Cholesterol Side-chain Cleavage Activity Research Articles

Purification and characterization of rat adrenodoxin

Adrenodoxin was purified from the rat adrenal gland. The A 414 A 280 value of the purified rat adrenodoxin was 0.90 and the oxidized spectrum showed absorption maxima at 320, 414 and 455 nm, similar to those of bovine adrenodoxin. On SDS-PAGE, the rat adrenodoxin showed a single band with a molecular mass of 11.2 kDa, while the apparent molecular mass by gel filtration through Sephadex G-75 equilibrated with 10 mM K-phosphate (pH 7.5) was 27 kDa. In the reconstituted system, V max of NADPH-cytochrome c reduction activity and the K m for the rat adrenodoxin were much the same as those for recombinant bovine adrenodoxin. In the case of cholesterol side-chain cleavage activity, however, these values of the rat adrenodoxin were about half of those of the bovine adrenodoxin. The CD spectrum of the rat adrenodoxin was similar to that of the bovine adrenodoxin but showed a significantly lower ellipticity value in the 195–205 nm region than that of the bovine adrenodoxin. The structural differences may possibly explain differences in the enzymic properties between rat and bovine adrenodoxins.

Development of cytochrome P-450 side chain cleavage mRNA levels in neonatal ovaries of normal and hypogonadal (hpg) mice

Cytochrome P-450 side chain cleavage (P-450scc) is vital for biosynthesis of gonadal steroids. In this study changes in P-450scc mRNA levels and cholesterol side-chain cleavage (CSCC) enzyme activity have been measured during development in the ovary of the normal mouse and the hypogonadal (hpg) mouse which lacks circulating gonadotrophins. Levels of P-450scc mRNA were measured using a semi-quantitative reverse transcription-polymerase chain reaction technique for which part of the 5' sequence of a mouse P-450scccDNA was sequenced. On the day of birth P-450scc mRNA was present at low levels in the mouse ovary. Thereafter there was no change in P-450scc mRNA levels for 5 days after which time levels increased significantly to reach a peak around day 10. Activity of CSCC showed a similar pattern of development although activity was not detectable on days 1 and 3. In the hpg mouse P-450scc mRNA levels were normal on day 1 but did not increase thereafter up to 15 days. These results show that there is gonadotrophin-independent expression of P-450scc mRNA in the mouse ovary at birth when only primordial follicles and stromal tissue are present. As the ovary develops after 5 days changes in P-450scc mRNA levels become gonadotrophin-dependent and coincide with maturation of secondary follicles.

Vasoactive intestinal peptide-induced expression of cytochrome P450 cholesterol side-chain cleavage and 17 alpha-hydroxylase enzyme activity in hen granulosa cells.

Experiments were conducted to determine whether vasoactive intestinal peptide (VIP) can regulate expression of cytochrome P450 side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase (P450 17 alpha-OH) mRNA levels and enzyme activity in granulosa cells from nonhierarchal (6-8-mm) follicles. Initial studies demonstrated that immunoreactive VIP is localized within the theca (but not granulosa) layer of both resting (< 0.5-mm follicles) and 6-8-mm follicles, thus providing a potential paracrine mechanism of action for VIP. While short-term (3 h) incubation of granulosa cells with VIP (0.001-1.0 microM) failed to stimulate progesterone production from 6-8-mm follicle granulosa cells, a 4-h culture period in the presence of VIP resulted in increased cyclic AMP (cAMP) accumulation, and a 24-h culture period resulted in progesterone synthesis and increased P450scc mRNA levels; control levels of each endpoint measurement were not altered within the period observed. By contrast, culture with the growth factor transforming growth factor alpha (TGF alpha) in the presence of VIP (1 microM) prevented increases in P450scc mRNA levels and progesterone production. Similar effects of VIP and TGF alpha in the presence of VIP were demonstrated for P450 17 alpha-OH mRNA levels and enzyme activity. Finally, there was an additive effect of VIP (0.1 microM) plus recombinant human (rh) FSH (100 mIU) on the initiation of progesterone production in cultured 6-8-mm follicle granulosa cells compared to the addition of VIP or rhFSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P-450 levels of immature rat ovaries during early follicular development: quantification, cellular and subcellular distribution, enzymatic activity and response to FSH in vivo and in vitro.

Absolute values of cytochrome P-450 (cyt. P-450) content were determined by dual beam spectrophotometry in mitochondrial and microsomal fractions of granulosa cells and stroma (theca and interstitial cells) obtained from ovaries of immature rats treated with diethylstilbestrol (DES) or with DES and FSH. Cholesterol side-chain cleavage activity and aromatase activity were also measured in relevant cell fractions. The cyt. P-450 content in the granulosa cell mitochondria was significantly increased in FSH-treated animals (cyt.P-450 in nmols/mg protein, mean +/- SE: DES-treated 0.027 +/- 0.00213, N = 9; DES/FSH-treated 0.0668 +/- 0.0120, N = 13, P = 0.014). The increased cyt. P-450 content was associated with an increase in cholesterol side-chain cleavage activity confirming the functional relevance of the spectrophotometric measurements. The increase in cyt. P-450 content in stroma after FSH treatment was not statistically significant but there was a significant increase in cholesterol side-chain cleavage activity. The cholesterol side-chain cleavage activity of intact granulosa cells from secondary follicles of DES-treated rats was significantly stimulated by in vitro exposure to FSH during 2 hr assay incubations. A significant cyt. P-450 content was detected in the microsomal fraction of granulosa cells from DES-treated rats which was not associated with aromatase activity. Granulosa cell microsomal cyt. P-450 content was not significantly altered by in vivo FSH treatment despite a dramatic increase in aromatase activity of such microsomal preparations.

Cholera toxin directly stimulates pregnenolone generation with increasing Ca2+ efflux in bovine adrenocortical mitochondria

The present experiments demonstrated that the holotoxin as well as the A- and B-subunits of cholera toxin were able to directly enhance pregnenolone synthesis when isolated intact mitochondria, prepared from bovine adrenocortical tissue, were incubated; they were not, however, able to enhance pregnenolone synthesis when the inner mitochondrial fraction was similarly incubated, suggesting that the conformational structure of mitochondria is very important for activation of cholesterol side-chain cleavage by cholera toxin. Data are also presented demonstrating that cholera toxin can enhance Ca2+ release from isolated mitochondria, while pertussis toxin could activate neither pregnenolone generation nor increase Ca2+ efflux from mitochondria. Thus it is suggested that cholera toxin may activate pregnenolone synthesis by regulating Ca2+ movement in mitochondria.

Maturational changes in steroidogenesis in the inner and outer zones of the guinea pig adrenal cortex

Studies were done to determine the effects of age on steroidogenesis in the inner (zona reticularis) and outer (zona fasciculta plus glomerulosa) zones of the guinea pig adrenal cortex. In 35-day-old animals, cortisol production by adrenal outer zone cells was approximately twice as great as that by inner zone cells. With aging, cortisol secretion by inner zone cells decreased to very low levels, but there was no detectable change in the capacity for cortisol production by the outer zone. However, the outer zone comprised a progressively decreasing fraction of the total adrenal mass in older animals. To determine the basis for the decline in cortisol production by inner zone cells with aging, the activities of several steroidogenic enzymes were determined. Microsomal 21-hydroxylase activity was greater in the inner than outer zone but was not significantly affected by age. By contrast, 17α-hydroxylase activity was greater in the outer zone at all ages, and decreased with aging in the inner but not the outer zone. Mitochondrial cholesterol sidechain cleavage and 11β-hydroxylase activities were also higher in the outer than inner zone and declined in the zone only in older animals. The decrease in inner zone cholesterol sidechain cleavage activity with aging was proportionately greater than the age-dependent changes in other enzyme activities. The results indicate that the effects of aging on steroidogenesis are both zone- and enzyme-specific. The overall decline in cortisol secretion by the guinea pig adrenal cortex with aging is attributable to both a decrease in cortisol production by the cells of the zone reticularis and a disproportionate increase in the mass of the gland comprised by this zone. The decrease in cortisol secretion correlates closely with a decline in cholesterol sidechain cleavage activity in the zona reticularis, and may be causally related.

Pregnenolone metabolism in rodent embryonic neurons and astrocytes.

The rat CNS has been previously shown to synthesize pregnenolone (PREG) and to convert it into progesterone (PROG) and some of its 5 alpha-reduced metabolites. However, the brain cell types involved in the metabolic conversions of PREG are poorly known. Selective conditions were used to obtain purified cultures of neurons and astrocytes from mouse or rat fetal striatum and cerebral cortex. Neurons converted PREG to only one identified metabolite, 20 alpha-dihydro PREG, whereas astrocytes converted PREG also to PROG, 5 alpha-dihydro PROG, and 3 alpha (3 beta)-5 alpha-tetrahydro PROG. Therefore, astrocytes can convert the neurosteroid PREG into the steroid hormone PROG and the neuromodulatory steroid 3 alpha, 5 alpha-tetrahydro PROG, whereas neurons lack the delta 5-3 beta-hydroxysteroid dehydrogenase isomerase activity (and cholesterol side-chain cleavage activity), necessary for the biosynthesis of PROG. Provision of steroid substrates is another example of cross-talk between glial cells and neurons.

Direct expression of adrenodoxin reductase in Escherichia coli and the functional characterization.

A plasmid for direct expression in Escherichia coli of the mature form bovine adrenodoxin reductase was constructed from the full-size cDNA for the enzyme [Y. Sagara, Y. Takata, T. Miyata, T. Hara, and T. Horiuchi, J. Biochem. (Tokyo), 102, 1333 (1987)] and an expression vector pCWori+. The recombinant adrenodoxin reductase was purified from the transformed E. coli cell lysates using adrenodoxin-Sepharose affinity chromatography [T. Sugiyama and T. Yamano, FEBS Lett., 52, 145 (1975)] with a yield of 2.5 mg/l of culture. The purified recombinant enzyme showed a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and migration was identical with that of the authentic enzyme purified from bovine adrenal cortex mitochondria. The recombinant enzyme had Ser at its amino-terminus and the sequence of the amino terminal 9 residues was identical with that of the authentic bovine enzyme. The absorption spectrum of the recombinant enzyme showed peaks at 270, 376, and 450 nm and shoulders at 425 and 475 nm. Flavin content of the recombinant enzyme was 0.8 mol FAD/mol. The apparent Km value for bovine adrenodoxin in NADPH-cytochrome c reductase activity using a reconstitution system was 16 nM, a value comparable with that of the authentic bovine enzyme (17 nM). The cholesterol side chain cleavage activity with a reconstitution system was about 75% of that obtained when the authentic enzyme was used.

Ontogenesis of cholesterol side-chain cleavage activity in the ovine adrenal during late gestation

The present study examined the activity of the cholesterol side-chain cleavage system, and the amount of cytochrome P450scc in adrenal glands of sheep fetuses and newborn lambs as well as the in vitro regulation of these parameters. Freshly isolated fetal adrenal cells incubated in the presence of 1 mM 8Br-cAMP or 25 μM 22R-OH cholesterol, produced 4- to 5-fold less pregnenolone than neonatal cells under similar conditions. Likewise, pregnenolone production by isolated fetal adrenal mitochondria was lower than that of neonatal mitochondria when endogenous cholesterol was used as a substrate or when 22R-OH cholesterol was added to the incubation medium. Also, the amount of P450scc, determined by immunoblot, was lower in fetal mitochondria than in neonatal mitochondria. In culture, ACTH, despite enhancing both the production of pregnenolone and the incorporation of [ 14C]acetate in cholesterol and its end-products by fetal adrenal cells, neither increased the amount of pregnenolone formed from 22R-OH cholesterol nor the amount of immunoreactive P450scc. By contrast, during the first 48 h of culture under standard conditions, there was a “spontaneous” increase in the activity of P450scc which reached values observed in neonatal adrenal cells. Such a development was inhibited when 5% ovine fetal serum was added to the culture medium. These results reinforce the view that in the ovine fetal adrenal gland, the development of P450scc is not ACTH-dependent but involves most probably a decrease in inhibitory factors present in fetal blood.

Decreased luteinizing hormone-stimulated progesterone secretion by preovulatory follicles isolated from cyclic rats treated with the progesterone antagonist RU486.

Since administration of the antiprogesterone RU486 to cyclic female rats at metestrus and diestrus results in increased serum levels of LH, estradiol, and testosterone at proestrus, we investigated whether RU486 affects follicular steroidogenesis. Female rats with a 4-day estrous cycle, induced experimentally by a single injection of bromocriptine on the morning of estrus, were given RU486 (2 mg) twice daily (0900 and 1700 h) on metestrus and diestrus. At proestrus the preovulatory follicles were isolated and incubated for 4 h in the absence and presence of LH. In the absence of LH, accumulation of estradiol, testosterone, and progesterone in the medium was not different for RU486-treated rats and oil-treated controls. In contrast, LH-stimulated estradiol, testosterone, and progesterone secretions were significantly lower in RU486-treated rats compared with controls. Addition of pregnenolone to the incubation medium resulted in a significantly lower increase of progesterone in follicles from RU486-treated rats compared with those from oil-treated controls. This suggests that 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity is decreased by administration of RU486 in vivo. Aromatase and 17 alpha-hydroxylase/C17-20 lyase activities were not affected: addition of substrate (androstenedione and progesterone respectively) did not affect differently the amount of product formed (estradiol and testosterone) in RU486- and oil-treated rats. However, LH-stimulated pregnenolone secretion was lower in follicles from RU486-treated rats compared with follicles from oil-treated controls, suggesting that either cholesterol side-chain cleavage activity or LH responsiveness is decreased. At proestrus the preovulatory follicles from RU486- and oil-treated rats were not morphologically different.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of prostaglandin F2 alpha inhibition of lipoprotein use by bovine luteal cells.

Prostaglandin F2 alpha (PGF2 alpha) inhibits lipoprotein-stimulated progesterone production by bovine luteal cells in vitro and the objective of this study was to localize the site of action of PGF2 alpha. Cultured bovine luteal cells were treated with PGF2 alpha for seven days, and then with either lipoproteins or 25-hydroxycholesterol in the presence of aminoglutethimide (which inhibits cholesterol side-chain cleavage) for the final 48 h. The effects of PGF2 alpha on progesterone production, cellular cholesterol content, mitochondrial cholesterol content and cholesterol side-chain cleavage activity were determined. As expected, PGF2 alpha inhibited (P less than 0.05) lipoprotein-stimulated progesterone production. However, PGF2 alpha did not inhibit low-density lipoprotein-stimulated, or high density lipoprotein-stimulated, increases in cellular cholesterol (P less than 0.05) or inhibit lipoprotein-induced increases in mitochondrial cholesterol content (P less than 0.05). Additionally, cholesterol content of mitochondria increased (P less than 0.05) in the presence of PGF2 alpha alone. To determine if the PGF2 alpha-induced inhibition of steroidogenesis occurred at, or after, the side-chain cleavage reaction, we treated cells with the readily diffusable sterol, 25-hydroxycholesterol. Prostaglandin F2 alpha did not inhibit 25-hydroxycholesterol-stimulated progesterone production (P less than 0.05). Prostaglandin F2 alpha may therefore exert its luteolytic effect at a site after cholesterol transport to the mitochondria but before cholesterol side-chain cleavage.

Steroidogenic enzyme activity in the hypogonadal ( hpg) mouse testis and effect of treatment with luteinizing hormone

The hypogonadal ( hpg) mouse, which lacks circulating gonadotrophins during development, has been used (a) to determine whether initial expression of steroidogenic enzyme activity is dependent upon gonadotrophins and (b) to examine the responsiveness of these enzymes to luteinizing hormone (LH) stimulation. Activities of 17α-hydroxylase, 17-ketosteroid reductase and 5α-reductase were very low but detectable in the hpg testis while cholesterol side-chain cleavage (CSCC) activity was undetectable. In contrast, 3β-hydroxysteroid dehydrogenase (3βHSD) activity was high (11% of normal testis). Treatment with LH increased CSCC and 17α-hydroxylase activity more than 11-fold within 24 h. 5α-Reductase activity was increased 3-fold after 3 days treatment while 17-ketosteroid reductase and 3βHSD activities did not respond until after 10 days of treatment. The overall increases in 5α-reductase (4-fold) and 3βHSD (6-fold) activities were low while changes in 17-ketosteroid reductase (20-fold) and, particularly, CSCC (> 130-fold) and 17α-hydroxylase (153-fold) were more marked. Results show (1) that expression of 3βHSD activity may be independent of gonadotrophins, (2) that activity of 17α-hydroxylase, 17-ketosteroid reductase and 5α-reductase is expressed, though at low levels, in the absence of gonadotrophins and (3) that prior exposure to gonadotrophins is not required for a rapid response to LH stimulation, particularly with respect to the cytochrome P-450 enzymes.

Steroidogenic enzyme activity in the rat testis following Leydig cell destruction by ethylene-1,2-dimethanesulphonate and during subsequent Leydig cell regeneration.

Ethylene-1,2-dimethanesulphonate (EDS) rapidly destroys Leydig cells in the rat testis, although repopulation occurs within 5-7 weeks. In this study we have examined the activity of testicular steroidogenic enzymes after Leydig cell destruction and during regeneration. This was designed to measure the contribution of cells, other than Leydig cells, to steroidogenic activity in the testis, and to determine whether changes in steroidogenic enzyme activity during Leydig cell regeneration after EDS parallel those which occur during normal Leydig cell development. The enzymes studied are those responsible for androgen synthesis and metabolism in the testis. Adult male Wistar rats (300-350 g) were injected with EDS (100 mg/kg, i.p.) and testicular steroidogenic enzyme activity was measured on days 0, 3, 7, 14, 21 and 35. On day 3, when no Leydig cells remain in the testis, cholesterol side-chain cleavage (CSCC) activity, per testis, declined to undetectable levels, while 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17 alpha-hydroxylase retained only 0.04 and 0.15% of control activity respectively. In contrast, 17-ketosteroid reductase (17-KSR) and 5 alpha-reductase retained 33 and 10% of control activity respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporin-Mediated Depression of Luteinizing Hormone Receptors and Heme Biosynthesis in Rat Testes: A Possible Mechanism for Decrease in Serum Testosterone*

The toxic side-effects of the immunosuppressive drug cyclosporin (CsA) include testicular dysfunction and a decline in circulating testosterone. However, mechanisms for the consistently observed CsA-mediated depression of serum testosterone levels are unclear because of conflicting reports concerning circulating gonadotropin levels and incomplete studies of intratesticular steroidogenesis. To elucidate these mechanisms, endocrine-regulated testicular steroidogenesis and heme metabolic parameters were studied in male rats given sc injections of either 25 or 40 mg/kg.day CsA for 6 days and then killed on the seventh day. Consistent with earlier reports, CsA treatment dramatically suppressed serum testosterone levels (less than 20% of control at both CsA doses). Additionally, the intratesticular testosterone content declined with the higher CsA dose. Serum LH and FSH levels were elevated up to 2- to 4-fold after the higher CsA treatment regimen. Measurement of decreases in testicular receptors for LH revealed for the first time that CsA treatment significantly reduced the ability of the testes to respond to normal or elevated circulating levels of LH. In animals receiving higher dose of the drug, cytochrome P-450-dependent mitochondrial cholesterol side-chain cleavage activity, which is the rate-limiting step in steroidogenesis, was markedly reduced to a mere 30% of the control value. Additionally, the activity of the microsomal cytochrome P-450-dependent 17 alpha-hydroxylase was decreased to less than half of the control value. Biotransformation of the prototype drug, benzo(a)pyrene, as well as microsomal cytochrome P450 levels declined significantly after the higher CsA dose, suggesting that CsA has an adverse affect on testicular cytochromes P-450 in general. In addition, CsA treatment altered heme metabolic parameters; significant increases in the activity of uroporphyrinogen-I synthetase and total porphyrin content were noted. Conversely, the activity of ferrochelatase, the enzyme that incorporates iron into porphyrin to form heme molecule, decreased significantly, as did the total heme levels. The latter was reduced to only 61% of control values. The findings suggest the likelihood that the observed inhibition of heme formation may contribute substantially to the reduced levels of microsomal cytochromes P-450 and steroidogenic activities that depend on them. Taken collectively, these data suggest a plausible mechanism by which CsA may induce testicular dysfunction; as the result of a combination of reduction in the number of LH receptors and a suppression of heme formation, the hemoprotein-dependent steroidogenic enzymes activities are compromised, leading to an impairment of normal testicular function.

Mechanism of action of spironolactone on cortisol production by guinea pig adrenocortical cells

Studies were done to determine the mechanism(s) of action of spironolactone (SL) and of its deacetylated metabolite, 7α-thio-SL, to inhibit cortisol secretion by guinea pig adrenocortical cells in vitro. Preincubation of cells at 37 ° C with SL or with 7α-thio-SL caused a time-dependent decline in subsequent ACTH-stimulated cortisol secretion. In the absence of a preincubation, neither compound affected cortisol production, indicating the need for production of an active metabolite. When the 17α-hydroxylase inhibitor, SU-10'603, was included during the preincubation period, neither SL nor 7α-thio-SL decreased cortisol secretion, indicating the involvement of the 17α-hydroxylase in the activation of both compounds. By contrast, neither the 11/gb-hydroxylase inhibitor, metyrapone, nor the cholesterol sidechain cleavage inhibitor, aminoglutethimide, diminished the effects of SL or of 7α-thio-SL on cortisol secretion. Preincubation of cells with SL or 7α-thio-SL also decreased the conversion of exogenous progesterone to cortisol, but did not affect cortisol production from the 17α-hydroxylated substrates, 17α-hydroxyprogesterone and 11-deoxycortisol, suggesting that only 17α-hydroxylation was impaired. In addition, there was a decline in 17α-hydroxylase activity in microsomes isolated from cells preincubated with SL or with 7α-thio-SL, but no change in microsomal 21-hydroxylase or in mitochondrial 11/gb-hydroxylase and cholesterol sidechain cleavage activities. The results indicate that the direct effects of SL and of 7α-thio-SL on the adrenal cortex to decrease cortisol production result from the selective inhibition of 17α-hydroxylation. Since 17α-hydroxylase activity is apparently required for the activation of both compounds, suicide inhibition of the enzyme may be the mechanism of action.

Steroidogenesis during postnatal development in the mouse ovary

In the mouse, follicular formation and development is largely postnatal. Changes in ovarian steroidogenesis during early postnatal life are likely, therefore, to reflect changes in follicular activity during early folliculogenesis. In this study, basal progesterone and androstenedione production and responsiveness to gonadotrophins, dibutyryl cyclic AMP (dbcAMP) and 22R-hydroxycholesterol have been measured following short-term in-vitro incubations of ovaries from mice aged 1, 3, 5, 7, 10 or 15 days. On the day of birth (day 1), basal and gonadotrophin-stimulated progesterone and androstenedione production were undetectable although a response to dbcAMP and a low level of cholesterol side-chain cleavage (CSCC) activity (measured using 22R-hydroxycholesterol) were present. On day 3 progesterone and androstenedione production were undetectable under all conditions. On day 5 only a low level of CSCC activity was detectable but on day 7 there was an increase in ovarian steroid production. Basal progesterone and androstenedione were detectable and LH, but not FSH, increased the production of both steroids. These changes were associated with a marked increase of more than 80-fold in CSCC activity. Basal steroid output increased from days 7 to 15 and LH continued to stimulate progesterone accumulation although no effect on androstenedione was seen. Addition of FSH had no effect on steroidogenesis on day 10 but significantly increased progesterone production on day 15. Ovaries from the mice used in these studies contained primordial follicles and stromal tissue on day 1. By day 5 primary and secondary follicles were present and the major increase in steroid production between days 5 and 7 was associated with an increase in secondary follicles and increased differentiation of the thecal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Human Chorionic Gonadotropin Treatment Prevents Depressed 17α-Hydroxylase/C17−20 Lyase Activities and Serum Testosterone Concentrations in 23,7,8-Tetrach Iorodibenzo-p-Dioxin-Treated Rats1

It is known that administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes decreased serum testosterone concentrations in the rat. Previous studies in this laboratory have shown that in rats TCDD exposure results in decreased 17 alpha-hydroxylase and C17-20 lyase activities. The decreases in these activities paralleled decreases in testicular microsomal heme and cytochrome P450 contents. As reported herein, neither testicular mitochondrial cytochrome P450 content nor the activity of cholesterol side-chain cleavage was altered in rats exposed to TCDD. Since the production of testosterone in the testis is dependent on LH, it is important to determine the early effects of TCDD on serum LH concentrations in the rat. Male Sprague-Dawley rats were given a single, oral dose of TCDD (50 micrograms/kg). Serum LH concentrations were determined by RIA on Days 1, 2, 3, 5, and 7 following TCDD treatment. Rat serum LH concentrations were decreased to 60% of controls as early as Day 1 and continued to be depressed on Days 2 and 3 at 53% and 59% of control values, respectively. Rat serum LH returned to control values by Day 5 in spite of continued depression of serum testosterone concentrations. The early depression in serum LH levels caused by TCDD may be related to the subsequent androgenic deficiency in the rat. Treatment of rats with hCG was found to be able to prevent the depression of the activities of testicular microsomal 17 alpha-hydroxylase and C17-20 lyase and serum testosterone concentrations caused by TCDD. These data indicate that TCDD decreases serum testosterone by decreasing P450(17 alpha) and C17-20 but not P450sec activities and that hCG treatment prevents the TCDD-induced decrease.

Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria: Effect of tween20 concentration

Cholesterol side-chain cleavage activities of cytochrome P-450ssc purified from bovine adrenocortical mitochondria were measured for various substrates, including cholesterol, 20[ S]-hydroxycholesterol, 22[ R]-hydroxycholesterol and 20[ R], [ R]-dihydroxycholesterol, in the reconstituted enzyme system at various Tween20 concentrations. The side-chain cleavage activity for cholesterol showed more than 10-fold enhancement upon addition of 0.1% Tween20, compared with that without the detergent. Addition of Tween20 did not cause any enhancement of the side-chain cleavage activities for 20[ S]-hydroxycholesterol and 22[ R]-hydroxycholesterol; rather, it resulted in an inhibition of the activities. The side-chain cleavage activity for 20[ R],22[ R]-dihydroxycholesterol showed a very high value even without the detergent. As the stimulatory effect of Tween20 was only specific for cholesterol, Tween20 seemed to enhance the rate of access of cholesterol to cytochrome P-450scc. These results are consistent with the suggestion that a transfer of substrate, cholesterol, in mitochondrial inner membrane, to the substrate-binding site of cytochrome P-450scc is the rate-limiting step in the cholesterol side-chain cleavage reaction.

2-Hydroxyestradiol Enhanced Progesterone Production by Porcine Granulosa Cells: Dependence onde novoCholesterol Synthesis and Stimulation of Cholesterol Side-Chain Cleavage Activity and Cytochrome P450scc Messenger Ribonucleic Acid Levels*

2-Hydroxyestradiol (2-OH-E2) stimulates progestin secretion by granulosa cells, but the intracellular locus of the stimulatory effect has not been clarified. The objectives of the present studies were to 1) determine the role of de novo sterol synthesis in the effect of 2-OH-E2 on progestin biosynthesis, and 2) examine the effects of 2-OH-E2 on cholesterol side-chain cleavage (SCC) activity and the level of messenger RNA (mRNA) for P450scc. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase with lovastatin (5 micrograms/ml) or mevinolin (5 micrograms/ml) reduced FSH- and 2-OH-E2-stimulated (but not E2-stimulated) progesterone production. Mevalonate (20 mM) enhanced basal progesterone production and reversed the inhibitory effect of lovastatin but did not affect progesterone biosynthesis in the presence of 2-OH-E2. As an index of the activity of cholesterol SCC enzyme, granulosa cells were exposed to 25-hydroxycholesterol (10 micrograms/ml) for 24 h and progesterone secretion monitored. Conversion of 25-hydroxycholesterol into progesterone was stimulated 2- to 3-fold by maximally effective concentrations of 2-OH-E2, E2, and FSH. 2-OH-E2 and/or E2 further enhanced 25-hydroxycholesterol conversion in the presence of FSH, LH, and epinephrine. Aminoglutethimide, an inhibitor of SCC, reduced 2-OH-E2- and 2-OH-E2 plus FSH-stimulated progesterone production by 97% and 95%, respectively. 2-OH-E2 also increased basal (by 2 to 3-fold) and FSH-stimulated (to 3.5-fold of FSH-treated controls) levels of mRNA for cytochrome P450scc. Collectively, our studies support the hypothesis that 2-OH-E2-enhanced progesterone biosynthesis by porcine granulosa cells is dependent on de novo cholesterol synthesis and is associated with increased levels of the mRNA encoding cytochrome P-450scc, which leads to increases in basal and gonadotropin-induced SCC activity.

Heterogeneous pools of cholesterol side-chain cleavage activity in adrenal mitochondria from adrenocorticotropic hormone-treated rats: Reconstitution of the isocitrate response with succinate and low concentrations of isocitrate

Cholesterol side-chain cleavage in isolated adrenal mitochondria requires unique energy requirements that may determine not only electron transport to P450 but also cholesterol availability. In mitochondria from ACTH-treated rats, two approximately equal pools of reactive cholesterol are indicated by the partial effectiveness of succinate (SU; Type A), and the metabolism of residual cholesterol by 1 m m isocitrate (IC; Type B). Type A metabolism is associated with relatively few initial cholesterol-P450 scc complexes and is rapidly and selectively lost when mitochondria are preincubated without an energy source. We now show that cholesterol metabolism supported by IC resolves into equal high and low affinity components (EC 50 = 10 and 250 μ m) exhibiting, respectively, Type A and Type B characteristics. SU and 50 μ m IC, in combination, provided nearly the same activity characteristics as 1 m m IC, including resistance to preincubation and increased turnover of cholesterol-P450 scc complexes. Much higher (three to six times) and more sustained pregnenolone formation was seen, with all reductants, following either enhancement of the reactive cholesterol pool or addition of 20-α-hydroxycholesterol, indicating that adrenocorticotropic hormone-mitochondria are limited by substrate availability. ATP generation was most effectively supported by SU, and IC was maximally active at 50 μ m, emphasizing differences between respiratory and steroidogenic energy requirements. ATP production and the maintenance of uniform suppression after in vivo cycloheximide treatment indicate the integrity of the mitochondrial interaction with all reductants. Inhibitors of SU oxidation (KCN, malonate) strongly inhibited SU-supported cholesterol metabolism but had little effect on SU synergism with IC. Fumarate (but not α-ketoglutarate or oxaloacetate) was equally effective as a synergist, but was totally ineffective as a reductant. SU or fumarate, therefore, act by a nonreductive pathway to boost NADPH production from low concentrations of IC. This decrease in apparent K m for IC may be mediated by stimulation of mitochondrial uptake of the reductant through the specific transporters.