The Development of Bovine Fetal Ovary and Its Relationship with Reproductive Disease

Abstract

The development of fetal ovary is a complex process involving the communication between primordial germ cells (PGCs), Gonadal Ridge Epithelial-Like (GREL) cells and stroma. Many studies in ovarian development have focused on follicles, making other features, such as stroma and ovarian surface the least studied. Additionally, perturbation of ovarian development has been suggested to lead to many reproductive diseases, such as Polycystic Ovary Syndrome (PCOS) and ovarian cancer. Studies have shown that prenatally androgenised female mammals had ovaries with PCOS features, such as small follicles and increased ovarian stromal volume. Similarly, ovarian cancer has been suggested to be able to arise from non-ovarian tissue, such as fallopian tubes and endometrium. This thesis discussed the changes of ovarian stroma (chapter II) and linked the changes to PCOS (chapter III). Furthermore, this thesis quantitated changes of non-stromal areas containing GREL cells and follicles (chapter IV) as well as identified the changes in ovarian surface during fetal development (chapter V). Early in ovarian development, the surface epithelium of the mesonephros differentiates into GREL cells and proliferates, forming the ovarian primordium into which PGCs migrate. The stroma from the mesonephros penetrates the developing ovary and then branches. At this time, a true surface epithelium is located only at the base of the developing ovary. When the stroma reaches just underneath the surface of the developing ovary, it spreads laterally. The branching and spreading of the stroma encloses some GREL cells and PGCs into ovigerous cords. At this point, the ovigerous cords are still open to the ovarian surface. The stroma keeps on spreading, dividing the ovigerous cords into smaller units and eventually into primordial follicles. GREL cells located on the ovarian surface then start to differentiate into surface epithelium. At the last stage of development, a single layer of surface epithelium is formed and the stroma underneath the surface epithelium changes its phenotype into tunica albuginea, a collagen rich layer. In this thesis, I analysed the stromal and non-stromal areas morphometrically and linked ovarian development with expression of a number of extracellular matrix genes. My results show that the volume of the ovarian cortex and medulla increased throughout gestation. The stromal proportion and total volume in the cortex were significantly increased (p>0.05), whereas the proliferation index and numerical density of proliferating cells in the stroma decreased significantly (p>0.05). There was no change in numerical density of stromal cells in the cortex. Twelve extracellular matrix genes were highly expressed later in the development and positively correlated with each other and with gestational age. The total volume of non-stromal areas in the ovarian cortex significantly increased and then levelled off. The proportion of non-stromal areas in the cortex decreased significantly. The proliferation index of the non-stromal area peaked in early gestation and then decreased significantly and then remained low. The numerical density of the stromal area remained constant throughout ovarian development. My morphometric data of the stromal area as well as the gene expression have been published in Reproduction Fertility and Development. The data of the non-stromal area have been combined with other data and published in PLoS ONE. Since many reproductive diseases might have a fetal origin, we studied the linkage between fetal ovarian development and PCOS. Recent studies have recognised 18 PCOS candidate genes identified by genome wide associated study (GWAS) analyses. Using qRTPCR, I analysed the expression of these genes, as well as three other genes (androgen receptor (AR), Transforming Growth Factor Beta 1 induced transcript 1 (TGFB1I1), fibrillin3 (FBN3)) in the bovine fetal ovaries across gestation. To assess the regulation of these genes in the bovine fetal ovary, I analysed the expression of these genes in bovine fetal fibroblasts which had been treated with cAMP regulators, growth factors and hormones in vitro (24 treatments in total) during a previous honour project. FBN3, GATA4, HMGA2, TOX3, DENND1A and LHCGR were highly expressed in the early development, whereas INSR, FSHR and LHCGR, including 3 PCOS-related genes (AMH, AR and TGFB1I1) were highly expressed in the late development. These eleven genes were strongly correlated to each other, although some of them expressed in different cell types. Treatment of fetal stromal cells with TGFβ induced the expression of INSR, AR, C8H9orf3 and RAD50 and inhibited expression of TGFB1I1. The data have been submitted to Scientific Reports. In the attempt of investigating the changes of ovarian surface, I analysed Scanning Electron Microscope (SEM) images and compared them with immunohistochemistry images, as well as determined the proportions of different types of cells in the ovarian surface epithelium. Early in development, the cells at the base of the developing ovary were cuboidal whereas the remaining surface appeared more irregular. Around 10 weeks of gestation until 5 months of gestation, the surface was covered by a stratified or simple epithelium of cuboidal cells. During mid-gestation clefts could be observed on the surface coinciding below with open ovigerous cords. Later in development, most of the ovary was covered by a simple surface epithelium. There appear to be two origins of ovarian surface epithelium – at the base/hilum originating from the mesonephros and on the remainder from the GREL cells.. The data have been submitted to Journal of Histology and Cytochemistry. Together, this work has shown the behavioural and structural changes of stroma and ovarian surface during fetal development. Since some of the PCOS candidate genes are expressed during fetal ovarian development, any potential disruption during ovarian development might have implications for the development of PCOS phenotype in the adult life.