Abstract
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 either 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 factor(s) in this
induction processs were investigated. An activator or component of the cholesterol
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 and certain experiments suggested
that substrate depletion also limited steroidogenesis in vivo. Therefore the
interaction of cholesterol with the mitochondrial cholesterol side chain 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 ester
hydrolase 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.
