Neurosteroids: oligodendrocyte mitochondria convert cholesterol to pregnenolone.

Oligodendrocyte mitochondria from 21-day-old Sprague-Dawley male rats were incubated with 100 nM [3H]cholesterol. It yielded [3H]pregnenolone at a rate of 2.5 +/- 0.7 and 5-[3H]pregnene-3 beta, 20 alpha-diol at a rate of 2.5 +/- 1.1 pmol per mg of protein per hr. Cultures of glial cells from 19- to 21-day-old fetuses (a mixed population of astrocytes and oligodendrocytes) were incubated for 24 hr with [3H]mevalonolactone. [3H]Cholesterol, [3H]pregnenolone, and 5-[3H]pregnene-3 beta, 20 alpha-diol were characterized in cellular extracts. The formation of the 3H-labeled steroids was increased by dibutyryl cAMP (0.2 mM) added to the culture medium. The active cholesterol side-chain cleavage mechanism, recently suggested immunohistochemically and already observed in cultures of C6 glioma cells, reinforces the concept of "neurosteroids" applied to delta 5-3 beta-hydroxysteroids previously isolated from brain.     

The Stoichiometry of the Conversion of Cholesterol and Hydroxycholesterols to Pregnenolone (3β-Hydroxypregn-5-en-20-one) Catalysed by Adrenal Cytochrome P-450

The stoichiometry of the side-chain cleavage of cholesterol (cholest-5-en-3beta-ol), 20alpha-hydroxycholesterol (cholest-5-ene-3beta,20alpha-diol), and 20alpha,22-dihydroxycholesterol (cholest-5-ene-3beta,20alpha,22-triol) has been examined with respect to TPNH, oxygen and H(+). Side-chain cleavage of cholesterol can be described by the following equation:Cholesterol + 3TPNH + 3H(+) + 3O(2) --> Pregnenolone (3beta-hydroxypregn-5-ene-20-one) + isocapraldehyde + 3 TPN(+) 4H(2)OStoichiometry of 20 alpha-hydroxycholesterol is 2TPNH and 2O(2) per mole of cleavage and with 20alpha, 22-dihydroxycholesterol the values are 1:1:1. In addition 1 mole of H(+) is consumed per mole of TPNH oxidized in each of these reactions. These observations are in keeping with a mechanism previously proposed in the literature, namely:Cholesterol --> 20alpha-OH-Cholesterol --> 20alpha, 22-di-OH-Cholesterol --> pregnenoloneDiscordant observations are reviewed and it is concluded that insufficient data are available to sustain objections to this pathway.     

Induction of progesterone receptor by androgens in the mouse uterus

The comparative abilities of 3 naturally occurring androgens to compete for [3H]estradiol-17 beta ([3H]E2)-binding sites in uterine cytosol and to induce uterotrophic responses in immature female mice have been investigated. 5 alpha-Androstane-3 beta,17 beta-diol (3 beta-diol), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5-androstene-3 beta,17 beta-diol (delta 5-diol) are all effective at diminishing cytoplasmic [3H]E2 binding. Their relative effectiveness are in the order of 3 beta-diol greater than delta 5-diol = 3 alpha-diol. When these steroids were injected intramuscularly (two 100-200 micrograms injections) into immature female mice (21 days old), they induced similar elevations of uterine dry weight, water content, cytosolic estrogen (ER) and progesterone (PR) receptor contents, and nuclear ER content as did estradiol-17 beta (E2). 3 beta-Diol was the most effective steroid at inducing cytosolic and nuclear ER, and PR production in mouse uterus. Followed in effectiveness was delta 5-diol and 3 alpha-diol. Unexpectedly, 3 beta-diol was found to be more potent than E2 in stimulating uterine ER and PR increases. Nonetheless, the androgens are poorer stimulators of uterine dry weight increase and water retention than E2 is. These results indicate that the androgens may interact with the ER system in the uterus to bring about the uterotrophic responses and 3 beta-diol is a more estrogenic steroid than delta 5-diol and 3 alpha-diol in the mouse uterus.     

Probucol reduces plasma and aortic wall oxysterol levels in cholesterol fed rabbits independently of its plasma cholesterol lowering effect.

To understand further the antiatherogenic mechanism of probucol, the antioxidant effect of this agent was studied on specific cholesterol oxidation products in plasma and aortic wall in equally hypercholesterolemic New Zealand white rabbits. In order to maintain equal plasma total cholesterol levels, five control rabbits (C group) received a 1% followed by a 0.5% cholesterol enriched diet, while the probucol treated rabbits (C+P group) received a graded increase in the cholesterol supplemented diet from 1% to 3%; probucol supplementation was constant at 1%. After 9 weeks of feeding, the plasma oxysterols, cholest-5-ene-3 beta,7 alpha-diol, cholest-5-ene-3 beta,7 beta-diol, 5,6 beta-epoxy-5 alpha-cholestan-3 beta-ol, 5,6 alpha-epoxy-5 alpha-cholestan-3 alpha-ol and 5 alpha-cholestane-3 beta,5,6 beta-triol significantly increased over baseline levels in both experimental groups. However, the increase in all these products in plasma was 20-60% less in the C+P group than the C group (P < 0.05). Furthermore, the C+P aortic wall cholesterol oxide concentrations were 50-90% less than the C group (P < 0.05). The oxysterol pattern of the aortic wall was similar to plasma. Additionally, the aortic wall cholesterol content in the C+P group was 50% less than the C group (P < 0.05). The plasma cholesterol levels were not significantly different at any time point during the study and the cholesterol oxide content in the diets was the same. These results are consistent with the contention that the antioxidant properties of probucol serve as the basis for its antiatherogenic effects in vivo.     

Effects of transforming growth factor-beta on the production of immunoreactive insulin-like growth factor I and progesterone and on [3H]thymidine incorporation in porcine granulosa cell cultures

Transforming growth factor-beta (TGF beta) has been reported to enhance many FSH-stimulated functions in rat granulosa cell cultures. We therefore, investigated the actions of TGF beta on cultured porcine granulosa cells. We evaluated the production of immunoreactive insulin-like growth factor I (iIGF-I) and progesterone in short term (3-day) and in longer term (7-day) cultures using porcine TGF beta 1 (pTGF beta 1). TGF beta had a biphasic effect on epidermal growth factor (EGF)-stimulated iIGF-I production in short term cultures. A modest stimulatory effect was apparent at 10 pg/ml; however, this end point was completely inhibited by 1-10 ng/ml. TGF beta also had a slight stimulatory effect on basal iIGF-I production at 1 pg/ml, but not at higher levels. In longer term cultures TGF beta did not have a significant effect on either basal or FSH-stimulated iIGF-I production. In both short and longer term cultures TGF beta markedly inhibited basal and FSH-stimulated progesterone production. We also evaluated the effects of TGF beta on the incorporation of [3H]thymidine into DNA and found that basal and growth factor-stimulated [3H]thymidine incorporation were inhibited. No stimulatory effects of TGF beta on progesterone production or [3H]thymidine incorporation could be detected over the dose range tested (1 pg/ml to 10 ng/ml). The effects of human TGF beta 1 and pTGF beta 2 were compared with those of pTGF beta 1 on basal and EGF-stimulated [3H]thymidine incorporation. Effects of the peptides were qualitatively similar, but pTGF beta 2 was somewhat less inhibitory to EGF-stimulated [3H]thymidine incorporation than pTGF beta 1. The present studies show that in contrast to the well documented stimulatory actions of TGF beta in cultured rat granulosa cells, this growth factor is a predominantly negative regulator of porcine granulosa cells. With the exception of a modest stimulation of iIGF-I production at very low doses, the effects of TGF beta were to potently inhibit both growth and differentiated function. The inhibitory nature of TGF beta should not be overlooked when considering the possible role of this peptide in ovarian development and differentiation.     

Formation of steroids by the pregnant mare. VI. Metabolism of [14C]farnesyl pyrophosphate and [3H]dehydroepiandrosterone injected into the fetus.

A mixture of [4,8,12-14C]farnesyl pyrophosphate and [3H]dehydroepiandrosterone was injected into a horse fetus im during laparotomy, after which maternal urine was collected for 6 days. Steroid conjugates in the urine were extracted with Amberlite XAD-2 resin, hydrolyzed, and separated into phenolic and neutral fractions. Estrone, 17 alpha-estradiol, equilin [3-hydroxy-1,3,5(10),7-estratetraen-17-one], and 17 alpha-dihydroequilin [1,3,4(10),7-estratetraene-3,17 alpha-diol] were isolated from the phenolic fraction and their radiochemical purities were established. Only estrone and 17 alpha-estradiol contained both 3H and 14C, while the B ring unsaturated estrogens, equilin and 17 alpha-dihydroequilin, contained only 14C. From the neutral fraction, 14C-labeled 3 beta-hydroxy-5 alpha-pregnan-20-one, 5 alpha-pregnane-3 beta-20 beta-diol, and 5 alpha-pregnane-3 beta, 20 alpha-diol were isolated. These results together with our previous findings demonstrate that the route of biosynthesis of both the ring B saturated and unsaturated estrogens is the same up to the stage of farnesyl pyrophosphate. Thus, the bifurcation in the classical pathway of steroid biosynthesis is occurring at a point after the formation of farnesyl pyrophosphate and before the formation of squalene and cholesterol.     

Synthesis of 20 alpha-hydroxylated steroids by ovaries of the dab (Limanda limanda)

Dab (Limanda limanda) ovarian fragments were incubated in vitro with either [4,7-3H]pregnenolone or 17 alpha-hydroxy[1,2,6,7-3H]progesterone to investigate the pattern of steroidogenesis. A major enzyme found in the dab ovary was 20 alpha-hydroxysteroid dehydrogenase. Among the steroids that were tentatively identified in ovarian incubates were 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P). 17 alpha,20 alpha-dihydroxy-5 beta-pregnan-3-one, 3 beta, 17 alpha,20 alpha-trihydroxy-5 beta-pregnane (3 beta,17,20 alpha-P-5 beta), and 3 alpha,17 alpha,20 alpha-trihydroxy-5 beta-pregnane. The presence of these steroids in plasma of mature female and male dabs was studied by radioimmunoassay. The antiserum was raised against 17,20 alpha-P. The 17,20 alpha-[3H]P label was produced by incubating place milt with 17 alpha-hydroxy [3H]progesterone. The radioimmunoassay was shown to have a high cross-reaction with the 5 beta-reduced analogues of 17,20 alpha-P and was therefore used, in conjunction with thin-layer chromatography, to measure the steroids. High concentrations of both 17,20 alpha-P and 3 beta,17,20 alpha-P-5 beta found in female and male dab plasma. The possible role of these steroids is discussed.     

Biosynthesis and secretion of follicle-stimulating hormone beta-subunit from ovine pituitary cultures: effect of 17 beta-estradiol treatment

An assay was developed to detect tritium-labeled ovine FSH beta-subunit [( 3H]oFSH beta) secreted from primary ovine pituitary cultures. This procedure used affinity-enriched antibodies raised against reduced and carbamylmethylated oFSH beta (RCM-oFSH beta) in a two-cycle immunoextraction procedure. A discrete species with an apparent mol wt of 21,000 was detected in sodium dodecyl sulfate electrophoretic patterns of immunoextracts from culture medium. This species was identified as RCM-[3H]oFSH beta by its comigration with highly purified RCM-oFSH beta, its reduction in culture media after cultures were treated with 17 beta-estradiol, which normally decreases radioimmunoassayable oFSH; and its displacement from the extracting antibodies by excess unlabeled RCM-oFSH beta. The assay was used in a pulse-chase study to determine that [3H]oFSH beta is secreted within 1-2 h of its synthesis. Prior treatment of cultures with 17 beta-estradiol did not change this timing of secretion.     

Cholesterol feeding increases plasma and aortic tissue cholesterol oxide levels in parallel: further evidence for the role of cholesterol oxidation in atherosclerosis.

To determine the relationship between plasma and arterial wall oxysterols, plasma and aortic tissue from 7 New Zealand White rabbits fed a high cholesterol (1%) diet for 6 weeks was compared to plasma and aortic tissue from 7 normocholesterolemic rabbits fed standard rabbit chow. Cholesterol and cholesterol oxide fractions were isolated and analyzed by gas chromatography. Normocholesterolemic plasma and aortic tissue contained low levels of cholest-5-ene-3 beta, 7 alpha-diol, cholesta-3,5-dien-7-one, 5,6 alpha-epoxy-5 alpha-cholestan-3 alpha-ol, cholest-5-ene-3 beta, 7 beta-diol, and 5 alpha-cholestane-3 beta, 5,6 beta-triol while hypercholesterolemic plasma and atherosclerotic aorta contained significantly higher levels (P less than 0.05) of these products. Furthermore, 5,6 beta-epoxy-5 alpha-cholestan-3 beta-ol not found in normocholesterolemic plasma or aortic tissue was present in substantial amounts in both hypercholesterolemic plasma and atherosclerotic aortic tissue. Cholest-5-ene-3 beta,25-diol and 3 beta-hydroxycholest-5-ene-7- one not present in normocholesterolemic aorta were present in the atherosclerotic aorta. The oxysterol chromatographic patterns of normocholesterolemic plasma and normocholesterolemic aortic tissue were similar to each other as were the oxysterol chromatographic patterns of hypercholesterolemic plasma and atherosclerotic aortic tissue. The chromatographic patterns between the normocholesterolemic and hypercholesterolemic samples differed however. Possible absorption of the low levels of cholesterol oxides present in the cholesterol feed could account for the elevation of only some of the oxysterols. We conclude that cholesterol oxides exist at some basal level in normocholesterolemia and that these levels are increased by cholesterol-feeding which results in hypercholesterolemia. Our findings demonstrate that there is a strong relationship between plasma and aortic arterial wall levels of cholesterol oxides and suggest that in addition to exogenous sources, formation of cholesterol oxides proceeds via free radical oxidation acting upon elevated cholesterol levels resulting in the accumulation of these potentially cytotoxic and atherogenic products.     

Transforming growth factor-alpha and -beta differentially regulate growth and steroidogenesis of bovine thecal cells during antral follicle development

The actions and interactions of transforming growth factor-alpha (TGF alpha) and TGF beta on growth and differentiation of bovine thecal cells were investigated. Bovine thecal interna cells were isolated from small (less than 5 mm), medium (5-10 mm), and large (greater than 10 mm) antral follicles and cultured in the presence or absence of TGF alpha and/or TGF beta. Both [3H]thymidine incorporation and changes in cell number (i.e. DNA levels) were evaluated to determine effects on thecal cell growth. Short term treatment of cells with TGF alpha (18-24 h) stimulated thymidine incorporation, and longer term treatments (4 days) increased cell number. TGF beta suppressed thymidine incorporation below that observed in untreated cultures, but had no effect on cell number. When combined with TGF alpha, TGF beta suppressed the ability of TGF alpha to stimulate thymidine incorporation and increase cell number. The response to these growth factors was similar for cells isolated from the different stages of antral follicle development. The effects of TGF alpha and TGF beta on thecal cell differentiation were evaluated by quantitating changes in androstenedione and progesterone accumulation in cultures treated with TGFs in the absence (basal) or presence of hCG, estradiol (E2), or a combination of hCG and E2. E2 and hCG were included in this study because previous research has demonstrated that these hormones alter thecal cell steroidogenesis. Treatment with TGF alpha resulted in a suppression of basal and hormonally stimulated accumulation of androstenedione during days 0-3 of culture, whereas TGF beta did not significantly alter androstenedione accumulation. TGF alpha also suppressed progesterone accumulation during days 0-3 of culture in the absence or presence of hormones. In contrast, TGF beta stimulated accumulation of progesterone in cultures that did not contain E2, which suppressed progesterone during this period. Therefore, during days 0-3 of culture, TGF alpha appears to have suppressive effects on androstenedione and progesterone production, whereas TGF beta can stimulate progesterone production in the absence of E2. During days 3-6 of culture, thecal cell differentiation changes, and the capacity to produce androstenedione dramatically declines, while the capacity to produce progesterone increases. During this period, either TGF alpha or TGF beta slightly increased basal progesterone accumulation and partially suppressed the ability of hCG to stimulate progesterone. The effects of TGFs on thecal cell steroidogenesis were similar with cells isolated from the different stages of antral follicle development. Results from these studies provide evidence that THF alpha and TGF beta can modulate thecal cell growth and differentiation (i.e. steroidogenesis).(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of androsterone and 5 alpha-androstane-3 alpha,17 beta-diol in human lung tissue and in pulmonary endothelial cells in culture

The metabolism of [3H]androsterone and [3H] 5 alpha-androstane-3 alpha,17 beta-diol ( [3H] 3 alpha-diol) was studied in slices of human lung tissue and cultures of human pulmonary artery endothelial cells. Lung tissue metabolized [3H]androsterone (0.25 microM) to 5 alpha-androstane-3,17-dione (30.3 pmol 100 mg-1 tissue h-1), isoandrosterone (0.7 pmol 100 mg-1 tissue h-1), 5 alpha-dihydrotestosterone (5 alpha-DHT; 0.1 pmol 100 mg-1 tissue h-1), 3 alpha-diol (0.1 pmol 100 mg-1 tissue h-1), and two polar metabolites. Pulmonary arterial endothelial cells produced the same metabolites of [3H]androsterone (0.083 microM), with the exception of the polar compounds [5 alpha-androstane-3,17-dione (1.3 pmol mg-1 protein h-1), isoandrosterone (0.1 pmol mg-1 protein h-1), 5 alpha-DHT (0.2 pmol mg-1 protein h-1), and 3 alpha-diol (0.2 pmol mg-1 protein h-1)]. Thus, the principal metabolite of [3H]androsterone in both lung tissue and endothelial cells was 5 alpha-androstane-3,17-dione. Human lung tissue metabolized [3H]3 alpha-diol (0.28 microM) to 5 alpha-DHT (8.8 pmol 100 mg-1 tissue h-1), androsterone (2.2 pmol 100 mg-1 tissue h-1), 5 alpha-androstane-3,17-dione (0.8 pmol 100 mg-1 tissue h-1), isoandrosterone (0.1 pmol 100 mg-1 tissue h-1), and four polar metabolites (0.2 pmol 100 mg-1 tissue h-1). 5 alpha-DHT was the principal metabolite of [3H]3 alpha-diol within the first hour of incubation, but the concentration of this androgen declined thereafter to 3.6 pmol 100 mg-1 tissue after 4 h of incubation. This decline was correlated with increased 5 alpha-androstane-3,17-dione synthesis (6.7 pmol 100 mg-1 tissue 4 h-1). Androsterone formation from [3H]3 alpha-diol, however, was linear with time of incubation for 4 h (8.9 pmol 100 mg-1 tissue 4 h-1). The formation of these products demonstrates that the principal 5 alpha-reduced-C19-steroid-metabolizing enzymes in human lung are 3 alpha-hydroxysteroid oxidoreductase.     

In situ subcellular distribution and metabolism of progesterone, estradiol and androstenedione in the vascularly separated and isolated hypothalamus of the female rhesus monkey

A neurosurgical procedure has been developed for the vascular isolation of the hypothalamus-thalamus region of the rhesus monkey brain. The circulation to the left and right halves of the hypothalamus was also isolated and each half of the hypothalamus was perfused simultaneously, but separately, with a dextran-blood solution which contained radioactive gonadal steroids. The hypothalamus in situ efficiently converted [3H]androstenedione to [3H]estrone and this aromatization was inhibited by the presence of androsta-1,4,6-triene-3,17-dione (ATD) in the perfusate. [3H]Progesterone was metabolized predominantly to 5 alpha-pregnane-3,20-dione (5 alpha-DHP) and 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-OHP). Subcellular fractionation of the hypothalamus after the in situ perfusion with [3H]-progestin or [3H]estradiol to the hypothalamus of estrogen-treated ovariectomized monkeys or oil-treated ovariectomized monkeys, respectively, indicated that the retention of [3H]estradiol in the nucleus was a saturable, limited-capacity phenomenon. No saturable subcellular distribution of [3H]progesterone or [3H]R 5020 was observed. This latter observation might be attributable to the presence of a progesterone receptor in too small a concentration to be detected by the methods used.     

Follicle-stimulating hormone regulates low density lipoprotein metabolism by swine granulosa cells

FSH amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine granulosa cells in a synergistic manner. The mechanisms subserving this synergism included the following. 1) FSH increased by 2.1-fold the number of specific high affinity low density lipoprotein (LDL) receptors on granulosa cells, with no change in apparent binding affinity. 2) FSH augmented by 1.8- and 3.6-fold the maximal rates of [125I]iodo-LDL internalization and degradation, respectively, without altering half-maximally effective concentrations of LDL supporting these processes. 3) FSH increased significantly the mass (micrograms) of free and esterified cholesterol (measured by fluorometry) contained in granulosa cells. 4) FSH stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester from exogenous [3H]cholesteryl linoleate-labeled LDL and amplified [3H]progesterone secretion by granulosa cells exposed to this source of lipoprotein-borne sterol. 5) The stimulatory effects of FSH on LDL degradation and cholesterol side-chain cleavage activity were mimicked by other activators of granulosa cell cAMP production, e.g. forskolin and cholera toxin. We conclude that FSH and LDL synergistically enhance progesterone biosynthesis by swine granulosa cells via cellular mechanisms that result in significantly augmented binding, internalization, and degradation of LDL in concert with increased effectual utilization of sterol substrate in the cholesterol side-chain cleavage reaction. These observations are consistent with a key trophic role for FSH in regulating the sterol-metabolizing ability and steroidogenic differentiation of granulosa cells and indicate that such actions of FSH can be accounted for by activation of the cAMP-effector pathway.     

Steroid profile in serum: increased levels of sulphated pregnenolone and pregn-5-ene-3 beta,20 alpha-diol in patients with adrenocortical carcinoma

A serum steroid profile was determined in 11 patients with adrenocortical carcinoma, 5 with adrenocortical adenoma and 10 healthy controls. Seven of the patients with carcinoma had different forms of endocrine symptoms, and of those with adenoma 3 had Cushing's syndrome and 2 primary hyperaldosteronism. Sulphated steroids dominated in serum from both patients and controls, whereas the levels of free and glucuronated steroids were low. All patients with adrenocortical carcinoma had increased levels of sulphated pregn-5-ene-3 beta-ol-20-one (pregnenolone) and pregn-5-ene-3 beta,20 alpha-diol compared with healthy controls and patients with adrenocortical adenoma. Serum levels of 11-deoxycortisol and/or its glucuronated metabolite tetrahydro-11-deoxycortisol were clearly elevated in 8 of the patients with carcinoma. The results are in agreement with those previously found for conjugated urinary steroids in patients with adrenocortical carcinoma. Thus, an impaired function or deficiency of 3 beta-hydroxysteroid dehydrogenase/delta isomerase and in some cases also of 11 beta-hydroxylase could explain the findings. The serum levels of sulphated pregnenolone and/or pregn-5-ene-3 beta,20 alpha-diol, possibly together with free 11-deoxycortisol seem to be useful for preoperative discrimination between malignant and benign adrenocortical tumours.     

Stimulation of progesterone synthesis in luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-adenosine 3',5'-monophosphate: the effect of low density lipoprotein

Primary cultures of luteinized human granulosa cells reduced progestin secretion when taken from serum- and gonadotropin-containing medium into serum- and hormone-free medium. When added individually hCG, 8-bromo-cAMP and low density lipoprotein (LDL) stimulated progestin secretion by the cells after they spent 48 h in serum- and hormone-free medium. However, combinations of hCG or 8-bromo-cAMP and LDL were most effective in increasing steroidogenesis. The effects of hCG in enhancing steroidogenesis in the presence of LDL were first detectable after 3 h, but were most marked after 20 h of culture. hCG and 8-bromo-cAMP increased the conversion of [3H]cholesteryl linoleate, which had been incorporated into the core of LDL, into [3H]progesterone. hCG also stimulated cellular accumulation of LDL cholesterol, as assessed by incorporation of [1-14C]oleic acid into sterol esters or by measurement of total cellular cholesterol in the presence of amino-glutethimide to block steroidogenesis. In contrast to progesterone secretion, estradiol secretion was not affected by the addition of LDL in the absence or presence of 8-bromo-cAMP. We conclude that LDL cholesterol is required for maximal rates of progestin synthesis by human luteinized granulosa cells. When granulosa cells are stimulated by hCG, uptake of LDL cholesterol is promoted, and there is increased utilization of LDL cholesterol for steroid synthesis.     

Specific effect of oestrone sulphate on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium

Patterns of induced protein synthesis and secretion in guinea-pig endometrial epithelial cell cultures in response to oestrone sulphate alone and oestrone sulphate plus progesterone were investigated. Epithelial cells were cultured for 3 days in growth medium, then washed three times in a steroid-free medium. For each experiment, anticytokeratin immunostaining was used to discriminate the epithelial cells from the stromal cells. Only experiments in which the control dishes displayed more than 80% of anticytokeratin-immunostained cells were further processed. After this period oestradiol-17 beta (20 nmol/l; control), oestradiol-17 beta (20 nmol/l) plus progesterone (0.5 mumol/l), oestrone sulphate (1 mumol/l) or oestrone sulphate (1 mumol/l) plus progesterone (0.5 mumol/l) were added to the medium for 48 h. An immunocytochemical progesterone receptor assay showed that oestradiol-17 beta increased the progesterone receptor content of cells, and progesterone added to cultured cells in the presence of oestradiol-17 beta induced a significant increase in oestrogen sulphotransferase activity assessing the hormone responsiveness of the cultured cells. In these culture conditions and after 16 h of incubation, oestradiol-17 beta induced a 1.7-fold increase in [3H]thymidine incorporation into DNA, and [35S]methionine incorporation into cellular proteins was linearly increased up to 8 h. Biochemical changes induced by the different hormone treatments were studied by labelling the proteins with a 6-h pulse of [35S]methionine. The proteins present in the medium and in cells were analysed by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pregnenolone binds to microtubule-associated protein 2 and stimulates microtubule assembly

Fetal or adult rat-brain cytosol and fetal rat-brain microtubules contain a high-affinity, low-capacity pregnenolone-binding protein. The equilibrium dissociation constant is in the 30-50 nM range. The best competitors (in decreasing order) are pregnenolone sulfate, progesterone, Delta5-pregnene-3beta,20alpha-diol, and 3beta-hydroxy-5alpha-pregnan-20-one. It was hypothesized that the pregnenolone-binding protein pertained to microtubule-associated proteins (MAPs). Indeed, partial purification of fetal brain cytosol by fast pressure liquid chromatography with sequential ion-exchange and gel-filtration columns yielded two fractions, one of very high molecular mass, >200 kDa, and the other of 40-60 kDa, enriched in [(3)H]pregnenolone-binding activity and in proteins immunolabeled with monoclonal anti-tubulin and anti-MAP2 antibodies. Because many proteins are associated with microtubules, binding assays were repeated with purified calf-brain tubulin, MAP2, and Tau protein. Only the MAP2 fraction showed saturable [(3)H]pregnenolone binding with an affinity very close to that of rat-brain microtubules, but with a much larger concentration of binding sites (16 pmol/mg MAP2), which was increased more than 8-fold after copolymerization of MAP2 with tubulin. Finally, steroid effects on microtubule-assembly kinetics were assayed. Pregnenolone induced a large, dose-related increase of both the rate and extent of MAP2-induced tubulin assembly, whereas progesterone, inactive per se, counteracted the stimulatory effect of pregnenolone. Electron microscopic analysis confirmed that pregnenolone-increased assembly of microtubules produced a completely normal structure. The stimulatory effect on MAP2-tubulin interaction was also observed in fetal rat-brain neuron cultures. Therefore, we propose a mechanism of neurosteroid action, the control of microtubule or, more generally, of neural cytoskeleton dynamics, with potential roles in brain development, plasticity, and aging.     

Monoclonal antibody to chicken oviduct progesterone receptor.

Antibodies to molybdate-stabilized chicken oviduct progesterone receptor were raised in a Wistar rat and detected by interaction with homogeneous radioiodinated progesterone receptor. Spleen cells of this rat were then fused with mouse Sp2/0-Ag14 myeloma cells and the antibodies produced by the hybrid cells were detected by double immunoprecipitation using the 125I-labeled receptor. Cells of one of the positive cultures were then cloned by limiting dilution and one hybridoma cell line was studied. The monoclonal antibody produced was an IgG2b, and it reacted with the molybdate-stabilized "8-9S" form of the chicken oviduct progesterone receptor, labeled with either [3H]progesterone or [3H]ORG 2058 (a high-affinity synthetic progestin). Kd for the 8-9S progesterone receptor was approximately equal to 1 nM. Progesterone receptor-monoclonal antibody complexes were labeled with radioactive progesterone, suggesting that antibody does not prevent hormone binding. By using a [35S]methionine-labeled antibody, we were able to detect the progesterone receptor independently of its characteristic function of binding radioactive hormone. No crossreaction with human progesterone receptor was detected.     

The insulin-like growth factor, somatomedin-C, modulates low density lipoprotein metabolism by swine granulosa cells

Insulin-like growth factor I (IGF-I) synergistically amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine ovarian cells. The mechanisms subserving this facilitative interaction included the following. IGF-I's synergism with LDL was associated with a decrease in the mean half-maximally stimulatory concentration of LDL from 20-3.5 micrograms/ml. IGF-I increased by 3- to 6-fold the number of specific high affinity LDL receptors on ovarian cells, with no change in apparent binding affinity. IGF-I augmented by 3- and 18-fold the maximal rates of [125I]iodo-LDL internalization and degradation, respectively, without altering half-maximally effective concentrations of LDL supporting these processes. IGF-I increased by 2- to 2.5-fold the total mass of free and esterified cholesterol contained in granulosa cells. IGF-I stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester from exogenous [3H]cholesteryl linoleate-labeled LDL, and amplified [3H]progesterone secretion by granulosa cells exposed to this source of lipoprotein-borne sterol. These actions of IGF-I were demonstrated at 30- to 100-fold lower concentrations of IGF-I than insulin. We conclude that IGF-I and LDL synergistically enhance progesterone biosynthesis by ovarian cells. This synergism occurs in part via mechanisms that regulate the effectual delivery of lipoprotein-borne cholesterol substrate into cellular sterol pools that participate in steroid hormone biosynthesis.     

Steroidogenic pathways and trophic response to adrenocorticotrophin of cultured adrenocortical cells in different states of differentiation.

Adrenocortical cells obtained from adult rats were propagated in monolayer culture. Depending on culture conditions, they grew either as lipid-containing epithelial-like cells with a high level of steroid production, or as fibroblast-like cells with a low level of steroid production. The major fluorogenic steroid secreted by both morphologic forms of adrenal cortical cell was corticosterone as determined by chromatography and acid fluorometry. Basal fluorogenic steroid production per 10(6) cells over 24 h was: epithelial-like cells, 5.0-mug; fibroblast-like cells, 0-014 mug. Stimulation with ACTH for 5 days increased fluorogenic steroid production and induced morphologic changes in both adrenal cell forms. ACTH stimulation of fluorogenic steroid production by both cell forms reached a maximum after 3 days, then dropped to a refractory state after 5 days. With maximal ACTH stimulation, production increased 25-fold in fibroblast-like cells and five-fold in epithelial-like cells. The latter rate of corticosterone production is similar, per cell, to ACTH-stimulated adrenal glands in vivo. Progressive morphologic changes were observed with ACTH stimulation: epithelial-like cells retracted from the substratum and lost lipid inclusions; fibroblast-like cells became more epithelial-like. Both adrenal cell types formed intermediates from [4-(14)C] pregnenolone including pregn-5-ene-3 phi, 20 alpha-diol and 20 alpha-hydroxy-pregn-4-en-3-one. Control cultures of muscle fascia fibroblasts did not produce corticosterone or intermediates from [4-(14)C[ pregnenolone and did not respond to ACTH functionally or morphologically.