Cholesterol Side-chain Cleavage Activity Research Articles

Purification and immunological characterization of human placental mitochondrial cytochrome P-450

Human placental mitochondrial cytochrome P-450 was purified to electrophoretic homogeneity by hydrophobic, anion exchange and cation exchange column chromatography. The specific content of the purified protein was 15.7 nmol/mg protein and it showed a single band mol. wt 48,000 D in SDS-gel electrophoresis. When reconstituted with bovine adrenal adrenodoxin reductase and adrenodoxin it converted cholesterol to pregnenolone (cholesterol side-chain cleavage activity, CSCC) at the rate of 1 pmol/min/pmol P-450. Antibodies against the purified protein were raised in rabbits. Inhibition studies demonstrated 85% inhibition of placental CSCC activity at an antibody/protein ratio of 10:1. Placental microsomal aromatase activity was inhibited by 47% at the same antibody/protein ratio. The antibody inhibited bovine mitochondrial CSCC activity by 87% at the same antibody/protein ratio. Placental microsomal 7-ethoxycoumarin O-deethylase, aryl hydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities were not significantly inhibited by the antibody. The results indicate that (1) the purified protein catalyzes cholesterol side-chain cleavage reaction, (2) human placental microsomal aromatase and bovine adrenal mitochondrial P-450scc may share common antigenic determinants with placental P-450scc, but (3) the placental microsomal xenobiotic-metabolizing cytochrome(s) is (are) distinctly different.

The stimulation by calcium and its inhibition by adp of cholesterol side-chain cleavage activity in adrenal mitochondria

Cholesterol side-chain cleavage activity (cholesterol SCC) in a mitochondrial preparation is increased by calcium in a sigmoidal manner. A 5–10-fold increase is obtained and an effect may be seen at 20 μM CaCl 2. ADP inhibits the stimulation by calcium with a shift of the sigmoid curve to the right and 10 μM ADP results in a 4-fold increase in the amount of CaCl 2 required to obtain one-half the maximal stimulation value. The inhibition is specific for ADP and inhibition by ATP is due to the formation of ADP. The characteristics of the calcium-ADP modulation are such that a suitable ADP-inhibited level of cholesterol sidechain cleavage activity will be stimulated by a given increment of calcium to a greater extent than in the absence of the added ADP. Steroid 11β-hydroxylation is also stimulated in a sigmoidal manner by calcium and this stimulation is inhibited by ADP. The 11β-hydroxylation, however, is less sensitive to calcium and ADP so that changes in cholesterol SCC are obtained at concentration of calcium and ADP where minimal effects on 11β-hydroxylation are found. Calmodulin-like activity is present in the mitochondrial preparation. No evidence, however, for a role for calmodulin in the calcium-ADP effects could be obtained, but the possibility of its involvement cannot be excluded. The calcium-ADP modulations are of a magnitude and take place at sufficiently low concentrations to suggest a physiological role in the regulation of mitochondrial steroidogenesis.

The insulin-like growth factor, somatomedin C, induces the synthesis of cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin in ovarian cells.

The actions of insulin and somatomedin C (insulin-like growth factor I) on cholesterol side-chain cleavage activity and the synthesis of cytochrome P-450scc and adrenodoxin were investigated in primary cultures of swine ovarian (granulosa) cells. Nanomolar concentrations of pure human somatomedin C stimulated biosynthesis of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Moreover, in the presence of exogenous sterol substrate for cholesterol side-chain cleavage, somatomedin C significantly enhanced pregnenolone biosynthesis in a time- and dose-dependent manner. This augmentation of functional cholesterol side-chain cleavage activity was accompanied by a dose-dependent (2-16-fold) increase in [35S]methionine incorporation into specific immunoprecipitable cytochrome P-450scc and adrenodoxin. Micromolar concentrations of insulin (but not proinsulin or desoctapeptide) also induced synthesis of cholesterol side-chain cleavage constituents by 4-7-fold. These results demonstrate that an insulin-like growth factor, somatomedin C, exerts discrete differentiating effects on ovarian cells characterized by increased synthesis of immunospecific cytochrome P-450scc and adrenodoxin. Thus, we infer that somatomedin C may serve a critical role in the differentiation of steroidogenic cells in the mammalian ovary.

Control of Gene Expression of Adrenal Steroid Hydroxylases and Related Enzymes

Utilization of cDNA probes specific for various components of the bovine adrenocortical steroidogenic pathway have led to the conclusion that there are three levels of regulation of steroid hydroxylase gene expression. In each case it is postulated that specific classes of proteins bind to regulatory regions of these genes and modulate their transcription. Throughout adult life, cAMP-dependent regulation via SHIP protein(s) is the predominant mechanism by which optimal steroidogenic capacity is maintained. A second type of regulation is tissue-specific. One subclass of tissue-specific expression is the "all-or-none" type whereby steroid 21-hydroxylase and 11 beta-hydroxylase gene expression occur only in adrenal cortex and not in other steroidogenic tissues. A second subclass of tissue-specific expression is the "variable" type whereby 17 alpha-hydroxylase and cholesterol side chain cleavage (SCC) activity are both expressed in ovarian thecal cells but only SCC activity is expressed in corpus luteum. The third type of regulation is cAMP-independent and leads to fetal-imprinting (initial expression of steroid hydroxylase genes during fetal life).

Restoration effects of exogenous luteinizing hormone on the testicular steroidogenesis and Leydig cell ultrastructure.

In the present study, we explored the restoration effects of exogenous LH on Leydig cell ultrastructure and testicular steroidogenesis in rats that were deprived of endogenous LH via treatment with testosterone-17 beta-estradiol-filled Silastic implants for 10 days. Exogenous LH was supplied continuously via Alzet miniosmotic pumps at the rate of 1 microgram/h for 3, 6, 12, and 24 h or 1, 2, 4, 8, and 12 days. Testes were then perfused in vitro with medium containing 1) LH, 2) 20 alpha-hydroxycholesterol, or 3) pregnenolone substrate, which allowed us to assess LH-stimulated testosterone secretion, cholesterol side-chain cleavage activity, or the conversion of pregnenolone to testosterone, respectively. Other testes were perfusion fixed via the testicular artery for morphometric measurement of Leydig cell number and volume per testis and the surface area of Leydig cell cytoplasmic smooth endoplasmic reticulum (SER), inner mitochondrial membrane, and outer mitochondrial membrane. The results verified that Leydig cell smooth endoplasmic reticulum and inner and outer mitochondrial membrane surface areas are drastically diminished (P less than 0.05 vs. intact controls) by LH withdrawal. Also, the results verified that exogenous LH administered in situ restores Leydig cell ultrastructure and capacity to biosynthesize testosterone. However, the recovery of Leydig cell structure and steroidogenic reactions occurred at strikingly different rates upon restoration of LH after 10 days of the treatment with testosterone-17 beta-estradiol implants. For example, the restoration of testicular capacity to synthesize progesterone in response to LH stimulation or 20 alpha-hydroxycholesterol substrate was completed within 24 h. In contrast, the restoration of Leydig cell SER and testicular capacity to synthesize testosterone from pregnenolone was completed only after 8 days of continuous LH treatment (P greater than 0.05 vs. intact controls). Thus, our results show that LH rapidly restores Leydig cell post-LH receptor steroidogenic events up to and including cholesterol side-chain cleavage activity. Interestingly, there is no temporal association between the recovery of cholesterol side-chain cleavage activity and the surface area of inner mitochondrial membrane surface area. In contrast, 8 days are required to coincidentally restore SER surface area and the capacity of Leydig cells to synthesize testosterone from pregnenolone. We conclude that different cellular mechanisms are involved in the LH-dependent restoration of inner mitochondrial cholesterol side-chain cleavage activity and SER-associated conversion of pregnenolone to testosterone.

Mitochondrial steroid metabolism in the inner and outer zones of the guinea-pig adrenal cortex

Previous investigations have demonstrated that cells isolated from the outer zone (zona fasciculata + zona glomerulosa) of the guinea-pig adrenal cortex produce far more cortisol than those from the inner zone (zona reticularis). Studies were carried out to compare mitochondrial steroid metabolism in the two zones. Protein and cytochrome P-450 concentrations were similar in outer and inner zone mitochondria. However, the rate of 11β-hydroxylation was significantly greater in the outer zone despite the fact that substrates for 11 β-hydroxylation (11-deoxycortisol, 11-deoxycorticosterone) produced larger type I spectral changes in inner zone mitochondria. The apparent affinities of 11-deoxycortisol and 11-deoxycorticosterone for mitochondrial cytochrome(s) P-450 were similar in the two zones. In both inner and outer zone mitochondria, 11β-hydroxylation was inhibited by metyrapone but unaffected by aminoglutethimide. Cholesterol sidechain cleavage activity, measured as the rate of conversion of endogenous cholesterol to pregnenolone, was far greater in outer than inner zone mitochondria. Addition of exogenous cholesterol or 25-hydroxycholesterol to the mitochondrial preparations did not affect pregnenolone production in either zone. Addition of pregnenolone to outer zone mitochondria produced a reverse type I spectral change (ΔA 420−390 nm), suggesting displacement of endogenous cholesterol from cytochrome P-450. In inner zone mitochondria, pregnenolone induced a difference spectrum (ΔA 425−410 nm) similar to the reduced vs oxidized cytochrome b 5 spectrum. A b 5-like cytochrome was found to be present in the mitochondrial preparations. Prior reduction of the cytochrome with NADH eliminated the pregnenolone-induced spectral change in inner zone mitochondria but had no effect in outer zone preparations. The results suggest that differences in mitochondrial steroid metabolism between the inner and outer adrenocortical zones account in part for the differences in cortisol production by cells in each zone.

Bipotential actions of estrogen on progesterone biosynthesis by ovarian cells. II. Relation of estradiol's stimulatory actions to cholesterol and progestin metabolism in cultured swine granulosa cells.

Although both inhibitory and stimulatory actions of estradiol on swine granulosa cells have been described, the bases for these inconsistent effects are not clear. We have tested properties of ovarian follicles and in vitro culture conditions that result in consistently stimulatory effects of estradiol on progesterone biosynthesis. Stimulatory actions of estradiol (in contrast to inhibitory effects) were critically dependent upon the density of granulosa cells in culture and the size and maturational status of the parent Graafian follicles. Granulosa cells isolated from small, rather than medium or large sized, swine follicles exhibited the greatest peak response to estradiol, although half-maximally stimulatory concentrations (ED50) of estradiol were similar (mean, 81 ng/ml). Granulosa cells from atretic follicles also secreted increased quantities of progesterone in response to estradiol, but the ED50 for estrogen stimulation was significantly higher (ED50 = 322 ng/ml estradiol) than that of comparable healthy follicles (ED50 = 109 ng/ml). This estrogen-responsive system was used to test the mechanisms subserving estrogen's trophic actions on granulosa cells. Estradiol significantly enhanced the activity of 3 beta-hydroxysteroid dehydrogenase with consequently increased production of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Estrogen also augmented functional cholesterol side-chain cleavage activity in a dose- and time-dependent fashion with a resultant increase in pregnenolone biosynthesis. Moreover, parallel observations documented concordant dose responses for the synthesis of all three major progestins by pig granulosa cells. The trophic actions of estrogen on the steroidogenic pathway were associated with enhanced hydrolysis of endogenous cholesteryl ester stores but were not significantly antagonized by inhibition of de novo cholesterol biosynthesis. We conclude that suitable follicle selection and appropriate in vitro culture conditions provide a consistently estrogen-responsive granulosa-cell system, in which estradiol modulates certain key aspects of progestin and cholesterol metabolism. These trophic actions of estrogen are likely to prepare granulosa cells for the increased rates of progesterone biosynthesis ultimately required by fully differentiated luteal cells.

The Golgi apparatus and adrenal function: The effects of monensin on adrenocorticotropic hormone-stimulated steroidogenesis

It has previously been shown that the steroidogenic action of adrenocorticotropic hormone (ACTH) is accompanied by characteristic alterations in cell ultrastructure. These include hypertrophy of the Golgi complex associated with increased vesicle formation and striking elevations of acid phosphatase activity in the Golgi complex and lysosomes. To investigate a possible relationship of these phenomena to steroidogenic function in monolayer cultures of murine adrenal tumor cells, monensin, a carboxylic ionophore which disrupts the ordered structure and transport function of the Golgi complex, was used, Monensin, at a concentration of 1.2 μ m, causes massive vacuolization and hypertrophy of the Golgi complex. No effect on mitochondrial structure was seen. Monensin, 0.6–1.2 μ m, inhibits both ACTH-stimulated and basal steroidogenesis by approximately 50% in incubations of 4 h or less. Dibutyryl-cAMP-stimulated steroidogenesis was inhibited to a similar degree. Incubations were carried out in serum-free media to eliminate possible effects due to exogenous cholesterol transport into the cell. There were no direct inhibitory effects of monensin on cholesterol side-chain cleavage (SCC) activity in isolated mitochondria. In contrast, mitochondria isolated from cells previously treated with monensin had a reduced capacity for this activity. These experiments suggest that monensin inhibits transport of cholesterol from the Golgi complex to the mitochondrial site of steroidogenesis action or interferes with the transport of key mitochondrial proteins synthesized on cytoplasmic ribosomes.

Are polyphosphorylated phospholipids involved in the hormonal control of cholesterol side-chain cleavage activity in tumour leydig cells?

The possible role of LH or dcAMP induced changes in polyphosphorylated phospholipid metabolism in the regulation of cholesterol side-chain cleavage activity has been studied in tumour Leydig cells. Mitochondria isolated from LH-stimulated Leydig cells were 400% more active in pregnenolone production than mitochondria from control cells. Steroid production in isolated mitochondria from control cells could be stimulated only 25% by cytosol fractions from stimulated cells and 100μM phosphatidyl inositol-4'-phosphate (PtdIns4P). Other polyphosphorylated phospholipids were either inactive or showed aspecific effects. During a preincubation period tumour cells were labelled with [ 32P]phosphate and steady-state labelling was obtained for the pholyphosphorylated phospholipids after 40–60 min. [ 32P]Phosphate incorporation in Ptd Ins4P, phosphatidyl inositol (Ptdlns), phosphatidyl choline (PtChl), phosphatidyl ethanolamine (PtdEtn) and cardiolipin (CL) was not affected by treatment of the Leydig cells with LH which stimulated (6-fold), or with cycloheximide which suppressed (4-fold) steroid production. A 25% increase of phosphate incorporation by LH was observed only in phosphatidyl inositol-4',5'-biphosphate (PtdIns4,5P 2). 32P Incorporation in PtdIns4,5P 2, PtdIns,PtdEtn and CL was stimulated by quinacrine 50 μM. Under these conditions the LH-stimulated pregnenolone production but not the 25-hydroxycholesterol dependent pregnenolone production, was completely inhibited. The results obtained with isolated mitochondria and intact cells indicate that increased levels of polyphosphorylated phospholipids are not consistently correlated with increased mitochondrial pregnenolone production. This argues against an important role of polyphosphorylated pohospholipids in the hormonal regulation of cholesterol side-chain cleavage activity in tumour Leydig cells.

Bipotential Actions of Estrogen on Progesterone Biosynthesis by Ovarian Cells. I. Relation of Estradiol's Inhibitory Actions to Cholesterol and Progestin Metabolism in Cultured Swine Granulosa Cells*

In short term, serum-free cultures of granulosa cells, estradiol inhibited basal progesterone production by more than 84% in a time- and dose-dependent fashion. Half-maximal inhibition (ID50) was observed at a mean estradiol concentration of 70 ng/ml. The acute inhibitory effects of estradiol were independent of cell density or maturational status of the parent follicle (small, medium, or large follicles). Estrogen decreased total progesterone biosynthesis, rather than merely progesterone secretion by granulosa cells. In addition, the production of progesterone and its 20 alpha-reduced metabolite were suppressed in parallel over a 30-fold range of inhibitory effects. Moreover, estradiol inhibited progesterone production from both endogenous and exogenous (25-hydroxycholesterol) substrate. In contrast to its inhibition of progesterone production, estradiol acutely enhanced pregnenolone accumulation and led to a 14- to 20-fold increase in the ratio of pregnenolone to progesterone. Inhibition of 3 beta-hydroxysteroid dehydrogenase activity was confirmed by demonstrating estrogen's ability to antagonize the conversion of exogenously supplied pregnenolone to progesterone in intact granulosa cells (ID50, 74 ng/ml) and to directly inhibit enzymic activity in ovarian homogenates (ID50, 79 ng/ml). On the other hand, estradiol did not alter the incorporation of [14C]acetate into cholesterol and specific progestins or influence the time-dependent hydrolysis of prelabeled cholesteryl ester stores in granulosa cells. In summary, in short term cultures of swine granulosa cells, estradiol significantly inhibits basal progesterone biosynthesis in a time- and dose-dependent fashion, encompassing estradiol concentrations attainable in vivo. Estrogen's inhibition is accomplished by mechanisms that are independent of cytotoxicity, cell density, and maturational stage of the parent follicle. The predominant locus of estrogen action is blockade of pregnenolone's conversion to progesterone, rather than accelerated metabolism of progesterone to its 20 alpha-reduced metabolite. Moreover, estradiol simultaneously enhances functional cholesterol side-chain cleavage activity without suppressing de novo cholesterol biosynthesis or impairing the mobilization of cellular cholesteryl ester stores. Thus, we infer that increasing concentrations of estradiol attained in antral follicular fluid during the later stages of follicle maturation could effectively limit the premature secretion of large quantities of progesterone before ovulation without impairing the cell's capacity for de novo cholesterol biosynthesis or cholesteryl ester hydrolysis.

Imidazole antimycotics: Inhibitors of steroid aromatase

Miconazole and clotrimazole, members of a class of imidazole agents which have broad spectrum antimycotic activity, were shown to be potent inhibitors of steroid aromatase activity of human placental microsomes. The I 50 values for the inhibition of aromatase activity by miconazole, clotrimazole, ketoconazole, and aminoglutethimide were 0.6, 1.8, 60 and 44 μM respectively. The most effective compound, miconazole, exhibited competitive kinetics with respect to androstenedione, the aromatase substrate. The apparent inhibitory constant ( K i ) was 55 nM, under assay conditions where the apparent K m for androstenedione was 220 nM. The inhibition of aromatase activity by miconazole was shown to be reversible by dilution. Miconazole was a relatively poor inhibitor of the cholesterol side chain cleavage activity of a placental mitochondria-enriched fraction, while both clotrimazole and ketoconazole markedly inhibited this mitochondrial monooxygenase activity. Spectrophotometric studies revealed that miconazole bound to the cytochrome P-450 component of the placental microsomal aromatase complex and had negligible effect on NADPH-cytochrome c (P-450) reductase activity. These results strongly support direct interaction of miconazole with microsomal cytochrome P-450 in human placental microsomes with high affinity resulting in the inhibition of aromatase activity.

Lutropin stimulates de novo synthesis of short-lived proteins required for lutropin-dependent steroid production in tumour leydig cells

Continuous protein synthesis is required for the hormonal regulation of cholesterol side chain cleavage activity. A protein with a short half-life ( t 1 2 = 2–13 min ) is believed to play an important role, but the regulation of the synthesis of this putative rapidly-turning-over protein is largely unknown. The steroid production rate in tumour Leydig cells can be increased more than 4-fold after addition oflutropin. However, steroid production by cells preincubated for 60 min with medium containing cycloheximide (89 μM) could not be stimulated when lutropin was added to the medium. Thus, the putative protein with the short half-life is apparently not derived from a stable precursor protein. Moreover, in tumour Leydig cells incubated with low concentrations of cycloheximide (0.2–0.8 μM), inhibition of steroid production was significantly greater in lutropin-stimulated cells than in control cells. These results support the hypothesis that lutropin regulates the de novo synthesis of rapidly-turning-over proteins by increasing the rate of initiation of the translation step of protein synthesis.

An early steroidogenic defect in hormone-induced Leydig cell desensitization

To define the nature of the lesion of the early steroidogenic pathway (prior to pregnenolone formation) in gonadotropin-induced desensitization of rat testicular Leydig cells, we evaluated cholesterol side-chain cleavage activity in isolated mitochondria by measurement of pregnenolone synthesis and [ 14C]isocaproic acid formation from [26- 14C]cholesterol. The enzyme activity was shown to be reduced after in vivo treatment with 10 μg hCG when compared to that of mitochondria from control animals only when measured in the presence of limiting NADPH concentrations (100 μM). Sonication of mitochondria from control and hCG-treated rats caused complete loss of cholesterol side-chain cleavage activity. When acetone-powdered adrenal cell mitochondria were employed as the source of the enzyme, the addition of sonicated Leydig cell mitochondria from control and hCG-treated animals caused the same differences as those observed with intact Leydig cell mitochondria in the presence of low concentration of NADPH. The K m value of the adrenal enzyme for NADPH incubated with Leydig cell mitochondria increased from 0.111 mM in control to 0.37mM after hCG, with no changes in V max . Moreover, cholesterol side-chain cleavage activity of adrenal mitochondria assayed in the presence of 100 μM cholesterol was progressively inhibited by increasing amounts of acetone powder from Leydig cell mitochondria of control and hCG-treated rats, with ID 50 of 500 and 280μg protein, respectively. The inhibiting factor was not a lipid or steroid but a heat-labile protein, with an approximate Stokes radius of 4.8 nm and an isoelectric point of 5.05 ± 0.23 SD ( n = 8). The inhibitory effect was confined to the Leydig cell mitochondrial membrane, and was not related to changes in oxidative phosphorylation. NADPH was not directly oxidized or immobilized by the mitochondrial factor, and this inhibiting substance was not adsorbed on 2',5'ADP-Sepharose 4B. These results have demonstrated that a heat-labile inhibiting protein factor is present in mitochondria from normal Leydig cells and is markedly activated or increased by hCG treatment. This substance that competitively modulates cholesterol side-chain cleavage activity could contribute to the early steroidogenic lesion, and also serve as an endogenous modulator of steroid hormone biosynthesis.

Differences between the regulation of cholesterol side-chain cleavage in Leydig cells from mice and rats

A Leydig cell culture system has been used to study the in vitro modulation by luteinizing hormone (LH) of steroidogenesis in Leydig cells isolated from mice and immature rats. Mouse Leydig cells precultured for 24 h in the presence of increasing concentrations of LH (1 ng–1 μg/ml) showed a dose-dependent decrease of the maximal LH-stimulated testosterone production. After pretreatment with 1 μg LH/ml, maximal LH-stimulated testosterone production and testosterone production in the presence of excess 20α-hydroxycholesterol (a cholesterol side-chain cleavage substrate) were reduced to approx. 50% of control values. The possible site of action of LH is probably prior to pregnenolone, because testosterone production in the presence of excess pregnenolone was not affected by the LH pretreatment. Immature rat Leydig cells showed no decrease of maximal steroid production after 24 h culture in the presence of 1 μg LH/ml. These results indicate that the regulation of the cholesterol side-chain cleavage activity during long-term LH action is different in mouse and rat Leydig cells. The properties of the cholesterol side-chain cleavage enzyme in mouse and rat Leydig cells were further investigated with different hydroxylated cholesterol derivatives as substrates. Steroid production by mouse Leydig cells in the presence of (22R)-22 hydroxycholesterol was similar as in the presence of LH. In contrast, steroidogenesis in rat Leydig cells in the presence of (22R)-22 hydroxycholesterol was at least 10-fold higher than in the presence of LH. It is concluded that the cholesterol side-chain cleaving enzyme in the mouse Leydig cell operates at its maximal capacity during short-term LH stimulation and can be inhibited after long-term LH action, whereas in the rat Leydig cell only a fraction of the potential activity is used during short-term LH stimulation, which is not affected during long-term LH action.

Characterization of the acute stimulation of steroidogenesis in primary bovine adrenal cortical cell cultures.

The early events of steroidogenesis, following adrenocorticotropin (ACTH) stimulation, were investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated 4-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide, even though cytochrome P-450scc only increases after 12 h. Mitochondrial free cholesterol levels increased during the same time period (16.5 to 25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. It is concluded that early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial free cholesterol. The maximum rate of cholesterol transport to mitochondria in aminoglutethimide-blocked cells (8.6 micrograms/mg of protein/h) was consistent with both the maximum rate of mitochondrial cholesterol SCC and cellular steroidogenesis (6.0 micrograms of pregnenolone/mg/h and 5.5 micrograms of steroid/mg of mitochondria/h, respectively). Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial free cholesterol. The distribution of steroid products fell into three distinct phases during a 24-h period following ACTH stimulation: an initial increase in SCC activity (0-4 h), elevation of androstenedione in place of corticosterone (4-12 h), and then in place of cortisol (12-24 h). The changes from 4 to 24 h result from a progressive stimulation by ACTH of 17 alpha-hydroxylase activity (but not 21-hydroxylase or C17:20 lyase activities) that is maintained even when stimulation of total steroidogenesis has stopped.

Cholesterol side-chain cleavage activity in human placenta and bovine adrenals: An one-step method for separation of pregnenolone formed in vitro

Cholesterol side-chain cleavage (CSCC) activity towards exogenous cholesterol was quantified by an one-step reversed-phase minicolumn method for the separation of pregnenolone formed in the reaction. The assay is rapid and reproducible. The method is linear for up to 2 mg of placental mitochondrial protein and up to 1 mg of bovine adrenal mitochondrial protein in the incubate over 30 min and 5 min reaction times, respectively. Average Km and Vmax values were 14.1 μM and 3.4 pmol/min/mg for the placental preparation and 1.5 μM and 20.7 pmol/min/mg for the bovine adrenal mitochondrial preparation. In human placenta, the mitochondrial fraction contained most of the CSCC activity. Inhibition studies showed that aminoglutethimide (500 μM) inhibited both placental and bovine adrenal activities at the same level (about 80–90 % inhibition) but androstenedione (500 μM), metyrapone (500 μM), benzo(a)pyrene (800 μM) and Emulgen 911 (0.05 %) were more effective in human placental preparations. Neither of the activities were inhibited to any great extent by α-naphthoflavone (500 μM), SKF 525A (500 μM) or 7-ethoxycoumarin (1 mM).

Cholesterol side-chain cleavage activity in the outer and inner zones of the adrenal cortex

Cholesterol side-chain cleavage activity in mitochondria isolated from the outer and inner zones of the guinea pig adrenal cortex was evaluated in order to clarify the role of the zona reticularis in steroidogenesis. It was found that side-chain cleavage activity was three times higher in the outer zone. In addition, ether stress increased side-chain cleavage activity in the outer zone but not the inner zone. The concentration of total and free cholesterol was also found to be higher in the outer zone. However, when exogenous cholesterol was added to mitochondria, there was no enhancement in side-chain cleavage activity in either zone.

Evidence for sterol carrier protein 2-like activity in hepatic, adrenal and ovarian cytosol

Purified sterol carrier protein 2 (SCP 2) from rat liver stimulated utilization of endogenous cholesterol for pregnenolone synthesis by adrenal mitochondria. Cytosolic preparations of rat liver, adrenal and luteinized ovary were also stimulatory in mitochondrial pregnenolone synthesis to different extents. Treatment of all preparations with rabbit anti-rat SCP 2 IgG neutralized the stimulatory effects, and immunoprecipitated proteins gave similar patterns on SDS-gradient polyacrylamide gel electrophoresis. Treatment with rabbit pre-immune IgG had no effect on these parameters. Thus, proteins which are immunochemically compatible with hepatic SCP 2 appear to be present in steroidogenic tissues and may play a role in control of mitochondrial cholesterol side chain cleavage activity.

Mechanisms subserving the trophic actions of insulin on ovarian cells. In vitro studies using swine granulosa cells.

Direct actions of insulin on gonadal tissues have been difficult to demonstrate in vivo. We have developed an in vitro system in which swine ovarian cells remain highly responsive to trophic actions of insulin. Purified porcine insulin significantly augmented the biosynthesis and secretion of progesterone by cultured granulosa cells. These stimulatory actions of insulin were dose- and time-dependent and saturable. Under serum-restricted conditions, insulin also significantly amplified the capacity of estradiol and 8-bromo cyclic AMP to stimulate progesterone production. Inhibitors of protein and RNA synthesis (cycloheximide, actinomycin D, and alpha-amanatin) inhibited insulin action. The stimulation of progesterone production by insulin was attributable to increased biosynthesis of pregnenolone, rather than diminished catabolism of progesterone to its principal metabolite, 20alpha-hydroxypregn-4-en-3-one. Insulin also enhanced progesterone production in the presence of a soluble sterol substrate, 5-cholesten-3beta,25-diol, which readily gains access to the mitochondrial cholesterol side-chain cleavage system. Moreover, exposure of granulosa cells to insulin produced a three- to sevenfold increase in mitochondrial content of cytochrome P-450 measured by difference spectroscopy, with a corresponding increase in mitochondrial cholesterol side-chain cleavage activity. The capacity of insulin to facilitate progesterone biosynthesis by ovarian cells was mimicked by the insulinlike somatomedin, multiplication stimulating activity, but not by epidermal growth factor, fibroblast growth factor, or porcine relaxin. Insulin's augmentation of progesterone production reflected a selective action on progestin biosynthesis, since insulin significantly suppressed estrogen biosynthesis by granulosa cells.Thus, our investigations indicate that insulin acts on ovarian cells selectively to stimulate pregnenolone (but not estrogen) biosynthesis. The actions of insulin are exerted by processes that require protein and RNA synthesis, and by mechanisms that augment mitochondrial cytochrome P-450 content and facilitate the utilization of cholesterol in the side-chain cleavage reaction. The striking mimicry of insulin effect by multiplication stimulating activity suggests that insulin action may be mediated through somatomedin receptors. Moreover, in view of the high concentrations of somatomedin in ovarian follicles in vivo, our in vitro observations suggest that specific trophic actions of insulin or insulinlike growth factors are likely to significantly regulate the differentiated function of the Graafian follicle in vivo.

Evidence for the presence of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in fresh granulosa cells. Effects of follicle-stimulating hormone and cyclic AMP on cholesterol side chain cleavage cytochrome P-450 synthesis and activity.

The synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and adrenodoxin was studied both in freshly harvested bovine granulosa cells and in granulosa cells maintained in primary monolayer culture. In addition, the action of follicle-stimulating hormone (FSH) and cyclic AMP analogs to stimulate the synthesis of cytochrome P-450scc was investigated in cultured cells. Precursor forms of cytochrome P-450scc and adrenodoxin were immunoisolated from a cell-free translation system directed by RNA prepared from freshly obtained granulosa cells that were not luteinized. Furthermore, the presence of cytochrome P-450scc in lysates of granulosa cells freshly obtained from very small follicles (containing less than 0.1 ml of follicular fluid) and in mitochondria of freshly obtained granulosa cells was demonstrated by using an immunoblotting technique. Continuous treatment of cultured granulosa cells with FSH or with cyclic AMP analogs (dibutyryl cyclic AMP or 8-bromo cyclic AMP) for 72 h increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc. Moreover, FSH, dibutyryl cyclic AMP, and 8-bromo cyclic AMP stimulated pregnenolone production by cultured granulosa cells (2.3-, 4.0-, and 7.5-fold increase over control, respectively), indicative of an increase in cholesterol side chain cleavage activity. The results of this study demonstrate for the first time the presence of two components of the cholesterol side chain cleavage system in freshly obtained granulosa cells, and provide direct evidence for the trophic effect of FSH and its presumed mediator, cyclic AMP, on the synthesis of cytochrome P-450scc in granulosa cells.