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We previously showed that progesterone rapidly down regulates nuclear estrogen receptor (Re) in the estrogen-primed rodent uterus. We have now extended these studies to test the response of the Re system in sheep uterus to progesterone withdrawal. Since the estrogen-Re complex is believed to regulate hormone-dependent gene expression, it was of interest to determine whether withdrawal of progesterone under constant estrogen stimulation would lead to the recovery of nuclear Re levels and estrogen action, i.e. oxytocin receptor (ROT) synthesis. Ovariectomized ewes were primed with estradiol-17 beta and serum steroid levels were maintained by constant infusion of estradiol (0.5 microgram/h) and progesterone (500 micrograms/h) for 5 days. The animals were anesthetized with fluothane/O2, and uterine samples were excised 1 h before and 3, 6 and 12 h after progesterone withdrawal. Estradiol infusion was continued during the experiment in order to maintain estrogen levels at a steady state (14 pg/ml plasma). Re, ROT and progesterone receptor (Rp) were measured in endometrium and myometrium using standard 3H-hormone binding assays. Following progesterone withdrawal, the nuclear Re concentration increased in both uterine compartments, and the nuclear Re level was correlated significantly with the ROT concentration in the membrane fraction of both uterine tissues (endometrium, r = 0.79; myometrium, r = 0.86). Although cytosol Re rose between 6 and 12 h in the endometrium, cytosol Re levels remained unchanged in myometrium. Cytosol Rp appeared to increase in endometrium but not in myometrium. Uterine tissue sampled from a control animal before stopping the progesterone infusion revealed that the observed changes in receptor concentration following progesterone withdrawal were not due to regional differences in receptor levels. These results demonstrate that the recovery of nuclear Re in the ovine endometrium and myometrium following progesterone withdrawal represents a selective effect on Re retention in the nucleus rather than on cytosol Re availability or Re activation which was controlled by constant estrogen infusion. Thus, these results are consistent with the hypothesis that progesterone induces an Re regulatory factor which acts to down regulate nuclear Re, and that the activity of this factor diminishes rapidly after progesterone withdrawal.

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ACTH produced a 75% increase in pregnenolone biosynthesis from endogenous precursors in isolated cells prepared from the rat Snell adrenocortical carcinoma 494. On the addition of 24- and 25-hydroxycholesterol to the tumor cells, the rate of pregnenolone synthesis increased 10-fold but was insensitive to the presence of ACTH. Addition of lipoprotein cholesterol resulted in increased pregnenolone biosynthesis when ACTH was present. High density lipoprotein cholesterol appeared to be internalized and used for steroidogenesis preferentially to low density lipoprotein cholesterol. The cholesterol ester hydrolase activity of the cytosolic fraction of the tumor was found to be extremely low compared to that of the normal adrenal cell. These results, noting also the low cholesterol content of the tumor cells, suggested that the lack of availability of cholesterol was the factor responsible for the poor steroidogenic response of the cells to ACTH. The major steroid product of the tumor cells was determined to be deoxycorticosterone. This correlated with the low levels of steroid 11-beta-hydroxylase activity detected in the adrenal tumor mitochondria compared to the mitochondrial cholesterol desmolase activity. Little of the mitochondrial cytochrome P-450 appeared to function in a steroid 11-beta-hydroxylase complex.

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Adrenocorticotrophin (ACTH) produced an insignificant stimulation of pregnenolone biosynthesis from endogenous precursors in isolated cells prepared from the rat Snell adrenal carcinoma 494. On the addition of 25-hydroxycholesterol, the rate of pregnenolone synthesis increased 10-fold. These results, noting also the very low cholesterol content of the tumor cells, suggested that lack of cholesterol was responsible for the poor steroidogenic response of the cells to ACTH. Endogenous pregnenolone production was sensitive to cytochalasin B as well as cycloheximide. However, pregnenolone synthesis after the addition of 25-hydroxycholesterol was not affected by these inhibitors. Removal of cycloheximide from the cells resulted in the immediate restoration of the initial rate of pregnenolone synthesis from endogenous precursors. This suggested that cycloheximide was interfering with the action of a stable activated intracellular messenger.

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Electron microscope studies were carried out with the adrenocortical carcinoma 494 and normal adrenal cortex tissue. The mitochondria of the tumor cells showed marked differences when compared with mitochondria from fasciculata cells of the normal adrenal cortex. These differences were primarily related to mitochondrial number and crista structure. Corticosterone production in isolated tumor cells was extremely low and neither ACTH nor dibutyryl cyclic AMP had any stimulatory effect. Normal adrenal cells showed at least a tenfold increase under identical conditions. In the presence of corticosteroid precursors the amount of corticosterone produced by the tumor cells was much less than that produced by normal cells. The results indicate a reduced capacity for 11beta-hydroxylation in the tumor mitochondria and a possible reduced capacity for biosynthetic steps before the 11beta-hydroxylation reaction. Glycolysis in isolated tumor cells was also lower than in normal cells. Isolated tumor mitochondria oxidized succinate normally with a good degree of coupling with phosphorylation. However, unlike normal adrenal mitochondria, the tumor mitochondria showed little or no oxygen uptake with other Krebs cycle substrates. These data suggest that the tumor mitochondria may be lacking in the flavoprotein dehydrogenases responsible for the oxidation of NADH and NADPH, although other components of the respiratory chain may be intact.

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