Ovarian androgen synthesis: unravelling the roles of INSL3 and BMP signalling

Fertility of female animals and humans requires the correctly-timed release of one or more healthy eggs (oocytes) from the paired ovaries. Eggs develop within structures called ovarian follicles which also produce a number of key hormones, without which reproduction is simply not possible. This research will investigate how the production of these hormones is regulated. In particular, two related hormones called androgen and oestrogen must be produced by specialized follicle cells in the right amounts - and at the right time - of the female cycle. Androgen is produced by cells called theca cells (TC) while oestrogen is produced by cells called granulosa cells (GC). GC can only synthesize oestrogen when they are supplied with androgen by neighboring TC. GC cannot synthesize androgen themselves and androgen is the essential precursor for oestrogen synthesis. Follicles grow from microscopic structures into large multi-layered, fluid filled structures ~ 2cm across (in human & cow). Pulses of luteinizing hormone (LH) from the pituitary stimulate TC to produce androgen while pituitary follicle-stimulating hormone (FSH) stimulates GC to proliferate and convert androgen into oestrogen. Mature follicles protrude from the ovary surface awaiting a hormone trigger from the pituitary (called the LH surge) that causes them to ovulate and release the egg for possible fertilization. This LH surge, in turn, depends on the secretion of increasing amounts of oestrogen by the growing follicle. In animals, this oestrogen rise also triggers vital behavioural changes ('heat') without which mating would not occur. Oestrogen also plays a key role in preparing the reproductive tract for pregnancy and both androgen and oestrogen affect many other tissues and organs. Thus, disordered follicle development leading to an imbalance in production of androgen by TC or of oestrogen by GC can not only cause infertility, but can affect many other aspects of physiology. For instance, a very common cause of infertility/subfertility in women called polycystic ovarian disease syndrome (PCOS) is associated with excess ovarian androgen production; this condition leads to disturbed menstrual cycles and excess growth of facial and body hair. Follicle development is a very complex process that is still poorly understood. It has been recognised recently that numerous signaling molecules are produced within the ovary itself that enable different cell-types to communicate with each other. This 'local' cell-cell communication must work in concert with signals from the pituitary (LH, FSH) to coordinate follicle growth and steroid production. In this project we will investigate the actions and interactions of two different 'local' signaling systems in regulating LH-induced androgen production by TC - the INSL3 system and the BMP system. Our decision to focus on these two systems is based on our recent discovery that (1) BMP can switch off production of both androgen and INSL3 by theca cells (2) a GC product called inhibin can, in turn, switch off BMP signaling and raise androgen production. In the project we will map the distribution of INSL3 (and its receptor) and various BMP system components (ligands, receptors, binding proteins) in follicles at different growth stages. We will also carry out detailed in vitro studies on TC (isolated from cow ovaries from the abattoir) to find out whether adding (or removing) different components of the INSL3, BMP or inhibin systems affects androgen production. In this way we will identify functional interactions between these different systems. Finally, we will find out if blood levels of INSL3 vary during the cow's oestrous cycle and if so, whether these changes are linked to patterns of follicle growth and levels of pituitary (LH, FSH) and ovarian (steroids, inhibin) hormone secretion. Collectively, these studies will significantly advance understanding of ovarian physiology and provide insights into a major cause of infertility.

Synthesis of androgens by the ovary is obligatory for female fertility and wellbeing. Substantive evidence indicates that an intra-ovarian BMP system and inhibin-activin system are involved in the regulation of androgen and oestrogen synthesis. In a recent microarray study on cultured bovine theca cells (TC) we unexpectedly found that INSL3 was one of the most abundant transcripts and that its expression was virtually abolished by BMP treatment. We showed previously that BMPs potently suppress basal and LH-induced CYP17 mRNA and androgen synthesis. Moreover, our pilot data indicate that granulosa-derived inhibin-A behaves as a BMP antagonist and reverses BMP-induced suppression of TC androgen synthesis. This leads us to hypothesize that TC androgen production is tightly regulated by the coordinated actions of pituitary LH and two opposing intraovarian systems: the INSL3-LGR8 system (stimulatory for androgen synthesis) and the BMP system (inhibitory for androgen synthesis). We further propose that the latter system regulates the former, and is in turn negatively regulated by GC-derived inhibin and by TC and/or GC-derived BMP-binding proteins. To address this we will first conduct a detailed ex-vivo survey of mRNA and protein expression during follicle development to examine association between expression of INSL3 and its receptor (LGR8), BMP system components, steroidogenic pathway components and LH/FSH receptors. Next, we will undertake a series of functional in vitro studies using our bovine TC model (plus TC-GC co-culture).These will include 'gain' and 'loss' of function approaches to test a series of specific hypotheses on the proposed interactions between LH, INSL3, BMP & inhibin systems and steroidogenesis. Thirdly using an improved bovine INSL3 immunoassay we will measure plasma INSL3 levels during the bovine oestrous cycle and after manipulation of follicle wave growth, and relate changes to LH, FSH and ovarian steroid profiles.