary gene transcription 
Organisation University of Edinburgh
Department Centre for Cardiovascular Science, Queens Medical Research Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Reagents have been supplied as necessary at no cost, but we have benefited from the interactions in which we have used our knowledge of endocrine regulation of prolactin and its role in reproductive processes, and our knowledge pituitary function and control to design experiments. We have also provided expert knowledge in imaging and hormone assays and how to run the animal experiments involved.
Collaborator Contribution The collaboration has educated us in the application of mathematics and systems biology approach to our research, and state of the art real time imaging.state of the art real time imaging.generation of transgenic rats
Impact The group have described for the first time regulation of transcription in real time in tissues in relation to individual cells and the evolution of this control during development. Results showed that transcription is an on and off system within individual cells.More recently the input of mathematical modelling has proved to be immensely useful in interpretation of the data, and added to our potential for our own research.
Start Year 2006
 
Description Temporal analysis of the dynamic regulation of pituitary gene transcription 
Organisation University of Liverpool
Department School of Biological Sciences Liverpool
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Reagents have been supplied as necessary at no cost, but we have benefited from the interactions in which we have used our knowledge of endocrine regulation of prolactin and its role in reproductive processes, and our knowledge pituitary function and control to design experiments. We have also provided expert knowledge in imaging and hormone assays and how to run the animal experiments involved.
Collaborator Contribution The collaboration has educated us in the application of mathematics and systems biology approach to our research, and state of the art real time imaging.state of the art real time imaging.generation of transgenic rats
Impact The group have described for the first time regulation of transcription in real time in tissues in relation to individual cells and the evolution of this control during development. Results showed that transcription is an on and off system within individual cells.More recently the input of mathematical modelling has proved to be immensely useful in interpretation of the data, and added to our potential for our own research.
Start Year 2006
 
Description Temporal analysis of the dynamic regulation of pituitary gene transcription 
Organisation University of Manchester
Department School of Medicine Manchester
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Reagents have been supplied as necessary at no cost, but we have benefited from the interactions in which we have used our knowledge of endocrine regulation of prolactin and its role in reproductive processes, and our knowledge pituitary function and control to design experiments. We have also provided expert knowledge in imaging and hormone assays and how to run the animal experiments involved.
Collaborator Contribution The collaboration has educated us in the application of mathematics and systems biology approach to our research, and state of the art real time imaging.state of the art real time imaging.generation of transgenic rats
Impact The group have described for the first time regulation of transcription in real time in tissues in relation to individual cells and the evolution of this control during development. Results showed that transcription is an on and off system within individual cells.More recently the input of mathematical modelling has proved to be immensely useful in interpretation of the data, and added to our potential for our own research.
Start Year 2006
 
Description Public lecture series 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I organise a series of 5 public lectures each autumn and winter from the Centre for Reproductive Biology. Each lecture consist of a clinician presenting a pathology as presented in the clinic and a researcher describing the type of research we are undertaking to help treat the pathology.

Reports in the local press and in MRC news. Feedback extremely positive and raising the profile of the MRC and university.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012
 
Description talk to prospective medical students 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Discussed the alternative path of a research scientist for the school students if they did not get into medical school but still wanted to be involved in health care.

Very positive feedback from the students who attended this meeting organised by the Pathways to the Professions of Edinburgh University
Year(s) Of Engagement Activity 2008