Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The regulation of gonadotropin releasing hormone (GnRH) receptor numbers in the pituitary is a
crucial control point in reproduction. Pituitary sensitivity to GnRH can be directly correlated with
GnRH receptor levels, which can be regulated at transcriptional and post-transcriptional level. The
proximal promoter of the mouse GnRH receptor gene contains two cis elements bearing the
consensus sequence for a Steroidogenic Factor-l (SF -1) binding site. The distal site has previously
been shown to be involved in basal and tissue-specific transcriptional regulation, whereas the
function of the proximal site was not established. SF-I, a member of the nuclear receptor
superfamily of transcription factors, is involved in the transcriptional regulation of a large number
of genes involved in steroidogenesis and reproduction. The consensus SF-I binding site can serve
as a binding site for several members of the nuclear receptor superfamily. The aim of this study was
to investigate the binding of SF-I protein from the aT3-1 gonadotrope cell line to the two putative
SF-I binding sites in the mouse GnRH receptor promoter in vitro, in order to provide supporting
evidence for their functional roles in GnRH receptor gene regulation. It was shown by Western
blotting that SF-I and Nur77, another nuclear receptor transcription factor, are both expressed in
aT3-1 cells, in a manner that is influenced by cell culture conditions. Gel mobility shift assays
using specific antibodies showed that both SF-I and Nur77 protein in aT3-1 nuclear extracts bind
to both sites in a mutually exclusive fashion. As shown by competition assays using mutated
versions of the two sites, Nur77 protein had different base pair requirements than that of SF-I
protein for binding to the sites. Additionally, SF-I mRNA was shown by Northern blotting to be
increased in aT3-1 cells in response to stimulation of the Protein Kinase A (PKA) pathway by
forskolin. These results highlight unexpected degeneracy in so-called "consensus" nuclear receptor
binding sites. Furthermore, since Nur77 protein is involved in the stress response of the
hypothalamic-pituitary-adrenal (HPA) axis, the unexpected presence of Nur77 protein in a
gonadotrope cell line has potentially important implications for cross-talk between the HPA and
hypothalamic-pituitary-gonadal (HPG) axes. / AFRIKAANSE OPSOMMING: Daar bestaan 'n direkte verband tussen pituïtêre sensitiwiteit vir gonadotropien-vrystellingshormoon
(GnRH) en GnRH-reseptorvlakke Die regulering van GnRH-reseptorvlakke op transkripsionele en
post-transkripsionele vlak in die pituïtêre klier is belangrik by die beheer van voortplantingsfunksies.
Die proksimale promotor van die GnRH-reseptorgeen in die muis bevat twee cis elemente met die
konsensus volgorde vir 'n Steroidogenic Factor-l (SF-I) bindingsetel. Die distale element is betrokke
by basale en weefsel-spesifieke transkripsionele regulering, maar die funksie van die proksimale
element is nog nie vasgestel nie. SF-1 is 'n lid van die superfamilie van selkernreseptore en is betrokke
by die transkripsionele regulering van gene verantwoordelik vir steroïedogenese en voortplanting. Die
konsensus SF-I bindingsvolgorde kan dien as bindingsetel vir verskeie selkernreseptore. Ten einde 'n
beter insig ten opsigte van die regulering van die GnRH reseptorgeen te verkry, is ondersoek ingestel
na die binding van SF-I-proteïen, afkomstig van die aT3-1 pituïtêre gonadotroopsellyn, aan die twee
moontlike SF-l bindingsetels in die GnRH-reseptor promotor, in vitro. Die Western-klad metode het
getoon dat beide SF-l en Nur77, 'n ander selkernreseptor-transkripsiefaktor, in die aT3-1 sellyn
uitgedruk word. Die uitdrukking is afhanklik van selkultuurtoestande. Elektroforetiese mobiliteitsessais
met spesifieke antiliggame het getoon dat SF-l en Nur77 proteïene in aT3-1 selkernproteïenekstraksies
eksklusief aan beide bindingsetels bind. Nur77 proteïen benodig ander basispare as SF-l
proteïen om aan die bindingsetels te bind. Hierdie resultate dui op onverwagse degenerasie in
sogenaamde "konsensus" selkernreseptor-bindingsvolgordes. Die Northern-kladmetode het ook getoon
dat SF-l mRNA vlakke in aT3-1 selle styg wanneer die proteïenkinase A (PKA) pad gestimuleer word
met forskolin. Aangesien Nur77 proteïen betrokke is by die stres-respons van die hipotalamus-pituïtêre
klier-adrenale (HP A) aksis, hou die onverwagse teenwoordigheid van Nur77 proteïen in 'n
gonadotroop-sellyn potensieel belangrike inplikasies in vir kommunikasie tussen die HPA-aksis en die
hipotalamus-pituïtêre klier-gonadale (HPG) aksis.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52307 |
| Date | 12 1900 |
| Creators | Sadie, Hanel |
| Contributors | Hapgood, J. P., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 104, [15] p. : ill. |
| Rights | Stellenbosch University |
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