The studies described in this thesis have been concerned with several aspects of follicular development in the mammalian ovary. Chapters 2, 3 6 4 deal with mathematical modelling of ovarian follicle dynamics in normal animals and comparisons with experimentally manipulated animals. Chapter 5 describes a novel aethod for estimating the clonal origin of the mouse ovarian follicle. In the final two chapters, the comparative physiology and anatomy of follicular numbers and sizes and the incidence of polyovular follicles are described for a number of species. The unifying theme of these studies is that they reveal patterns existing in follicular development and utllisation by detailed examination of one species, (CBA/ca mouse), and broadly by interspeci,fic comparisons (with relation to scaling). A detailed mathematical description of the follicular dynamics of virgin CBA/ca mice up to 98 days of age has been obtained by the application of compartmental modelling to differential follicle counts. The rates of follicle growth (migration) and death have been estimated for five ?stages of development (primordial to Graafian). The model predicts age changes in follicle growth and death rate, there being transitions in the parameters at 20 days second at 60 days. The parameters for normal animals have been compared with those of anilllals under two experimental conditions: 1) by unilateral ovariectomy at 4-2 days of age, which abruptly halves the numbers of ovarian follicles and alters the ratio of large : small follicles. 2) by blocking ovulation using progesterone implants. The dynamics of follicle growth were altered by both treatments in comparison with the controls. Follicles at all stages of development were affected by unilateral ovariectomy and differences may exist with time. The compensatory response by the remaining ovary was due to a combination of an increased preantral growth rate and a decrease in atresia at antral stages. Earlier stages of follicle development were affected this may have been incidental to the compensatory response. In progesterone treated animals follicles developed through to antral stages when they un~erwent atresia. The effects of treatment were observed at three levels of development: 1) The initiation of growth from the primordial pool, 2) Growth rate of small follicles and 3) deaths at larger stages of follicular development. Longer term observations indicated that these effects may not be constant. The modelling studies have looked at numerical changes in the follicle population with time but a greater understanding of the develomental biology of the follicle is required in order to explain the changes in growth and death rates observed. This problem has been tackled initially by studying the clonal origin of the follicular epithelium. The technique used is based on the principle that cells in females. are generally mosaic as a result of X-chromosome inactivation the use of X linked cell markers phospho-glycerate kinase-1 (PGK-1). Granulosa cells were found to be polyclonal in origin with the number of progenitor cells numbering 5 on average. Analysis of cumulus and mural granulosa cells showed that substantial cell mixing had occurred and cuaulus cells were generally founded by more than one clone. Finally, comparative studies have been conducted to look at scaling of follicle sizes and numbers and of polyovular follicles. Ovarian follicle and oocyte sizes were scaled according to body weight (ranging from .005-500Kg) using data from 22 species. Primordial and Graafian follicle sizes varied with body weight but closer correlations for the latter were obtained when the sum of the surface areas or volUiles for a preovulatory set were considered as opposed to the values for individual follicles. The numbers of nongrowtng follicles 1n reserve at young adult ages were correlated with maximum longevity of the species and related to body weight. The frequency of polyov~lar follicles varied 1n species studied and were most abundant 1n the domestic bitch. The overall incidence of polyovular folUcles 1n young bitches was 14 S, being reduced to 5~ 1n bitches at 7-11 years. The frequency of the various types of polyovular preantral folUcle varied inversely with the numbers of oocytes per follicle.

No description available.

Ovarian cancers often develop resistance mechanisms against the standard platinum and taxane chemotherapy, which indicates the need for novel therapeutics to improve patient outcome. In vitro assays were performed to assess the effects and mechanism of action of a novel peptide, GAP-107B8, on ovarian cancer cell viability. Xenograft models were used to determine GAP-107B8’s effects on tumour burden in immune-incompetent mice. GAP-107B8 significantly reduced cell viability in ovarian cancer cell lines, although no synergistic effects with carboplatin were observed. This reduction in cell viability was due in part to apoptosis and may involve mechanisms leading to decreased pAKT, but without any change in pPKC levels. In vivo, GAP-107B8 had no effect on ovarian tumour burden, but significantly reduced ascites volume. The findings suggest that GAP-107B8 can reduce some malignant characteristics of cancer cells in vitro and in vivo and should be evaluated further as a potential therapeutic for ovarian cancer.

PAX2 is a transcription factor that is essential for development. Aberrant expression of PAX2 in adult tissues is associated with carcinogenesis and experimental evidence shows that PAX2 generally exhibits oncogenic properties. Although PAX2 is not expressed in normal ovaries, it is highly expressed in low malignant potential and low-grade epithelial ovarian tumors, suggesting that PAX2 induction in ovarian surface epithelium (OSE) may contribute to transformation. Herein, we provide evidence that expression of PAX2 in normal murine OSE (mOSE) cells enhances their proliferation and survival and, when combined with loss of P53, induces tumorigenicity. In addition, OSE cells are known to gain an epithelial phenotype and express epithelial markers prior to their transformation. This study revealed that PAX2 induction in mOSE cells results in an enhanced epithelial phenotype associated with reduction of the epithelial-mesenchymal transition markers, SMA-α and COX-2. Furthermore, PAX2 inhibits the mesenchymal phenotype induced by TGF-β and reverses the TGF-β-mediated induction of both SMA-α and COX-2, in mOSE cells. Toward tumor progression, we found that when PAX2 was expressed in murine ovarian cancer cells, it enhanced or inhibited their aggressiveness, depending on the model system. In OSE cells transformed by K-RAS and MYC, PAX2 inhibited P53 accumulation and increased the level of pERK1/2 and COX-2. In addition, PAX2 inhibited apoptotic induction in these tumors, while increasing angiogenesis, both of which are enhancers of tumor aggressiveness. However, in a murine model of high-grade serous ovarian cancer, PAX2 expression reduced tumor mass and improved animal survival, likely via reduced proliferation and metastasis. Mechanistic studies showed that PAX2 increased Htra1 and decreased COX-2 in those tumors. Both HTRA1 and COX-2 are novel downstrream targets for PAX2 that are identified in the current study. These results suggest that PAX2 may not act as a classical oncogene or tumor suppressor in ovarian cancer; rather, it modulates tumorigenesis differently, depending on the tumor context. The observation that PAX2 targets distinct biological and molecular pathways might help to guide future studies to different therapeutic targets in low-grade vs. high-grade cancers.

Estrogenic hormone replacement therapy increases the risk of developing ovarian cancer, and 17-β estradiol accelerates tumour growth in mouse models of this disease. One possible mediator of estrogen action is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1), an estrogen receptor alpha (ESR1)-upregulated protein. GREB1 is required for hormone-driven proliferation of several breast and prostate cancer cell lines, but its role in tumour progression is unknown and its mechanism of action remains unclear. To determine the role of GREB1 in ovarian cancer, its expression and function were examined in ovarian cancer cell lines and mouse models. GREB1 was upregulated by estradiol, and overexpression and/or knockdown of GREB1 showed that GREB1 promotes proliferation and migration in ovarian cancer cells. GREB1 knockdown also slowed tumour growth and prolonged survival in mice engrafted with ovarian cancer cells. GREB1 expression was detected in human ovarian tumours of all major histotypes, with moderate to strong expression in 75-85% of serous, endometrioid, mucinous, and clear cell carcinomas. GREB1 mRNA levels correlated with ESR1 transcripts, but protein levels of GREB1 and ESR1 did not show a correlation in tumours. Serous, endometrioid and mucinous ovarian cancers were almost always positive for either ESR1 or GREB1 (59/60 tumours), which may imply a dependence on estrogen signalling pathways. GREB1 expression in normal tissues is mainly confined to the reproductive tract, suggesting that it may be useful as a diagnostic biomarker. GREB1 combined with PAX8 showed high efficacy in differentiating mucinous tumours of gastrointestinal vs. ovarian origin. Targeting GREB1 may inhibit tumour-promoting pathways downstream of ESR1 and could therefore prove more effective than current anti-estrogen therapies.

Retrotransposons constitute about a third of our genome. It is challenging to identify the causes and consequences of retrotransposon expression in human disease due to hundreds of active genomic copies and poor conservation across species. We profiled genomic insertions of retrotransposons in ovarian cancer. RNAs exhibiting Bayesian correlation with retrotransposon RNA were analyzed to identify potential causes and consequences of retrotransposon expression in ovarian and breast cancers. This strategy found divergent inflammatory responses associated with retrotransposon expression in ovarian and breast cancer. It identified new factors inducing the expression of endogenous retrotransposons, including anti-viral responses and the tumor suppressor BRCA1. In cell lines, mouse ovarian epithelial cells and patient-derived tumor spheroids, BRCA1 promoted the accumulation of retrotransposon RNA and facilitated transcription of active families of retrotransposons and their insertion into the genome. Intriguingly, elevated retrotransposon expression predicted survival in ovarian cancer patients. Retrotransposons are part of a complex regulatory network in ovarian cancer, including BRCA1 contributing to patient survival. The above-described analysis strategy could also be used to identify the regulators and impacts of retrotransposons in various contexts of biology and disease in humans.

No description available.

Ovarian cancer, primarily diagnosed at advanced stages of the disease, is the most lethal of all gynaecological malignancies, with a 5-year survival of only 45%. Increasing age and number of ovulations are the primary non-hereditary risk factors, with the median age of onset being 63 years. Considering that risk peaks upon onset of menopause and that 50% of all cases of ovarian cancer have non-ovarian origins, it is believed that the physiological aging of the ovary renders it an appealing pre-metastatic niche. Mutations in the tumor suppressor genes BRCA1 and BRCA2 are the primary hereditary risk factors, accounting for 20-25% of all cases. We have preliminary data showing that BRCA1 mutation carriers tend to develop ovarian fibrosis, a phenomenon that naturally accompanies ovarian aging, at premenopausal ages, whereas age-associated fibrosis becomes evident after menopause in non-carriers. Consistently, BRCA1/2 mutation carriers are at elevated risk for premature cessation of ovarian function. With the median age of cancer onset decreasing from 63 to 50 years of age in BRCA1 mutation carriers, these data collectively suggest accelerated ovarian aging in these women and highlights an association between ovarian aging and increased risk for cancer. As such, we hypothesized that loss of Brca1 in murine ovarian fibroblasts (MOFs) may accelerate the onset of ovarian fibrosis through fibroblast hyperactivation, contributing to ovarian aging. Using primary MOFs isolated from Brca1 LoxP/LoxP mice and adenoviral cre mediated recombination, we generated Brca1 deficient MOFs. RNA sequencing was used to characterize the transcriptomic changes associated with the loss of Brca1. Our findings suggest that Brca1 deficiency in MOFs induces cellular senescence and enhances their myofibroblastic function, likely yielding increased stiffness of the ovarian extracellular matrix due to the dysregulated synthesis and degradation of its constitutive components, contributing to accelerated ovarian aging.

Fibrinogen-like protein 2 (FGL2) expression is associated with tumour progression and poor survival in a number of different cancers. In these cancers, FGL2 promotes tumour progression via an immunosuppression-mediated mechanism or by promoting angiogenesis. Querying single-cell RNA sequencing (scRNA-seq) human cancer data shows FGL2 is produced by immune and stromal cells in the tumour microenvironment (TME), while cancer cells have minimal expression of FGL2. We therefore studied the role of FGL2 produced by cells in the TME in tumour progression in two models of cancer in which the role of FGL2 has not been previously studied: epithelial ovarian cancer (EOC) and melanoma. EOC is the most lethal gynecologic cancer with an imperative need for new treatments. Before testing novel immunotherapies, we first characterized the TME of six syngeneic ovarian cancer models. ID8-p53-/- and ID8-C3 tumours were most highly infiltrated by T cells, whereas STOSE and MOE-PTEN/KRAS tumours were primarily infiltrated by tumour associated macrophages and were unique in MHC class I and II expression. This panel of well-defined murine EOC models can serve as a valuable resource for studies that aim to test immunotherapies. We next studied the role of FGL2 in tumour progression in melanoma and ovarian cancer models. Using wild-type and FGL2 knockout mice, we found that absence of FGL2 led to a more activated TME, including activated dendritic cells (DCs - CD86+, CD40+) and T cells (CD25+, TIGIT+), as well as demonstrating for the first time that the absence of FGL2 led to more activated Natural Killer (NK) cells (DNAM-1, NKG2D) in the TME. The absence of FGL2 prolonged survival in the B16F10 model of melanoma, and synergized with oncolytic virus to prolong survival in the ID8-p53-/-Brca2-/- model of ovarian cancer.

Chemotherapy treatment in premenopausal women has been linked to premature ovarian failure (POF), and hence infertility, through ovarian follicle loss. The exact mechanisms that lie behind this loss are unclear and so the action of two commonly used chemotherapeutic agents were compared here. Cisplatin is a DNA cross-linking agent commonly used in the treatment of ovarian, lung and bladder cancers, while the anthracycline doxorubicin is commonly used to treat leukaemia and breast cancer. Neonatal mouse ovaries were cultured in vitro and exposed to cisplatin or doxorubicin in order to determine their effects on primordial and early growing follicles. Both drugs caused a dose dependant follicle loss but targeted different cell types. Cisplatin caused a significant increase in follicles with unhealthy oocytes; furthermore primary stage follicles were the follicle class most affected (up to 98% classified as unhealthy compared with 13% in control, p<0.001). In contrast, doxorubicin caused a significant increase in follicles with unhealthy granulosa cells and affected all follicle stages present. When the mechanism of cell death was further investigated, apoptosis was the main pathway through which these drugs cause ovarian cell death. Doxorubicin in particular caused a significant increase in apoptosis of ovarian somatic cells including the granulosa cells and stroma. Imatinib mesylate, a tyrosine kinase inhibitor which is also used as a chemotherapeutic agent, has been implicated as a potential therapy to block the ovotoxic effects of cisplatin. Results here confirm this finding (29% of follicles classified as unhealthy in the cisplatin only group compared to 8% in the cisplatin and imatinib co-treatment group, p<0.001) and found further, that imatinib was unable to protect against doxorubicin-induced damage (28% of follicles classified as unhealthy in the doxorubicin treated group compared to 19% in the doxorubicin and imatinib cotreatment group). Imatinib treatment alone in newborn ovaries caused a significant increase in the number of follicles present at the end of culture compared to control (402±43 in the imatinib group compared to 188±34 in control, p<0.001), which is likely due to an effect on follicle formation. In conclusion, the work presented in this thesis demonstrates drug specific actions of cisplatin and doxorubicin on the mouse ovary. This suggests that any therapy designed to confer ovarian protection in the future may have to be tailored to be drug specific.