Comparison of two CYP17 isoforms : implications for cortisol production in the South African Merino

Hough, Denise (Stellenbosch : Stellenbosch University, 2012-03)

Thesis (PhD)--Stellenbosch University, 2012.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/19951
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