The role of BMP antagonists in ovarian follicle development

Female fertility declines with age and this has important implications for family planning in an era in which many women, often because of the demands of a career, delay attempting to conceive until their mid-late thirties. The basis of this well-described age-related reduction in fertility is the steady loss from the pool of resting follicles in the ovary. Female mammals are born with all the eggs they are ever going to have. The stock of eggs in the ovaries is maintained in structures called follicles. In the quiescent (primordial) stage each follicle consists of the egg (oocyte) enclosed by a single layer of flattened cells called granulosa cells. During reproductive life a steady trickle of follicles leave the quiescent stage and start to grow. This continues until the stock of eggs is exhausted and, in the human female, this results in the menopause, normally at the age of about 50 years. The progression of follicles from the quiescent to the growing phase has to be tightly regulated to ensure a normal reproductive lifespan. Premature depletion of oocytes, leading to an early menopause, is a common cause of infertility in women. Little is known about the factors that control the start of follicle growth. Growth factors produced locally in the ovary seem to have an important role but there are several possible candidates and it is not clear what the key factors are. In a recently published study (that resulted from work funded by a previous BBSRC Grant) we showed clear evidence that follicles in the ovary are much less likely to start growing if they have one or more quiescent (primordial) neighbours. This strongly suggests that primordial follicles produce an inhibitory signal. This is important because most of the growth factors so far identified in the ovary stimulate, rather than restrict, follicle growth. However we know that the action of certain growth factors (known as bone morphogenetic proteins - BMPs - because they were first discovered in bone) which are important in follicle development can be modified by proteins that inactivate BMP growth factors, and therefore inhibit their action. They are therefore very good candidates to be the 'missing' inhibitor(s) that we are looking for. To date little is known about these BMP antagonists in the ovary. In our preliminary studies we have been able to show that many of these proteins are produced in the ovary. In this project, using the mouse ovary as a model, we will focus on where and when these BMP antagonists are produced in the ovary and how they are regulated by other factors within and outside the ovary. We have developed, in our laboratory, techniques for the culture of whole mouse ovaries, individual follicles or cells isolated from these follicles, and we will use these models to study the action of BMP antagonists. We expect the findings from this series of experiments to shed new light on how the rate of entry of follicles from the resting to the growing stages (and hence reproductive lifespan) is controlled and provide the basis for therapies that can be used to improve fertility in both domestic animals and, particularly, in women.

Each oocyte in the ovary is enclosed by somatic granulosa cells (GCs) to form a follicle. The rate at which follicles start growing must be tightly regulated to ensure a normal reproductive lifespan. Regulation of early follicle growth is poorly understood, but many members of the TGF-beta superfamily have been implicated. Our recent quantitative spatial analysis of quiescent and growing follicles in mouse ovaries predicts that an inhibitor is produced by primordial (quiescent) follicles, which may be key to conserving follicle supply. While primordial follicles express receptors for TGF-betas, there are currently very few candidate inhibitors. BMP antagonists (extracellular proteins that bind and inactivate TGF-betas) play a crucial role in morphogenesis and patterning in many species. We have recently shown that 14 BMP antagonists are expressed in mouse ovary, with some of them demonstrating gradients in GCs. We propose to investigate the role of BMP antagonists in early follicle development. We will examine where and when BMP antagonists are expressed in mouse ovary using in situ hybridisation, immunofluorescence and RT-PCR of isolated follicles. We will investigate whether follicle expression of these antagonists is regulated by the hormonal environment and/or by the oocyte itself, and will also study the effects of BMP antagonists on follicle development using ovary and follicle culture systems. The effect of BMP antagonists on downstream TGF-beta signalling pathways will be investigated using luciferase reporter assays. We will use our system for quantitative spatial analysis to investigate the relationship between ligands and antagonists, and start mathematically modelling networks of these interactions. Increased understanding of the expression and action of BMP antagonists in the mouse ovary will contribute to our knowledge of the regulation of mammalian follicle development.