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Title: Studies on Sertoli cell function using in vivo and in vitro models
Author: Hooley, Robert P.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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SK11 cells had detectable expression of ERβ mRNA, and activated an ERE-luciferase reporter construct in response to a range of oestrogenic ligands. In contrast endogenous Ar expression was low, but transfection with mouse Ar cDNA resulted in sufficient Ar expression to activate a luciferase reporter under control of the Rhox5 proximal promoter in response to androgen treatment. However transfected SK11 cells could not stimulate endogenous Rhox5 mRNA expression under similar conditions. Infection of SK11 cells with an advanced construct in vitro resulted in efficient transgenic expression in 80-100% of cells, and was associated with excellent cell survival. In vivo infection in mouse testes with an adenoviral vector containing a GFP transgene, performed by injection via the efferent ductules, resulted in SC-only transgene expression. When a dose of 4x108 pfu was introduced the seminiferous tubule’s architecture was severely damaged, invasion by macrophages and neutrophils occurred, plus expression of markers of hypoxia and apoptosis was detected. Infection with lower doses (1x105 – 1x107 pfu) resulted in disruption to the seminiferous epithelium consisting of loss of pachytene germ cells and formation of intra-epithelial vacuoles. Formation of vacuoles may be due to interaction of adenovirus with the coxsackie/adenoviral receptor (CAR) in SC-SC junctions. Androgen-depletion in rats using a single dose of EDS to ablate the Lc population that synthesise testosterone caused reduced expression of Ar, Rhox5, espin and β3-tubulin mRNA and Ar protein 6 days after treatment, but the expression and distribution of the junctional proteins espin, Cx43, zona occludins 1, and N-cadherin were unaffected. We can conclude that SK11 cells lack vital factor/s required for activation of response elements by androgens in their endogenous promoter regions, which raises the possibility that association with other testicular cell types (GC or PTM) may be required for normal SC function to be maintained. Adenoviral vectors appear good for efficient introduction of transgenes into SC in vitro but not for use in vivo. Finally, testosterone depletion using the EDS-rat model revealed reduced mRNA expression of putative androgen responsive genes identified in previous array studies, and provides a valuable model for validation of other putative targets identified in other studies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available