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Title: Oestrogen regulation of gene expression in male germ cells and Sertoli cells
Author: Sneddon, Sharon F.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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The aims of this study were to investigate the role of steroid hormones, in particular oestrogens, in murine spermatogenesis. A major focus of these investigations was the role played by ERβ in the modulation of germ cell and somatic cell function. Studies were conducted both using a transformed murine Sertoli cell line (SK11), which has maintained a differentiated Sertoli cell phenotype and spermatogonial stem cells, which were successfully isolated and characterised. ERβ mRNA and protein were shown to be expressed in the SK11 cells both in the undifferentiated and differentiated states. Transient transfections using ERE or ARE-luciferase reporter constructs and stimulation with steroid ligands revealed that the cells contained functional steroid hormone receptors. Knockdown of ERβ mRNA and protein was achieved in the cells after targeted deletion using a short hairpin RNAi containing vector; this blunted the ability of the cells to respond to oestrogen. Isolation of spermatogonial stem cells was carried out using immunomagnetic beads. The stem cell population were shown to express Oct-4 and GFRα-1 mRNAs, both of which are stem cell markers, but not c-kit, which is a marker of differentiated germ cells. Taqman Q-RT-PCR demonstrated that the stem cell population expressed ERβ. Oct-4 mRNA expression was shown to be reduced by RNAi; this induced the cells to undergo differentiation in vitro characterised by increased expression of c-kit. In conclusion, the current studies have extended our understanding of the impact of steroid hormones on testicular function and have revealed for the first time that spermatogonial stem cells are ERβ positive. The SK11 cell line has been found to provide a suitable model system for the study of steroid regulation of Sertoli cell function. In addition, the use of RNAi has provided and exciting new avenue by which to manipulate gene expression levels in testicular germ and somatic cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available