Identification of human-specific regulatory mechanisms in female germ cell development

Human female fertility stretches over decades, but all the oocytes (eggs) necessary for this are formed before birth. Therefore at the time of her birth, a woman's ovary contains all the oocytes she will ever have. The number and also the quality of these oocytes then declines and when the supply of oocytes is exhausted at the menopause, women not only reach the end of their fertility, but are at increased risk of other health problems related to bone, cardiovascular and cognitive function. Having sufficient eggs to ensure normal fertility and age at menopause is therefore central to lifelong women's health. The type of cell division called meiosis, which is essential for the manufacture of oocytes that are capable of fertilisation in adult life, is initiated during fetal life and therefore events that occur before birth will determine a woman's fertility and reproductive lifespan.
Gene expression is a multi-step process where mRNA, which is made from DNA (itself the genetic blueprint) is used as a template to make proteins. In order to function properly, oocytes need to make proteins at the right time, place and in the correct amount, and this is regulated by RNA binding proteins. Oocytes have specific RNA binding proteins that are critical for their development and these proteins have specific mRNA targets which they recognise and bind to, thereby controlling gene expression. DAZL and BOLL are two RNA binding proteins that are specifically found in oocytes of all mammalian species and we have shown in the human, there is a switch from DAZL to BOLL during the early stages of meiosis. This switch does not happen in mice. Our previous research has revealed some of the mRNA targets that are regulated by DAZL in human fetal oocytes, and in this project we wish to identify the key mRNA targets regulated by BOLL, to find out how BOLL contributes to meiotic progression and follicle formation, thus to overall human oocyte development and setting up a woman's fertility. It is essential to do this in humans because of this switch that seems to be necessary for normal human, but not rodent, oocyte development. This work will shed light on the key steps necessary to make sufficient and good quality eggs that are the basis for women's life-long fertility.

Primordial germ cells undergo significant, overlapping and possibly interconnected processes on their journey to become functional oocytes, and it is these milestone events that lay the foundations of developmental competence required to form an oocyte that is capable of fertilisation in adult life. The initiation of meiosis is one of the defining features of germ cell differentiation, however during meiosis, the highly condensed state of chromatin is not easily accessible to transcription factors. Consequently, oogenesis is punctuated by periods of transcriptional silence, where homeostasis and development are dependent on the translation of pre-transcribed mRNAs, under the regulation of RNA binding proteins. Of particular interest are the RNA binding proteins DAZL and BOLL, which are specifically expressed in germ cells. We have identified key differences in the expression of DAZL and BOLL in mouse and human during early meiosis and hypothesise that unlike in mouse, where it is likely these two proteins have a level of redundancy, the switch from expressing DAZL to BOLL early in meiotic prophase reflects significant functional differences. This research plan aims to investigate this hypothesis by identifying mRNA targets of BOLL in the human fetal ovary to complement our previous work which has identified human DAZL mRNA targets. We then propose to demonstrate that regulation of DAZL and BOLL mRNA targets are separately necessary for entry into meiosis and sustained meiotic differentiation in human fetal oocytes. This work will not only benefit research in the fields of meiosis, germ cell biology and female reproductive lifespan, but may also provide insights into reproductive disorders that potentially have their origin during fetal life, which is well recognised to be a time of vulnerability to external influences and therefore has consequences for lifelong health.

1. Competitiveness of UK science. Establishment of the ovarian reserve, meiosis and RNA binding proteins are areas of intense international interest (see Academic Beneficiaries). Their importance in diverse pathologies means academic investigators spanning biology (e.g. development, human genetics), clinical research (infertility, developmental disorders) and pharma-based researchers stand to benefit from new knowledge, extending the scope of our impact far beyond the field. They may also benefit by taking advantage of our methodologies regarding RIP-seq on limited starting material, our 3'UTR-luciferase constructs, as well as our BOLL mRNA targets data set for secondary analysis, which will be made publically available on Gene Expression Omnibus (GEO).
2. Training and capacity building. Historically the UK is a leading presence in reproductive biology research, however this is threatened by increased international competition, and substantial capacity building is required to maintain this position. In particular, next generation scientists with multidisciplinary skill sets are needed. This proposal combines novel and unique approaches to studying the regulation of meiosis in human fetal oocytes, and will provide high quality specialised and multidisciplinary training to the Res Co-I (e.g. fetal gonad xenograft model). This work will have an immediate impact on the Res Co-I who will enlarge her area of expertise and the experienced gained will help her towards scientific independence. Training will also be extended to our basic/clinical PhD, MRes and undergraduate students, and knowledge transferred to others at national and international meetings, and local research clinics. These skills are highly transferrable between academic, clinical and industrial settings.
3. Health and pharma. The finite nature of human female fertility underpins many ovarian pathologies and reproductive disorders. This work aims to establish the clinical relevance of BOLL as a novel locus for human (in)fertility, and to confirm the importance of DAZL in human female fertility. Increased understanding in this area will shed light on the molecular basis of disease and will give the opportunity to develop in the future improved individualised assessment and advice/intervention. With a marked increase in the age at which women start families in recent decades, our work will increase understanding of infertility and subfertility due to insufficient formation/premature depletion of the ovarian reserve which affects not only reproduction, but also many physiological systems such as bone health, the cardiovascular system and others, thus can affect women's health across the life course. Thus, this work will impact clinicians involved in the care of women across a wide range of women's health related specialities and interests, particularly reproductive medicine but also obstetrics and sexual and reproductive/post-reproductive healthcare. This work may provide a basis for later commercially exploitable results such as molecular diagnosis and/or pharmacological treatment.
4. The UK livestock industry also stands to benefit from this work, since currently many female animals are culled at a relatively young age solely due to reproductive problems: wider application of the results obtained here could help improve the reproductive potential of female mammalian domestic species.
5. Charities and the general public. All pregnant women are concerned to optimise the health of their unborn children. The growing awareness of the importance of this period of development in lifelong reproductive and non-reproductive health will inform the provision of healthcare services and public health activities. We will inform charities which support couples suffering from infertility include Fertility UK and Fertility Network Scotland of our results. More immediately the general public will benefit from our public engagement strategies.