The role of BMP antagonists in ovarian follicle development
Lead Research Organisation:
Imperial College London
Department Name: Surgery and Cancer
Abstract
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.
Technical Summary
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.
Organisations
Publications
Fenwick MA
(2013)
Investigations of TGF-ß signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.
in Endocrinology
Fenwick MA
(2011)
Identification and regulation of bone morphogenetic protein antagonists associated with preantral follicle development in the ovary.
in Endocrinology
Hardy K
(2017)
Onset and Heterogeneity of Responsiveness to FSH in Mouse Preantral Follicles in Culture.
in Endocrinology
Hardy K
(2018)
Nuclear exclusion of SMAD2/3 in granulosa cells is associated with primordial follicle activation in the mouse ovary.
in Journal of cell science
Mora JM
(2012)
Characterization and significance of adhesion and junction-related proteins in mouse ovarian follicles.
in Biology of reproduction
Description | Understanding what regulates the number of follicles entering the growing phase, and the rate at which they grow are two key events that dictate how the ovary provides a steady supply of oocytes for ovulation, and how long a woman can remain fertile. The molecular mechanisms that regulate early follicle growth are not well understood, but a family of genes known as Transforming Growth Factor betas (TGF-betas) play a central role. TGF-betas are proteins secreted by one cell type (such as the egg) which can promote growth of neighbouring cells (such as the granulosa cells). Cells close to the egg will be exposed to a high concentration of a particular TGF-beta and may behave differently to cells further away and exposed to a lower concentration. Proteins that bind to, and inactivate TGF-betas (TGF-beta binding proteins), maintain these concentration gradients and block the stimulatory effect of TGF-betas on neighbouring cells. Since the early stages of follicle growth depend on TGF-betas for growth, we hypothesized that TGF-beta binding proteins could play a pivotal role in fine-tuning the growth of follicles. We initially carried out a comprehensive screen for known TGF-beta binding proteins in a wide range of organs and found many of these were expressed in the ovary. In particular, we were able to identify several binding proteins that were clearly present in granulosa cells of follicles that had begun to develop, implying that follicles are capable of modifying their own TGF-beta environment as they grow. Although TGF-beta signaling is essential for initial growth, our results also suggested that TGF-beta signaling plays an important role during further growth. To test this idea we isolated early developing follicles from ovaries and cultured them in dishes in the presence of different synthetic TGF-beta inhibitors. TGF-betas transmit signals from the cell surface to the nucleus (where they can stimulate genes involved in cell division) via intermediate proteins called SMADs. We discovered that by inhibiting one branch of the TGF-beta signalling pathway (termed SMAD2/3), follicles were unable to grow, while in contrast inhibition of a different branch (SMAD1/5/8) promoted rapid growth. In addition, we found that each specific pathway could be activated by a specific subset of TGF-beta factors. Another key finding was that primordial follicles express SMAD2/3 and only express SMAD1/5/8 once they have begun to grow. These results indicate that follicles are not only sensitive to TGF-beta signalling, but they also change the way they respond to different members as they develop. Finally, we tested whether TGF-beta binding proteins played a role in regulating early follicle development. By inhibiting the expression of Twsg1, a binding protein normally produced by developing follicles, we found evidence of increased SMAD1/5/8 signalling accompanied by a reduction in follicle growth. This indicated that Twsg1 normally acts by inhibiting BMPs, the sub-set of TGF-betas that activate SMAD1/5/8. Thus, we have confirmed our hypothesis that TGF-beta binding proteins play an important role in early follicle development. Overall these findings have enabled us to identify a mechanism that describes how the ovary is able to regulate the rate of early follicle development, allowing it to provide a steady supply of oocytes for later ovulation. |
Exploitation Route | These findings can help us understand the physiology of the normal ovary. This is of key importance in understanding pathological conditions of the ovary, such as Polycystic Ovarian Syndrome, Premature Ovarian Insufficiency (premature menopause) and Ovarian Cancer. Our results can therefore be taken forward by academic clinicians and research scientists who specialize in these disorders, and may be used in the future to develop therapies for these common ovarian disorders. |
Sectors | Agriculture, Food and Drink,Healthcare |