Major Research Programs

1. Ovarian Follicular Growth and Female Infertility.             
Ovarian follicular development and atresia is the culmination of complex actions and interactions of gonadotropins and intra-ovarian regulators. Although the importance of FSH, thyroid hormone, epidermal growth factors and transforming growth factor beta family members in the regulation of ovarian function is well established, how these ovarian regulators interact at the subcellular levels in determining the fate of granulosa cells (proliferation differentiation vs. apoptosis) and eventually follicle destiny (continual growth vs. atresia) is poorly understood. In addition, the regulation of preantral follicular growth is distinct from that in antral and preovulatory follicles. As the preantral/early antral development is most susceptible to dysregulation, understanding the molecular and cellular mechanisms controling preantral-early antral growth may provide important insight into ovarian pathophysiology.            
Polycystic ovarian Syndrome (PCOS), a multi-factorial heterogeneous syndrome with complex pathologies, affects up to 10% of women of reproductive age and accounts for 75% of anovulatory infertility.  PCOS is associated with follicle growth arrest at the early antral stage, chronic anovulation, minimal granulosa cell proliferation, hyperthecosis and hyper-androgenemia, and insulin resistance. PCOS is a disorder of reproduction and metabolism with potential systemic sequelae such as diabetes and obesity. Obese women often have more severe hyperandrogenism and anovulation than normal weight women with PCOS. In this research program, we examine the crosstalk between death and survival signaling pathways in the regulation of ovarian follicular growth and atresia by endocrine, paracrine and autocrine regulators and/or extracellular matrix protein-receptor activation.            
Our research plan will be focused primarily on two aspects: (1) Defining the physiologic actions and interactions of endocrine and intra-ovarian regulators (gonadotropins, the novel adipokine chemerin, GDF9, IGF-I) and their intracellular signalling pathways (prohibitin, PI3K/Akt, MAPK) and mechanisms (microRNAs) in the control of normal follicular growth; and (2) Understanding the complex pathophysiology of PCOS by studying the aberrant regulation of these cellular mechanisms, using an androgenised rat PCOS model and validating these findings by comparing their relative expression in normal and PCOS human follicles. We will also determine if elevated serum chemerin level is indeed associated with obesity and dysregulated steroidogensis, insulin sensitivity and lipid metabolism in PCOS.              
Our research strategies will involve in vivo (eCG- or DES-primed immature rats, mature normal and “PCOS” rats) and in vitro [granulosa cell and follicle cultures coupled to gene manipulation protocols (sense, siRNA, antisense, dominant negatives)] approaches. We will compare the ovarian expression of Chemerin, its receptor CMKLR1, IGF-I receptor, GDF9 and prohibitin in human preantral/early antral follicles and PCOS follicles and serum chemerin levels in normal and obese human subjects w/wo PCOS to validate the results from the rat “PCOS” model. Standard molecular and cellular techniques will be used to assess follicular growth and function (steroidogenesis). The role of microRNAs in the control of key steroidogenic enzymes will also be studied using specific precursors and inhibitors. (Funded by Canadian Institutes of Health Research).

       2. Human Ovarian Cancer Biology and Chemoresistance.
Ovarian cancer (OVCA) is the most lethal gynecological malignancy, which could be attributed primarily to late diagnosis and chemoresistance. Although its initial therapeutic response rate is high, many recur with a resistant phenotype. Chemoresistance is a major hurdle for successful treatment and the mechanism involved is multi-factorial and is partly due to defects in drug-induced apoptosis.  TP53 mutation is a frequent event in human OVCA and is often associated with decreased responsiveness to chemotherapy, suggesting that p53 is required for CDDP sensitivity. It induces apoptosis in a transcription-dependent and -independent manner. p53 is activated by CDDP through a Chk1-mediated phosphorylation of serine residues 15 and 20 which increases its pro-apoptotic properties. Our recent studies indicate that the protein phosphatase magnesium-dependent 1 D (PPMID) is important in the regulation of p53 activation and chemosensitivity in OVCA cells, although its underlying mechanism (s) is unknown. Gelsolin (GSN) is a cytoskeleton-associated protein that regulates actin dynamics and is aberrantly regulated in many tumor types. It also plays an important role in regulating apoptosis. We have shown higher levels of GSN in chemoresistant OVCA and head and neck cell than in their sensitive counterparts  and its level is significantly correlated with recurrent stage IV disease. Mitochondria are highly dynamic organelles, constantly elongating and dividing to form a network, undergo two opposing processes for maintaining mitochondrial function: mitochondrial fission and fusion. Whether and how the mitochondrial dynamics control of chemosensitivity and their dysregulation play a role in the pathobiology of chemoresistance in OVCA, is not known. The PI3K/Akt pathway promotes cell survival and chemoresistance and is frequently amplified/over-expressed in OVCA. If and how p53, GSN and Akt interact in eliciting changes in chemosensitivity is still poorly understood. Our overall objective is to better understand the mechanisms of chemoresistance in human OVCA by investigating p53, GSN and Akt function in the control of mitochondrial fission and fusion and nuclear function in these cells. Our overall hypothesis is that chemosensitivity is determined by complex interactions of intracellular intermediates which culminate in the regulation of apoptosis at different subcellular levels. Akt confers resistance in part by suppressing CDDP-induced p53 activation and apoptotic pathways. Specifically, in chemosensitive OVCA cells, CDDP treatment leads to I-GSN-Drp1 interaction and mitochondrial import. Activated p53 is translocated from the cytosol to the mitochondria and interacts with I- GSN-Drp1 complex, resulting in displacement of I-GSN. Drp1-p53 complex induces mitochondrial fission, Cytochrome c-AIF release and apoptosis. Secondly, we also hypothesize that the processing of Opa1 by protease Oma1, is necessary for mitochondrial fission and apoptosis. In chemoresistance, I-GSN, Drp1 and p53 are not activated nor targeted to the mitochondria, and Opa1 is not processed, resulting in suppressed mitochondrial fission. PPM1D modulates CDDP sensitivity by decreasing Chk1 and p53 activities, a phenomenon promoted by the PI3K/Akt survival pathway. Akt promotes PPM1D phosphorylation, and nuclear translocation, thereby protecting it from calpain-mediated degradation. Our specific objectives are: (1) To study the role of GSN and p53 in mitochondrial dynamics and chemoresistance; (2) To define the nuclear action of GSN: role in AIF-induced apoptosis;  (3) To examine the mechanism of p53 activation: PPM1D and modulation by Akt;  (4) To assess the role and regulation of PPM1D and Gelsolin in chemosensitivity in vivo (ovarian xenografts and tumour screening), (5) To examine the effectiveness of functional food compounds and natural products in sensitizing chemoresistant ovarian cancer cells to traditional chemotherapeutic agents. The program integrates in vitro, in vivo, and clinical approaches to dissect the mechanisms of chemoresistance in OVCA. The studies will demonstrate, for the first time, (i) role of GSN in the regulation of chemosensitivity, (ii) possible dysregulated mitochondrial fission/fusion in chemoresistance, (iii) regulation of PPMID by Akt in the control of p53 activation and CDDP sensitivity, and (iv) the complex interaction of GSN, p53, PPMID and Akt during CDDP-induced apoptosis. It will provide novel insights into the pathobiology of and possible new therapy for chemoresistant OVCA.  (Supported by Canadian Institutes of Health Research, National Cancer Institute of Canada, and National Research Foundation of Korea).  

3. Placental Apoptosis and the Regulation of Placental and Fetal Growth.               
The understanding of the regulatory mechanisms responsible for placental growth is crucial to further our knowledge of what controls fetal growth. Indeed, placental growth is directly correlated with fetal growth as the placenta is essential to provide adequate nutrient, blood and oxygen supply to ensure adequate fetal growth and development. Despite advances in perinatal care, fetal growth restriction, often seen in pre-eclampsia, remains a leading cause of perinatal morbidity and mortality. Infants from these pregnancies often suffer multiple complications such as asphyxia, respiratory distress syndrome, prematurity and metabolic disturbances. In collaboration with Drs. Andrée Gruslin and Ajoy Basak, we are testing the hypothesis that soluble Fas ligand is involved in the regulation of placental apoptosis and is dependent on expression and/or processing of mFasL as regulated by MMP-7 and its inhibitor, TIMP-3. The processing of mFasL is dysregulated in abnormal placental conditions such as pre-eclampsia and fetal growth restriction, which are characterized by hypoxia.            
The overall objective of this research program is to examine the role and regulation of soluble Fas ligand in the control of apoptosis in normal and pathological conditions of the human placenta. Specifically, we will: (a) determine the expression of the precursor protein of sFasL (mFasL) throughout placental development and its relationship to placental apoptosis, (b) assess the processing of mFasL and its significance in initiation of placental apoptosis throughout development by examining the expression and activities of MMP-7 and, TIMP-3, (c) examine the influence of hypoxia on mFasL expression and processing and assess the significance of the above mFasL-related changes in pathological placental development, (d) the interaction of EGF and Leptin in the regulation of trophoblast invasion; and (e) the role of proprotein convertase-4 and IGF-II terminal maturation in the pathophysiology of pre-eclampsia and intrauterine growth restriction (support by the Toronto Sick Children Hospital Foundation and Philip Morris Company).  
   
4. Reproductive and Mammary Tumourigenic Effects of Environmental Toxins.
The overall objective of this program is to investigate the influence of environmental toxins on female reproductive function and mammary tumourigenesis. In particular, the effects of physiologically relevant concentrations of organochlorine pollutants, e.g. dioxins and PCBs, on ovarian follicular development and atresia, ovulation and mammary tumour formation are studied (Funded by the Toxic Substances Research Initiative, Health Canada).

5. Molecular and Cellular Predictors for Outcome Assessment in Assisted Reproduction.  Despite considerable advances made in recent years in improving the outcome of assisted reproductive technologies, a significant hurdle yet to overcome in IVF-ET relates not only in the identification of the determinants for poor oocyte quality and embryo development and mortality, but also to defining strategies to ameliorate these conditions. In collaboration with Drs. M. Mbikay and M.C. Léveillé in The Ottawa Hospital IVF-ET Program, efforts are made to examine cumulus cell biomarkers and apoptosis and to correlate the observed changes with oocyte / cumulus cell morphology and the ability of the oocyte to develop into a viable embryo following fertilization. In addition, this research program should provide a better understanding the molecular and cellular mechanisms of human embryo fragmentation and their significance as determinants in IVF- ET outcome, as well as important insights for the development of diagnostic tests for oocyte quality for IVF-ET (Supported by Canadian Institutes of Health Research).