title: Analysis of the murine granulosa cell transcriptome during luteinisation
Creators: name: McRae, Robert Scott
Publisher: name: University of Glasgow
Year: 2005
Subjects: subject: Developmental biology, endocrinology.

description: The granulosa cells of the ovarian follicle surround the oocyte and support it during follicle development. Once exposed to the LH surge, the granulosa cells are characterised by the induction of genes necessary for cellular differentiation. The extensive morphological and functional changes which characterise luteinisation involve the regulation of gene and protein expression responsible for the cessation of proliferation and the induction of differentiation in the individual granulosa cells. The differentiating granulosa cell also functions in both an endocrine and paracrine manner mediating follicle and oocyte maturation and subsequent corpus luteum remodelling. The formation of the functional corpus luteum and secretion of progesterone is essential for the establishment of pregnancy following ovulation. Although much is known about the molecular mechanisms responsible for follicular development comparatively little study has been carried out to analyse the control of, and events which occur during, luteinisation. It is therefore pertinent to study gene expression changes to try to clarify and understand mechanisms which regulate and underpin ovarian granulosa cell luteinisation. In order to investigate the mechanisms underlying these processes we embarked on a time- and cell-specific analysis of gene expression in the granulosa cell during late follicle development and early luteinisation. Changes in gene expression during granulosa cell luteinisation were measured using serial analysis of gene expression (SAGE). Immature normal mice were treated with gonadotrophin to induce formation and luteinisation of ovarian follicles. SAGE libraries were generated from mRNA isolated from granulosa cells collected before and after induction of luteinisation. The combined libraries contained 105,224 tags representing 40,248 unique transcripts. Overall, 715 transcripts showed a significant difference in abundance between the two libraries of which 216 were significantly down-regulated by luteinisation and 499 were significantly up-regulated. Among transcripts differentially regulated, there were clear and expected changes in genes involved in modelling of the extracellular matrix, regulation of the cytoskeleton and intra and intercellular signalling. Also identified were transcripts relating to genes and cellular signalling pathways novel to the granulosa cell, including members of the E2F family of cell cycle regulators and the Notch signalling pathway as well as genes implicated in angiogenesis and cellular metabolism not previously associated with the granulosa cell. Further studies into an unmatched SAGE tag which was highly differentially expressed revealed that it represented a variable length non-coding transcript which showed a tissue- and temporal-specific expression pattern within granulosa tissue. This transcript is highly conserved across species and lies distal to the 3' end of the inhibin BA subunit. Highest levels of expression were found within the gonadotrophin-stimulated, mature antral follicle prior to the LH surge where it was the 6th most highly expressed transcript. After luteinisation there was a rapid downregulation of expression. It is suggested that this transcript may have involvement in regulating the transcription and/or translation of the inhibin BA subunit during follicle development and luteinisation. In conclusion, this thesis provides insight into some of the important mechanisms involved in the regulation of luteinisation, namely angiogenesis, differentiation, cell cycle control and the metabolic machinery within the granulosa cell. We have isolated a large number of candidate genes related to the cellular differentiation processes occurring within the granulosa cell during luteinisation. The data generated and presented here constitutes a new base for the testing of hypotheses in the field of follicle development and luteinisation.