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Research Projects Regulation of ovarian function Hormones produced by the anterior pituitary called gonadotrophins (follicle stimulating hormone, FSH and luteinising hormone, LH), play important roles in regulating the growth of ovarian follicles and the release of oocytes from the follicles at ovulation. Despite this knowledge, little is known about the factors and mechanisms operating within the ovary that determine the size of the follicle pool, which follicles will be recruited into the growth phase, or selects those follicles which will ovulate and those which will die. A better understanding of how the ovary is regulated would allow us to address issues related to fertility, contraception and cancer. 1. Regulation of follicle numbers Supervisors: Professor Jock Findlay and Associate Professor Jeff Kerr (Anatomy & Cell Biology, Monash University) Our knowledge of the factors that regulate the supply, activation and growth of ovarian follicles is important for an understanding of conditions such as premature ovarian failure, polycystic ovarian syndrome, and anovulation all of which impact fertility in women. This project will study the factors that regulate the establishment, activation, development, and growth of ovarian follicles in a range of animal models that alter gene activity, follicle supply, and the actions of estrogen in the fetal and postnatal ovary. The controversial notion that follicles may self-renew from as yet unidentified stem cell populations from intra- or extraovarian sources will form part of these studies. Techniques will include: gene expression, histology, immunocytochemistry, confocal and stereological analysis, electronmicroscopy, 2-D and 3-D biomapping, animal surgery, data analysis and publication. 2. Regulation of primordial and primary follicle growth Supervisors: Professor Jock Findlay and Dr Ann Drummond We will establish a model system for investigating the earliest stages of folliculogenesis ie, the recruitment of primordial follicles into the growth phase and their transition into primary follicles. An in vitro based model utilising ovarian slices would allow us to manipulate the environment either by the addition of hormones and/or growth factors. Gene expression studies, histological and stereological analyses will form the basis of projects in this area. 3. Role of TGF-beta superfamily members in folliculogenesis Supervisor: Prof Jock Findlay and Dr Ann Drummond TGF-beta superfamily members have been shown to influence steroid and inhibin production and the proliferation and differentiation of cells within the ovary. Little however, is known about the capacity of specific follicle populations to transmit these signals that influence biological function. We will use established cell culture models and develop new follicle based culture systems, employ immunohistochemistry, real-time PCR, microarrays, in situ hybridisation and hormone assays to investigate TGF-beta superfamily signalling pathways in follicle populations. 4. Role of estradiol in folliculogenesis Supervisor: Professor Jock Findlay Although estrogens are known to be important for fertility, their precise role and point of action in the development of ovarian follicles is still unclear. We can treat rats with estrogen in vivo and perform a range of analyses on the isolated ovaries and cells to investigate these questions. Ovaries isolated from these models will be subject to gene expression studies, including real-time PCR and microarrays, cell culture, histological studies including laser capture microscopy (LCM), and stereological analyses. Infertility and cancer 5. Gonad development in betaglycan knockout embryos Supervisor: Dr Kaye Stenvers The period when the foetal gonads first form is particularly vulnerable to disruption as a result of environmental and genetic factors. Insults at this time may perturb normal germ cell development, providing the platform for the subsequent development of cancers and infertility in adulthood. TGF-betas have been implicated in the regulation of many cellular events underlying gonad development and function. Betaglycan, a key TGF-beta and inhibin co-receptor, determines cellular sensitivity to these growth factors. We have found this receptor to be dynamically regulated during gonad development. We are now studying our betaglycan knockout mice to determine which specific developmental processes are affected by betaglycan deficiency. Cellular markers have been developed to discern particular cell types and key structures in embryonic gonads. We will use in situ hybridization and immunohistochemistry on betaglycan knockout and wildtype mouse gonad sections to assess whether the distribution or gross number of each gonadal cell type is perturbed when betaglycan is absent in the developing gonads. To investigate the function of betaglycan relative to growth factors known to regulate gonadogenesis, we will utilise primary cultures of explanted foetal gonads to examine cell migration, proliferation, aggregation, and differentiation. The student will gain experience in molecular and cellular biology, developmental biology, and anatomy. Techniques used include: basic histology techniques; immunohistochemistry; in situ hybridisation histochemistry; light, fluorescent, and confocal microscopy, imaging, and computer analysis; genomic DNA extraction; PCR; real-time PCR; and explant culture techniques. 6. Reproductive capacity of adult betaglycan mutant mice Supervisor: Dr. Kaye Stenvers Premature Ovarian Failure (POF or premature menopause) is a human clinical syndrome in which the ovaries stop producing viable eggs before the age of 40. Recently, POF was found to be associated with mutations in the human betaglycan gene (Dixit et al., 2006, Hum Reprod. 21: 2041-6), which suggests that this receptor plays important roles in determining female reproductive capacity. It is not yet clear, however, whether betaglycan is required at the level of the ovary and/or pituitary, as betaglycan is highly expressed in both tissues in adult mouse. We will assess adult betaglycan heterozygous female mice, which carry only one copy of the betaglycan gene and express reduced levels of betaglycan, in terms of their endocrine status, reproductive capacity, and the morphology/pathology of their ovaries. Finally, in order to determine the capacity of the adult betaglycan mutant mice to ovulate viable eggs, young adult females will be superovulated and mated to fertile males for the assessment of the rates of oocyte generation/fertilisation.This study will determine what impact reduced levels of betaglycan have on sexual maturation and adult reproductive capabilities. The student will gain experience in molecular and cellular biology, reproductive physiology, and anatomy. Techniques used include: basic histology techniques; immunohistochemistry; light and fluorescent microscopy, imaging, and computer analysis; genomic DNA extraction; PCR 7. Roles of betaglycan in ovarian and breast cancers Co-supervisors: Dr. Kaye Stenvers and Dr. Craig Harrison Betaglycan binds all three TGFß isoforms as well as the related factor, inhibin, and modulates the association of these ligands with their signalling receptors. Both TGF b and inhibin are multifunctional growth factors that regulate a number of important cellular functions, including proliferation, differentiation, and survival. Dr. Craig Harrison has created a number of betaglycan expression constructs which carry mutations in the binding domains of betaglycan. Dr. Harrison identified point mutations which largely eliminate either TGF b or inhibin- binding, thus generating ligand-specific binding mutants. Projects are available to test the function of these mutants in cultures of breast and ovarian cancer cell lines, many of which have little betaglycan on their cell surface and respond poorly to TGF b -mediated growth inhibition. The aim of these studies would be to determine whether specific TGF b or inhibin-binding betaglycan mutants restore growth factor-responsiveness and growth regulation in these tumour cell lines. We are also conducting additional rounds of site-directed mutagenesis in order to further refine our knowledge of betaglycan binding characteristics. Students would have the opportunity to create their own mutants and test their binding and functional characteristics. This project will give the student experience in a wide variety of cell and molecular biology techniques, including cell culture, transfection assays, DNA manipulation, site-directed mutatgenesis, PCR, cloning, luciferase reporter assays, proliferation assays, and apoptosis assays. 8. Kidney development in betaglycan knockout embryos Co-Supervisors: Dr. Kaye Stenvers and Professor John Bertram (Department of Anatomy and Developmental Biology, Monash University ) During nephrogenesis in the mouse, total nephron number is determined by multiple morphogenetic events. At the cellular level, these processes involve localised cellular proliferation, apoptosis, differentiation, and cell-cell interactions. A reduction in nephron number during foetal development as a result of environmental and/or genetic factors is frequently associated with a greater susceptibility to essential hypertension in adulthood. The existing data indicate that TGF b regulates nephron formation at more than one stage of development, yet it is currently unclear which specific cellular processes are affected by a loss or reduction in TGF b function. We hypothesize that modulation of TGF b signalling via betaglycan is vital for normal kidney development and renal health in adulthood. This hypothesis is supported by a recent study which shows that the presence of betaglycan on the surface of renal cells in vitro is necessary to confer optimal TGF b -mediated growth regulation. There are opportunities for students to help determine the in vivo impact of the loss of this receptor on nephrogenesis. We will utilise primary cultures of explanted embryonic kidney derived from our betaglycan knockout and wildtype embryos to determine betaglycan’s functions in particular cell types in the kidney. Wholemount immunohistochemistry using an antibody against calbindin will be used to visualise the ureteric tree as it develops. WT1 immunostaining will be performed to visualise the forming nephrons. Techniques used include: basic histology techniques, immunohistochemistry, light, fluorescent, and confocal microscopy, imaging, and computer analysis; sterology; genomic DNA extraction, PCR; mouse embryo microdissection; specialised kidney explant cultures; wholemount immunofluorescence Mechanisms of hormone actions Inhibin A and B are gonadal hormones synthesised by both the ovary and the testis to inhibit FSH production by the pituitary. Besides this important role in the negative feedback regulation of FSH, little is known of other physiological roles of inhibins on other target tissues. In addition, little is known on the molecular mechanisms underlying inhibin effects, or the genes that are differentially regulated by inhibins. Activins are closely related dimeric hormones that share common subunits with inhibins, and bone morphogenetic proteins (BMP) are more distantly related proteins. Inhibins can act as antagonists to activins, and some BMP forms. However, activins and BMPs additionally have diverse and widespread paracrine functions via the modification of cell proliferation, differentiation and apoptosis. 9. Modification of Sertoli cell proliferation and survival by inhibin Apart from the important role of inhibin in the negative feedback regulation of FSH, little is understood about other physiological roles of inhibin on peripheral target tissues, or of the molecular mechanisms underlying inhibin actions. The fact that “knockout” of the inhibin alpha-subunit gene leads to formation of testicular and ovarian tumours is as yet unexplained. The aim of this project is to study the ways that i) proliferation and apoptosis of testicular Sertoli cells are affected by activin and BMP isoforms, especially through interactions with the cytokine, oncostatin M, and ii) inhibin A and B alter such (inter)actions. These studies should provide us with new insights into the tumour suppressor role of inhibin. This project will involve cell culture, DNA transfections, proliferation and other functional assays, real-time RT-PCR. 10. Mechanisms underlying the common actions of activin, GDF-9, TGF-β and BMP in the adrenal cortex Supervisor: Dr Paul Farnworth Activins, GDF-9 and TGF-beta use distinct receptors but shared signalling pathways involving the intracellular Smad proteins 2 and 3, whereas BMPs share BMP type II receptors with GDF-9 and activin, but signal via Smads 1, 5 and 8. These ligands mostly regulate discrete sets of genes in other tissues. However, all four families of ligand suppress the expression of the Cyp17 gene in steroidogenic adrenocortical cells. This gene encodes an enzyme that is critical to the formation of androgens and estrogen. Preliminary evidence suggests that suppression of Cyp17 by these ligands may involve MAP/MEK kinases in addition to the different Smads. The aims of the study are to determine which kinase pathways are involved in mediating activin, GDF-9, BMP and TGF-beta actions on Cyp17 in adrenocortical cells, and comparing responses with those in Leydig-like cells. Pharmacological inhibitors will be used to provisionally identify which pathways are important, and critical kinases will then also be targeted using siRNA. This project will involve cell culture, DNA transfections, real-time RT-PCR, steroid analyses. 11. Structure/function analysis of betaglycan binding Co-supervisors: Dr. Kaye Stenvers an d Dr. Craig Harrison Betaglycan binds all three TGFbeta isoforms as well as the related factor, inhibin, and modulates the association of these ligands with their signalling receptors. Both TGF beta and inhibin are multifunctional growth factors that regulate a number of important cellular functions, including proliferation, differentiation, and survival. Dr. Craig Harrison has created a number of betaglycan expression constructs which carry mutations in the binding domains of betaglycan. Dr. Harrison identified point mutations which largely eliminate either TGFbeta or inhibin- binding, thus generating ligand-specific binding mutants. Projects are available to test the function of these mutants in cultures of breast and ovarian tumour cell lines, many of which have little betaglycan on their cell surface and respond poorly to TGFbeta-mediated growth inhibition. The aim of these studies would be to determine whether specific TGFbeta or inhibin-binding betaglycan mutants restore growth factor-responsiveness and growth regulation in these tumour cell lines. We are also conducting additional rounds of site-directed mutagenesis in order to further refine our knowledge of betaglycan binding characteristics. Students would have the opportunity to create their own mutants and test their binding and functional characteristics. This project will give the student experience in a wide variety of cell and molecular biology techniques, including cell culture, transfection assays, DNA manipulation, site-directed mutatgenesis, PCR, cloning, luciferase reporter assays, proliferation assays, and apoptosis assays.
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