Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The GnRH receptor is a G-protein-coupled receptor in pituitary gonadotrope
cells. Binding of its ligand, GnRH, results in synthesis and release of
gonadotropin hormones luteinizing hormone (LH) and follicle stimulating
hormone (FSH). Steroidogenic factor 1 (SF-1), a transcription factor, binds
to specific sites in the promoter region of gonadotropin genes, and thus
regulates transcription of these genes. The promoter region of the GnRHreceptor
gene contains two SF-1-like binding sites, one at -14 to -8 (site 1)
and another at -247 to -239 (site 2), relative to the methionine start codon.
The role played by these two SF-1-like sites in basal transcription of the
mouse GnRH receptor (mGnRH-R) gene in a pituitary precursor
gonadotrope cell line, aT3 cells, was the first area of investigation during this
study. Luciferase reporter constructs containing 580 bp of mGnRH-R gene
promoter were prepared, where SF-1-like sites were either wildtype or
mutated. Four such constructs were made, i.e. wildtype (LG), site 1 mutant
(LGM1), site 2 mutant (LGM2) and mutated site 1 plus site 2 (LGM1/2).
These constructs were transfected into aT3 cells to determine the effect of
mutations of sites 1 and/or 2 on the basal expression of the mGnRH-R gene.
Mutation of either site 1 or site 2 had no effect on basal expression of the
mGnRH-R gene. It was found that only upon simultaneous mutation of both
sites 1 and 2, a 50% reduction in basal transcription took place. The
implications of this is that SF-1 protein seems to only require one intact
DNA-binding site, to mediate basal transcription of the mGnRH-R gene,
suggesting that these two sites lie in close proximity during basal
transcription. The effect of the protein kinase A (PKA) pathway on the
endogenous mGnRH-R gene was also investigated by incubating non- ,
transfected aT3 cells with the PKA activators, forskolin and 8-Br-cAMP.
Similar incubations were also performed on the wild type and mutated site 1
constructs transfected into pituitary gonadotrope aT3 cells. It was found that
forskolin and 8-Br-cAMP were able to increase endogenous mGnRH-R mRNA levels in a concentration-dependent fashion, showing that
endogenous GnRH receptor gene expression is stimulated via a protein
kinase A pathway. Similar results were obtained with the wildtype promoter
construct, showing that the protein kinase A pathway stimulates transcription
of the promoter. This effect was only seen with wild type and not with the
mutated site 1. These results are consistent with a role for a SF-1-like
transcription factor in mediating the protein kinase A effect via binding to the
site 1 at position -14 in the GnRH receptor gene. A separate investigation
was performed to determine whether 25-hydroxycholesterol (25-0HC) is a
ligand for SF-1, by incubating aT3 cells transfected with the various
constructs with 25-0HC. Results show a dose-dependant response, with an
increase in gene expression at 1 μM and a decrease at higher
concentrations, for both mutant and wild type constructs. This suggests that,
if SF-1 is indeed the protein binding to sites 1 and 2, then 25-0HC is not a
ligand for SF-1 protein in aT3 cells and that the effect of 25-0HC on the
mGnRH-R gene is not mediated via site 1. The results indicate that these
decreases of expression at the higher concentrations may be due to
cytotoxic effects. Towards the end of the study the laboratory obtained a
luminoskan instrument with automatic dispensing features. Optimisation
studies on the luciferase and β-Gal assays were performed on the
luminoskan in a bid to decrease experimental error. It was found that
automation of these assays resulted in a decrease in experimental error,
showing that future researchers could benefit substantially from these
optimisation studies. / AFRIKAANSE OPSOMMING: Die GnRH reseptor is 'n G proteïen-gekoppelde reseptor in pituitêre
gonadotroopselle. Binding van die ligand, GnRH, lei tot die sintese en
vrystelling van die gonadotropien hormone, luteïniserende hormoon (LH) en
follikel stimulerende hormoon (FSH). Steroidogeniese faktor-t (SF-1) is 'n
transkripsie faktor wat aan spesifieke areas in die promotergebied van die
gonadotropien hormone bind, en dus transkripsie van hierdie gene reguleer.
Die promotergebied van die GnRH reseptor geen bevat twee SF-1 bindings
areas, een by -14 to -8 (area 1) asook by -247 to -239 (area 2), relatief to die
metionien beginkodon. Die rol wat hierdie twee SF-1 areas speel in basale
transkripsie van die muis GnRH reseptor (mGnRH-R) geen in 'n pituïtêre
voorloper gonadotroop sellyn, aT3 selle, was die eerste gebied van
ondersoek gedurende hierdie studie. Plasmiede bestaande uit die 580
basispaar mGnRH-R promoter verbind aan 'n lusiferase geen is vervaardig,
waar SF-1-soortige areas enersyds onveranderd gelaat is, of gemuteer is.
Vier sulke plasmiede is vervaardig, nl. onveranderd (LG), area 1 mutant
(LGM1), area 2 mutant (LGM2) en gemuteerde area 1 plus area 2 (LGM1/2).
Hierdie plasmiede is gebruik om aT3 selle te transfekteer om die effek van
mutasies van areas 1 en/of 2 op die basale ekspressie van die mGnRH-R
geen te ondersoek. Daar is gevind dat mutasies van areas 1 of 2 geen effek
op basale ekspressie op die bogenoemde geen gehad het nie. Slegs tydens
gelyktydige mutasie van areas 1 en 2 het 'n 50% vermindering in basale
transkripsie plaasgevind. Die implikasies hiervan is dat die SF-1 proteïen
blykbaar slegs een volledige DNA-bindingsarea benodig om basale
transkripsie van die mGnRH-R geen te reguleer. Dit wil dus voorkom of
hierdie twee areas baie na aan mekaar geposisioneer is tydens basale
transkripsie. Die effek van die proteïen kinase A (PKA) roete op die natuurlike
mGnRH-R geen is ook ondersoek tydens inkubasie van nie-getransfekteerde
aT3 selle met die PKA akiveerders, forskolin en 8-Br-cAMP. Soortgelyke
inkubasie is ook gedoen op die onveranderde en gemuteerde area 1
plasmiede wat in aT3 selle getransfekteer is. Daar is gevind dat forskolin en 8-Br-cAMP daarin geslaag het om die natuurlike mGnRH-R geen mRNA
vlakke op 'n konsentrasie-afhanklike wyse te vermeerder. Hierdie resultaat
dui daarop aan dat die natuurlike mGnRH-R geen se ekspressie gestimuleer
kan word via 'n proteïen kinase A roete. Soortgelyke resultate is verkry met
die onveranderde promoter plasmied en dit wys ook daarop dat proteïen
kinase A transkripsie deur die promoter kan stimuleer. Hierdie effek was
slegs aanwesig met die onveranderde en nie met die gemuteerde area 1
plasmied nie. Die resultate stem ooreen met 'n rol vir SF-1 transkripsie faktor
in die regulering van proteren kinase A effek deur middel van binding aan die
area 1 by posisie -14 in die GnRH-R geen. 'n Afsonderlike ondersoek is
gedoen om vas te stel of 25-hidroksiecholesterol (25-0HC) 'n ligand vir SF-1
is deur getransfekteerde aT3 selle met 25-0HC te inkubeer. Resultate toon 'n
dosis-afhanklike respons met 'n verhoging in geen ekspressie by 1 μM en 'n
verlaging met hoër konsentrasies vir beide onveranderde en gemuteerde
plasmiede. Dit impliseer dat, indien SF-1 wel die faktor is wat aan areas 1 en
2 bind, 25-0HC nie die ligand vir SF-1 proteren in aT3 selle is nie en dat die
effek van 25-0HC op die mGnRH-R geen nie gereguleer word via area 1 nie.
Die verlaging in ekspressie gevind by die hoër konsentrasies is dalk die
gevolg van sitotoksiese effekte. Teen die einde van die studie het die
laboratorium luminoskan toerusting met outomatiese pipettering verkry.
Optimiseringstudies van die lusifirase en β-Galtoetse is met die luminoskan
gedoen in 'n poging om eksperimentele foute te minimaliseer. Daar is gevind
dat outomatisering van hierdie toetse wel gelei het tot 'n verlaging in
eksperimentele foute. Toekomstige navorsers kan dus grootliks voordeel trek
uit hierdie optimiseringstudies.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52572 |
| Date | 03 1900 |
| Creators | Styger, Gustav |
| 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 | 106, [22] p. : ill. |
| Rights | Stellenbosch University |
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