Browsing Department of Biochemistry by browse.metadata.advisor "Cloete, Schalk W. P."
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- ItemComparison of two CYP17 isoforms : implications for cortisol production in the South African Merino(Stellenbosch : Stellenbosch University, 2012-03) Hough, Denise; Swart, Pieter; Cloete, Schalk W. P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.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.