The FecB gene may increase prolificacy by modulating multiple local regulatory pathways

In certain species (human, sheep, cattle), the number of offspring is tightly regulated to just one or two per pregnancy . The control of offspring number is an extremely important adaption to the physical limits of the mother to bear to term, give birth to and successfully rear young in these species under prevailing environmental conditions. The mechanism whereby offspring number is limited in these so-called monovulatory species involves selection of just one follicle to release an egg (ovulate) per reproductive cycle. The mechanisms controlling follicle development generally, and follicle selection in particular, have been the subject of intensive investigation for many years as they are central to our understanding of the causes and treatment of infertility. A mechanistic model has emerged from these studies that involves the action of local growth factor system (insulin/IGF, inhibin/activin, bone morphogenetic proteins [BMP]) that augment or attenuate the stimulatory role of the pituitary gonadotrophins, LH and FSH, during the terminal gonadotrophin-dependent stages of follicle development (when follicle selection takes place). The sheep has been extensively used as a model species for these studies due to the occurrence of a number of naturally occurring mutations in prolific breeds which lead to varying degrees of disregulation of these selection mechanisms. The Booroola or FecB mutation is one of the most dramatic of these mutations as it results in sheep having up to 6-8 lambs per pregnancy due to complete disregulation of the follicle selection mechanisms. Over recent years, we and others have been able to show that the FecB mutation occurs in the type 1B BMP receptor (BMPR1B). However, the ligands that bind to the BMPR1B and the effect of the FecB mutation on the response of cells within the ovary to stimulation, are not fully understood. On the basis of work we have carried out so it is thought that the BMPR1B interacts with a number of other factors known to be present in the ovary including anti-mullerian hormone (AMH), BMP15 and BMP6. Our mechanistic model of follicle selection therefore predicts that the effect of the Fec B mutation may be due to alteration of the inhibitory or stimulatory effects of one or more of these ligands of the BMPR1B receptor which normally act to augment (BMP-6) and attenuate (BMP-15, AMH) the activity of pituitary gonadotrophins in stimulating the development and selection of ovulatory follicles. This project will utilise a series of highly developed cell culture and whole animal models in combination with advanced molecular techniques to examine this idea by determining: (i) the identity and function of ovarian ligands to the BMPR1B receptor and the effect of the FecB mutation on receptor-ligand interactions and down-stream signalling events in cultured cells; (ii) the effect of increasing or decreasing the activity of ovarian ligands of the BMPR1B receptor on ovarian function in whole animals which do or do not carry the FecB mutation and (iii) if we can replicate the FecB phenotype of multiple small ovulatory follicles in sheep which do not carry the FecB mutation by modulation of the activity of multiple ligands of the BMPR1B. This works brings together the whole animal, cell culture and molecular expertise of experienced investigators in Edinburgh and Nottingham along with the unique availability of the FecB sheep mutant models in order to allow the elucidation of the complex homeostatic interactions between endocrine and local factors that act to regulate this complex and key physiological system. It is expected that this information will add to our fundamental understanding of ovarian function in order to address causes of ovarian disfunction and infertility and to devise innovative means to assess and treat these conditions

The FecB mutation in the BMP1B receptor (BMPR1B) results in precocious maturation of gonadotrophin-dependent follicles and the ovulation of multiple small follicles. Potential ligands for the BMPR1B include factors that have been shown to either augment (BMP-6) or attenuate (BMP-15, AMH) the actions of gonadotrophins in inducing differentiation of ovarian somatic cells and we hypothesise, on the basis of extensive preliminary in vitro and in vivo investigations, that the effect of the mutation is due to alteration of the inhibitory or stimulatory effects of one or more of these ligands. In order to test this hypothesis, technical developments in the micro-surgical preparation of in situ cannulation and ovarian autotransplant models have made it possible, for the first time, to allow in vivo exposure of the ovary to high local concentrations of factors designed to modulate the level of activity of local factors for prolonged periods, the monitoring of the endocrine and follicular response to treatment and subsequent recovery of ovarian tissue to examine the molecular basis of the response observed. The first experimental objective is to confirm the identity and function of ovarian ligands to the BMPR1B in this species and the effect of the FecB mutation on receptor-ligand interactions and down-stream signalling events in vitro. The second and third objective will utilise ovarian autotransplant and in situ cannulation models to determine the effect of exposure or knock-down (lentiviral transfection, passive immunisation) of ligands to the BMPR1B on the pattern of recruitment and development of gonadotrophin-dependent antral follicles in wild-type and FecB mutant animals in vivo. The final objective will determine whether the FecB phenotype can be induced in wild-type animals by modulation of multiple ligands of the BMPR1B. This work may therefore lead to the development of new methodolgies for the manipulation of ovarian function and the treatment of infertility.

The potential impact of this research is considerable, particularly as the concepts involved are relatively simple and the potential benefits wide ranging and easily appreciated by both industry, the general public and the scientific community. Thus the levels at which we foresee this research making an impact are: (i) Scientific: As discussed in more detail in the Case for Support, the mechanisms regulating follicle selection in monovulatory species is one of the key questions in ovarian physiology and the elucidation of the mechanisms underpinning the increase in prolificacy observed in FecB mutant would represent a major advance in our basic understanding of this mechanism. Thus we would expect these findings to be of significant interest to the scientific community in terms of the impact factors of the Journals willing to publish these results, the number of invitations to present our findings at international scientific meetings etc. Accordingly, we would plan to publish this work in as high an impact factor journal as possible and to utilize the profile of the investigators to disseminate this information in key note addresses internationally, as much as possible. (ii) Animal production and assisted reproduction: As illustrated clearly by the images presented in Figure 1 of the Case for Support, the FecB mutation we are studying results in a dramatic and easily quantifiable change in an economically important production characteristic for livestock production; prolificacy. Since the discovery of this mutation in Australia, it has been introduced into literally hundreds of sheep breeds around the globe as a means to boost prolificacy. These breeding programmes are expensive and time consuming but more importantly, once the mutation is introduced, it can actually be deleterious under adverse or marginal environmental conditions. The final objective of our programme of work seeks to devise an intervention in which we can test our central hypothesis by inducing the FecB phenotype in wild-type animals. Clearly, if we are successful in this objective then we will have devised a means by which significant increases in prolificacy could be induced as part of a production programme and this would therefore be of considerable commercial interest. Further, such an intervention could be adapted to replace gonadotrophin based superovulation protocols, which are notoriously variable and often result in poor oocyte quality, not only in sheep but also in cattle. (iii) Clinical medicine: Infertility in the human population affects 1 in 7 couples and although not life threatening, the impact of infertility on each individuals wellbeing and society in general is considerable. As mentioned above for production species, ovarian stimulation protocols for human ART are imprecise and emperical and generally result in a large numbers of oocytes, only a few of which are developmentally competent. Further, ovarian hyperstimulation syndrome is a common and potentially fatal complication of these gonadotrophin stimulation protocols. The results of these experiments in sheep therefore have a much wider potential impact in the field of assisted conception as a means to devise improved stimulation protocols which require much lower doses of gonadotrophic stimulation and which will yield better quality oocytes. Further, many of the local factors studied as part of this application such as AMH and BMP-15 have been implicated in the aetiology of anovulatory infertility in humans. This research therefore has potentially enormous impact in the field of reproductive medicine.