Investigation into the expression and localisation of c-kit and the regulation of kit ligand gene expression in the adult human ovary.

Abstract

Folliculogenesis is a complex process that is central to the ovary’s primary function, the production of healthy oocytes. One of the essential ligand/receptor pairs that mediates folliculogenesis is kit ligand (KITL), a granulosa-derived cytokine growth factor, and its receptor, c-kit. Since their discovery two decades ago, the KITL/c-kit system has been extensively studied in animal models, in particular the mouse, in which it has been demonstrated to be crucial for normal folliculogenesis and fertility. To date, little investigation into KITL and c-kit has been performed in the adult human ovary. Previously, this laboratory showed abnormally increased KITL protein levels in human polycystic ovaries (PCO) compared to non-PCO, suggesting that KITL may contribute to several PCO phenotypes according to the range of actions KITL has been shown to have in animal folliculogenesis. Thus, this thesis aimed to characterise KITL and c-kit expression and localisation in the adult human ovary, including polycystic ovaries, and examined regulation of KITL gene expression by endocrine and intraovarian factors. To perform these studies, human ovarian tissues were obtained. These included granulosa cell subtypes cumulus and mural granulosa cells from women undergoing assisted reproductive technology treatment at infertility clinics, fresh ovarian cortex from the Royal Adelaide Hospital and archival paraffin-embedded human ovarian tissue from the Institute of Medical and Veterinary Sciences. The human granulosa tumour cell line, KGN, was also used as a model. KITL and c-kit isoforms were demonstrated to be present in the human ovary throughout follicle development. KITL-2 was shown to be expressed primarily by granulosa cells representing preantral follicles, while KITL-1 was the predominant isoform expressed in preovulatory granulosa cells, suggesting that KITL-2 may play a greater role during early follicle development which then diminishes in preovulatory follicles with increased KITL-1 levels. Both c-kit mRNA isoforms were found to be present in human ovarian cortex. Examination of c-kit localisation throughout follicle development by immunohistochemistry revealed that all follicular cell types in preantral and antral follicles expressed c-kit protein. This may suggest that KITL has an unknown autocrine function in granulosa cells unique to the human ovary, as animals models have previously demonstrated c-kit staining to be confined to the theca layer and the oocyte. c-kit staining patterns were found to be different in PCO compared to non-PCO preantral and antral follicles, suggesting a potential involvement for c-kit in PCO pathology. Collectively these results suggest, as demonstrated in various animal models, that the KITL/c-kit system is present in the human ovary and may have some involvement in the mediation of human folliculogenesis. Regulation of KITL gene expression was examined using KGN and cumulus cells. Based on previous studies, the candidate regulatory factors that were examined included androgen receptor (AR), endocrine factor follicle-stimulating hormone (FSH), theca-derived factor keratinocyte growth factor (KGF) and oocyte-secreted factors bone morphogenetic factor-15 (BMP-15) and growth differentiation factor-9 (GDF-9). Of these candidate factors, GDF-9 was found to directly decrease KITL gene expression in KGN cells and cumulus cells via ALK 4/5/7 receptors. There was also some evidence for a slight reversal of the GDF-9 effect on KITL expression by the addition of the potent androgen 5α-dihydrotestosterone (DHT). The results of these studies indicated KITL gene expression is regulated by GDF-9 in human granulosa cells and are consistent with observations of negative regulation of KITL expression in mouse granulosa cells. Evidence shown in this thesis suggests that the ratio of KITL isoforms in granulosa cells changes throughout human folliculogenesis. Follicular target cells for KITL signalling were found to include granulosa cells, theca cells and the oocyte, suggesting that the KITL/c-kit system may have potential roles throughout human folliculogenesis as demonstrated in animal models. Furthermore, this thesis has demonstrated that GDF-9 directly regulates KITL gene expression in human granulosa cells. From these results, this thesis proposes an in vivo model in which abnormally low levels of GDF-9, shown by a previous study to be characteristic of PCOS oocytes, results in increased KITL levels and this effect may be further exacerbated by the reversal of GDF-9 inhibition by excess androgen. This thesis has provided a greater understanding of the molecular mechanisms involved in human folliculogenesis which may be of use in future therapeutic strategies and diagnosis in assisted reproductive technology, and provide a basis for understanding human ovarian function and ovarian disease.