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Title: Steroidogenesis and its control in rat corpus luteum
Author: Leaver, H. A.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1978
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The rat corpus luteum produces progesterone which is essential for the maintenance of pregnancy. This steroidogenesis is influenced and maintained by the pituitary trophic hormone luteinising hormone. However, the mechanism of the long-term and the acute activation of steroidogenesis by luteinising hormone is not known. The substrate of steroidogenesis, cholesterol, is hydroxylated by the cholesterol side chain cleavage enzyme in the mitochondria of the corpus luteum cell. This substrate cholesterol is derived from circulating extracellular cholesterol or as a product of intracellular cholesterol ester hydrolase activity. The object of the studies described was to investigate the control of steroidogenesis in relation to its substrate, cholesterol. The superovulated rat ovary was used in these studies as a model system. The long-term induction of steroidogenesis by gonadotrophins in the rat corpus luteum was initially studied. The rate-limiting factors induction processs were investigated. An activator or component of the enolesterol side chain cleavage enzyme appeared to be rate-limiting in the process of long-term activation. Characteristics of cholesterol side chain cleavage were examined in an isolated mitochondrial preparation. In such a preparation substrate cholesterol was found to limit the rate of steroidogenesis end certain experiments suggested that substrate depletion also limited steroidogenesis in vivo. Therefore the interaction of cholesterol with the mitochondrial cholesterol side chair, cleavage enzyme was investigated. Enzymological and spectrophotometric methods were used to study this interaction at the active site of the cholesterol side chain cleavage enzyme. These experiments showed that the access of substrate to the active site limited the rate of cholesterol side chain cleavage in mitochondria from normal and luteinising hormone- or cycloheximide-pretreated rats. The supply of cholesterol to the mitochondria was studied. Cholesterol esterhydrolase enzymes in the rat corpus luteum were characterised. In addition to the previously characterised supernatant enzyme, two particulate cholesterol ester hydrolases were identified. The pH dependence of these particulate enzymes' activity suggested that one was lysosomal and one microsomal in origin. Properties of the process of cholesterol uptake by rat corpus luteum mitochondria were then investigated. These studies suggested that cholesterol uptake by mitochondria was not rate-limiting in steroidogenesis. Finally, the control of steroidogenesis by factors other than substrate availability was studied. One regulatory factor which was investigated was the protein synthesis inhibitor, cycloheximide. The effect of other inhibitors of eukaryotic protein synthesis on steroidogenesis was examined. However, the action of the inhibitors did not resemble the effect of cycloheximide. Attempts were made to identify the subcellular distribution of the putative rapidly turning over protein factor which has been postulated to control steroidogenesis. No stimulatory factor was identified in any subcellular fraction. These experiments suggested that cycloheximide may inhibit steroidogenesis by some mechanism which does not involve the inhibition of protein synthesis. The control of steroidogenesis through alteration in the properties of the mitochondrial membrane was investigated. Mechanical disruption of the mitochondrial membrane and disruption by calcium ions increased the rate of steroidogenesis. This suggested that the control of steroidogenesis could be mediated by some effect on the mitochondrial membrane.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available