Role of thecal inhibin alpha subunit in the regulation of INSL3-dependent androgen production in the ovary

Fertility of female animals and humans requires the correctly-timed release of one or more healthy eggs (oocytes) from the ovaries. Eggs develop within structures called ovarian follicles that also synthesise important hormones, without which reproduction would fail. 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) whilst oestrogen is produced by granulosa cells (GC). GC can only synthesize oestrogen when supplied with androgen by neighbouring TC.

Follicles grow from microscopic structures into large multi-layered structures ~ 2cm across (in human & cow). Pulses of luteinizing hormone (LH) from the pituitary stimulate TC to produce androgen while follicle-stimulating hormone (FSH) stimulates GC to proliferate and convert androgen into oestrogen. Mature follicles protrude from the ovary surface awaiting a hormonal trigger from the pituitary (LH surge) that causes them to ovulate and release the egg(s) 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 around the body. 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 an intricate process that is still poorly understood. It is now clear 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 works in concert with hormonal signals from the pituitary (LH, FSH) to coordinate follicle growth and steroid production. In this project we will extend the findings of our previous BBSRC-funded grant that focused on the actions and interactions of two different 'local' signaling systems in regulating ovarian androgen production - the INSL3 system and the BMP system. In particular, we will investigate the functional role of another molecule called inhibin alpha subunit that we recently found to be produced in surprisingly large amounts by TC. While the role of this molecule in TC is currently unknown, we predict that its 'local' function is to 'protect' TC from stimulation by inhibin from neighbouring GC in the follicle. This is potentially a very important role since we have previously shown that inhibin can antagonise the suppressive action of BMPs on TC androgen production. BMPs, in turn, suppress TC expression of INSL3 that seems to be a critical requirement for TC androgen production. We predict that the relative 'balance' between the above signals varies during the course of follicle development and we will examine this experimentally. In addition, we will investigate the relationship between inhibin alpha subunit levels in TC, follicular fluid and peripheral blood samples from cows. By analysing cow blood samples collected for a previous study we will also test our prediction that circulating INSL3 and androgen levels are raised in cows immunized against inhibin alpha subunit. Collectively, these studies will significantly advance understanding of ovarian physiology and provide insights into a major cause of infertility.

Normative androgen biosynthesis by ovarian follicles is essential for female fertility and wellbeing. We recently showed that thecal (TC) production of INSL3 and androgen are potently suppressed by BMP treatment and that inhibin (from granulosa cells; GC) reverses this effect. Moreover, knockdown of INSL3 or its receptor (RXFP2) reduced TC androgen secretion while exogenous INSL3 raised androgen secretion. The hypothesis developed under previous BBSRC funding is that TC androgen production is tightly regulated by coordinated actions of pituitary LH and two opposing intraovarian systems: the INSL3-RXFP2 system (stimulatory) and the BMP system (inhibitory). The latter system regulates the former, and is in turn negatively regulated by inhibin and BMP-binding proteins. Based on new observations made during this study we now extend this hypothesis and propose that BMP attenuation of INSL3-dependent androgen production is augmented by 'free' inhibin alpha subunit expressed by TC. TC-derived inhibin alpha subunit effectively mutes the action of GC-derived inhibin on TC androgen production. The balance between these opposing signals shifts during follicle development allowing increased androgen production as follicles advance to the preovulatory stage. To address this we will evaluate the effects of betaglycan and inhibin alpha knockdown, overexpression, supplementation and neutralization on the ability of dimeric inhibin to (i) raise basal and LH-dependent INSL3 and androgen production (ii) antagonise BMP-induced suppression of INSL3 and androgen. We will also compare relative TC responses to BMPs, inhibin and antral fluid at pre- and post-selection follicle stages. Further, we will test our prediction that blood and antral fluid inhibin alpha levels more closely reflect TC-derived inhibin alpha than GC-derived inhibin alpha. Finally, using archived blood samples we will test the prediction that INSL3 and androgen levels are raised in cows immunized against inhibin alpha.

Potential beneficiaries of the research are (1) academic community; (2) commercial stakeholders in animal production; (3) clinical practitioners in reproductive medicine including NHS; (4) pharmaceutical industry; (5) postdoctoral researcher and students; (6) general public
Our research aims to advance fundamental knowledge of the underlying mechanisms that regulate mammalian ovarian function. Infertility/subfertility affect the lives of 1 in 6 couples and about half of these are attributed to female factors. The efficiency of farm livestock production (particularly dairy cattle) is also severely hindered by female subfertility.
Specifically, we seek to elucidate the roles of certain local signaling molecules (BMPs, INSL3, inhibin, inhibin alpha subunit) in regulating follicular androgen production. Androgen production is a vital aspect of ovarian function but is often disrupted giving rise to subfertility or infertility in animals and humans.
For example PCOS affects 6-10% of women of reproductive age and is associated with androgen excess, disrupted menstrual cycles and ovulation failure. Perturbed ovarian androgen production has also been implicated in premature ovarian failure (early menopause) that affects around1% of women <40 years of age.
Declining fertility in dairy cattle is a major issue for the UK and global dairy industry with typical conception rates now <40%. Important contributory factors in this decline are compromised follicle development, reduced preovulatory oestradiol output and poor oestrus expression leading to poor oestrus detection. Since the rise in oestrogen that induces oestrus (and the ovulation-inducing LH surge) is dependent on thecal androgen production, targeted manipulation of thecal androgen production could potentially provide a means to improve oestrogen output, oestrus expression and detection.
So, in the longer-term our research will inform and have potential impact on the following stakeholders:
1. The Academic community:
[Please see section: Academic Beneficiaries]

2. UK and global animal production industry: Our research focusing on the regulation of androgen production by the bovine ovary may lead to potential improvements in production efficiency, particulary in dairy cattle. These might accrue through increased cattle fertility, oestrus detection, reduced AI costs, reduced wastage/culling, improved animal welfare.

3. Clinical practitioners in reproductive medicine: By elucidating aspects of the underlying physiological systems regulating ovarian androgen biosynthesis our research may contribute to the development of new or improved methods to monitor and manipulate human ovarian function and treat ovarian dysfunction and infertility.

4. Pharmaceutical companies: Potentially our research may suggest new intervention-based approaches to modulate ovarian androgen production in livestock and/or humans. Such drug developments would clearly require significant long term commercial investment but could, ultimately, find application in 2 and 3 above.

5. Postdoctoral researcher and students: Societal impact will accrue through acquisition of project-specific and generic research skills by an early career researcher (the postdoctoral associate), as well as by postgraduate and undergraduate project students in the applicant's laboratory.

6. The general public: In terms of potential long-term societal and economic impact the public may ultimately benefit through (i) increased food supply and/or decreased cost of milk/beef associated with improvements in cattle fertility (e.g. due to better understanding of ovarian follicle development, improved expression and detection of oestrus, enhanced fertility); (ii) improvements in the treatment of human ovarian dysfunction and infertility (e.g. polycystic ovarian syndrome) associated with altered follicle development and androgen production; (iii) our contribution to sustaining the UK's competitiveness in R&D.