Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes:
• the comparison of the enzymatic activities of the two ovine cytochrome P450 17 -
hydroxylase/17,20-lyase (CYP17) isoforms expressed in non-steroidogenic COS-1 cells.
The Km and Vmax values for the metabolism of pregnenolone and progesterone were
determined, while time-dependent metabolism of pregnenolone, 17-hydroxypregenolone,
progesterone and 17-hydroxyprogesterone was also reported. The cloning and sequencing of
ovine cytochrome b5 is reported and was co-expressed with CYP17. The results showed that
the wild type 1 (WT1) isoform of ovine CYP17 produce more cortisol precursors than the
wild type 2 (WT2) isoform;
• the analysis of the frequency distribution of the CYP17 genotypes within a South African
Merino population, which were divergently selected for (H-line) or against (L-line) the
ability of a ewe to rear multiple offspring per birthing opportunity. It was observed that the
CYP17 frequency distribution was the same within the H- and L-line, with 78.3 %
heterozygous WT1/WT2 and 21.7 % homozygous WT1/WT1. No homozygous WT2/WT2
individuals were identified;
• the development of a UPLC-MS/MS method for the separation and quantification of all
thirteen adrenal steroids that are produced in the adrenal gland;
• the relative contribution of the CYP17 genotypes in the total steroidogenic output in adult
adrenocortical cells from the adrenal glands of H- and L-line sheep, with particular emphasis
on cortisol production. The adrenocortical cells from the H-line sheep showed a marked
higher cortisol production than the L-line, while adrenocortical cells from homozygous
WT1/WT1 sheep also produced more cortisol than heterozygous WT1/WT2 sheep;
• the blood cortisol responses upon the stimulation of the HPA axis by insulin induced
hypoglycaemia of the H- and L-line sheep with known CYP17 genotypes. It was observed
that the CYP17 genotype and selection line are important factors affecting the cortisol
responses of sheep, where L-line heterozygous WT1/WT2 sheep showed the lowest cortisol
response and glucose recovery; • the association of the CYP17 genotype with behavioural responses of H- and L-line sheep to
flock isolation stress, as well as the association of the CYP17 genotype with ewe
reproduction and lamb output. While reproduction seemed to be unaffected by the CYP17
genotype, the behavioural stress responses of sheep to flock isolation correlated with the
CYP17 genotype, where the heterozygous WT1/WT2 genotype was associated with a wilder
nature. / AFRIKAANSE OPSOMMING: Hierdie studie ondersoek:
• die vergelyking van die ensiemaktiwiteite vir twee isoforme van skaap sitochroom P450
17 -hidroksilase/17,20-liase (CYP17), wat uitgedruk was in nie-steroïed genererende COS-
1 selle. Die Km and Vmax waardes was bepaal vir die metabolisme van pregnenoloon en
progesteroon, terwyl die tyd-afhanklike metabolisme van pregnenoloon, 17-
hidroksiepregnenoloon, progesteroon en 17-hidroksieprogesteroon ook gerapporteer word.
Die klonering en volgorde bepaling van skaap sitochroom b5 was gedoen en gevolglik was
sitochroom b5 saam met CYP17 uitgedruk in COS-1 selle. Die resultate het gewys dat wilde
tipe 1 (WT1) meer voorlopers van kortisol produseer as wilde tipe 2 (WT2);
• die frekwensie distrubusie van die CYP17 genotipes in ‘n Suid-Afrikaanse Merino
populasie, waar skape in teenoorgestelde rigtings geselekteer was vir (H-lyn) of teen (L-lyn)
die vermoë van ‘n ooi om geboorte te gee aan veelvoudige lammers per lamgeleentheid. Die
frekwensie distrubusie van CYP17 was dieselfde in beide die H- en L-lyn, waar 78.3 % van
die populasie heterosigoties WT1/WT2 en 21.7 % homosigoties WT1/WT1 was. Geen
homosigote WT2/WT2 individue was geïdentifiseer nie;
• die ontwikkeling van ‘n UPLC-MS/MS metode vir die skeiding en kwantifisering van al
dertien steroïede wat natuurlik geproduseer word in die bynier van die skaap;
• die relatiewe bydrae van die CYP17 isoforme tot die totale steroïedale uitsette vanuit die
bynier kortex selle, vanaf die byniere van H- en L-lyn skape, waar klem geplaas word op die
produksie van kortisol. Die bynierselle van die H-lyn skape het aansienlik meer kortisol
produseer as die L-lyn, terwyl die bynierselle van die homosigotiese WT1/WT1 skape ook
meer kortisol produseer het as heterosigotiese WT1/WT2 skape;
• die bloed kortisol in reaksie tot die stimulering van die hipotalamus-hipofise-adrenale aksis,
deur insulien geïnduseerde hipoglisemiese stress, in skape van die H- en L-lyne met bekende
CYP17 genotipes. Dit was gevind dat die kortisol reaksie geaffekteer word deur beide die
CYP17 genotipe en seleksie lyn, waar L-lyn heterosigotiese WT1/WT2 skape die minste
kortisol geproduseer het en die stadigste herstel van glukose vlakke getoon het; • die assosiasie tussen die CYP17 genotipe en die gedrags reaksies op trop-isolasie, sowel as
ooi-reproduksie en lamuitset, van die H- en L-lyn skape. Die reproduksie parameters was
onafhanklik van die CYP17 genotipe, terwyl ‘n sterk assosiasie gevind was tussen die
CYP17 genotipe en gedrags reaksies op trop-isolasie. Die heterosigotiese WT1/WT2 skape
het ‘n wilder natuur getoon gedurende trop-isolasie in vergelyking met homosigotiese
WT1/WT1 skape.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/19951 |
| Date | 03 1900 |
| Creators | Hough, Denise |
| Contributors | Swart, Pieter, Cloete, Schalk W. 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 |
| Rights | Stellenbosch University |
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