Synthesis of the cholesterol side-chain cleavage enzymes in cultured rat ovarian granulosa cells: induction by follicle-stimulating hormone and dibutyryl adenosine 3',5'-monophosphate

The effects of FSH and (Bu)2cAMP on synthesis of the components of the cholesterol side-chain cleavage (SCC) enzyme complex, namely SCC cytochrome P-450 (P-450scc), the iron-sulfur protein adrenodoxin (ISP), and NADPH:ISP reductase (Red), were investigated in granulosa cells obtained from ovaries of immature estrogen-primed rats cultured for up to 72 h in defined medium in the presence or absence of FSH and (Bu)2cAMP. The cells were lysed, and proteins were subjected to polyacrylamide gel electrophoresis, followed by immunoblotting using antibodies specific to bovine adrenocortical P-450scc, ISP, and Red. A time-dependent increase was observed in the specific contents of these three components of SCC, but not of the reference mitochondrial protein, F1-ATPase, upon treatment with FSH or (Bu)2cAMP. The increase in the content of these three enzymes was accompanied by a rise in progesterone and 20 alpha-hydroxyprogesterone production. The synthesis of P-450scc, ISP, and Red increased 3- to 4-fold with time upon FSH or (Bu)2cAMP treatment respectively, as evidenced by pulse labeling of the cell proteins with [35S]methionine, followed by immunoprecipitation. Immunoprecipitation of P-450scc and ISP from an in vitro translation system programmed by RNA isolated from cultured cells revealed that treatment with FSH or (Bu)2cAMP resulted in an increase in the levels of translatable mRNA specific for these proteins, and that the initial products of translation were precursor forms of cytochrome P-450scc and ISP, similar to those observed in bovine adrenal and granulosa cells. It is concluded that in cultured rat ovarian granulosa cells, FSH induces the synthesis of cytochrome P-450scc, ISP, and Red by increasing the content of translatable mRNA coding for the precursor forms of these enzymes and that this action is mediated by cAMP. Furthermore, the effects of FSH and (Bu)2cAMP provide an explanation for the action of these compounds to stimulate progestin synthesis in cultured ovarian cells.     

Inhibition of iron-sulfur protein-mediated reduction of cytochrome P-450SCC by specific antibodies

The mechanism of inhibition of cholesterol side-chain cleavage by specific antibodies was studied systematically. The antibodies had no effect on substrate binding as determined by optical spectroscopy or on the heme environment of the cytochrome P-450 insofar as was detectable by electron paramagnetic resonance spectroscopy. They did not bind to either iron-sulfur protein or its reductase. The antibodies had no effect on chemical reduction of the P-450 or on P-450-CO complex formation. They did inhibit the NADPH-dependent reduction of P-450 and subsequent formation of the P-450-CO complex. This inhibitory effect was concentration dependent and was correlated with the inhibitory effect of the antibodies on enzymatic cholesterol side-chain cleavage. Similar results were obtained using Fab fragments. These results indicate that the antibodies inhibit side-chain cleavage by binding to a region close to the iron-sulfur protein-binding site, thereby preventing transfer of reducing electrons to the cytochrome P-450.     

Regulation of cytochrome P-450scc in immature porcine granulosa cells by FSH and estradiol

FSH, estradiol, or a combination of FSH and estradiol enhanced secretion of progesterone by primary cultures of immature porcine granulosa cells; they also increased the mitochondrial content of cytochrome P-450 and mitochondrial cholesterol side-chain cleavage activity. FSH or estradiol alone increased mitochondrial cytochrome P-450 levels and cholesterol side-chain cleavage activity to a similar extent. The combination of FSH and estradiol further increased cytochrome P-450 and side-chain cleavage activity. Our results demonstrate that FSH, estrogen and the combination of the hormones stimulate progestin synthesis by granulosa cells, at least in part, by increasing cholesterol side-chain cleavage enzyme levels. These observations suggest a dual hormonal regulation of the enzyme catalyzing the rate-limiting step in the biosynthetic pathway of steroid hormones.     

Effects of epidermal growth factor on the synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells in primary culture

The effect of epidermal growth factor (EGF) on the synthesis of the components of the cholesterol side-chain cleavage enzyme complex (SCC) was studied in rat ovarian granulosa cells. The cells were cultured for 48 h in the presence or absence of EGF (15 ng/ml) and/or FSH (50 ng/ml) after which proteins were radiolabeled by incubation with [35S]methionine followed by immunoprecipitation of newly synthesized P-450scc or adrenodoxin (ISP) with polyclonal antibodies directed against the corresponding proteins from bovine adrenal cortex. In addition the action of EGF on the level of translatable RNA for P-450scc was evaluated using a cell-free translation system programmed with RNA isolated from treated and untreated cells, followed by immunoisolation of newly synthesized proteins. Immunoisolated proteins were separated by polyacrylamide-gel electrophoresis, visualized by fluorography and quantified by densitometry. EGF stimulated progesterone formation by the cells 3-fold and potentiated the FSH-induced stimulation of progesterone formation, but had no effect on cAMP accumulation. EGF also stimulated the synthesis of P-450scc and ISP, and enhanced the FSH-induced synthesis of P-450scc and ISP in a concentration-dependent fashion with a maximal stimulation attained at concentrations ranging from 1.0 to 100 ng/ml. No appreciable changes in the induction pattern were observed when EGF and dibutyryl cyclic AMP (Bt2cAMP) were added together, as compared to when Bt2cAMP was added alone. Neither treatment affected the synthesis of the constitutive mitochondrial enzyme, F1-ATPase. Immunoisolation of P-450scc from the proteins synthesized in a rabbit reticulocyte in vitro translation system programmed with RNA isolated from EGF- and/or FSH-treated cells, revealed that EGF enhanced the FSH-stimulated synthesis of the precursor form of P-450scc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase stimulation of steroidogenesis in rat luteal cell mitochondria

The regulatory role of cAMP-dependent protein kinase in steroidogenesis was examined in luteal cell mitochondria prepared from heavily luteinized prepubertal rat ovaries. The cAMP-dependent protein kinase, localized in luteal mitochondria, comprised 5.5% of the total cellular protein kinase activity (cAMP-dependent). Intact mitochondria supported by a suitable electron-donor substrate and inhibited by isoxazole converted cholesterol to a single steroid product, pregnenolone. Neither (Bu)2 cAMP nor a crude preparation of cytosolic protein kinase stimulated pregnenolone production from cholesterol when added to intact luteal cell mitochondria; however, mitochondria treated with 10 mM CaCl2 became responsive to both (Bu)2 cAMP and protein kinase by showing increased pregnenolone production. Likewise, the addition of cytosol protein kinase to incubations of cholesterol and crude cholesterol sidechain cleavage enzyme (cytochrome P-450cscc) isolated from luteal mitochondria, also stimulated pregnenolone production. Cholesterol-poor mitochondria, depleted of endogenous sterol by prolonged preincubation, when subsequently incubated with Ca+2 plus (Bu)2 cAMP and protein kinase showed significantly increased pregnenolone production. Conversely, mitochondria with greatly increased intramitochondrial cholesterol after preincubation with 200 microM cholesterol and a cytochrome P-450cscc inhibitor (aminoglutethimide) synthesized pregnenolone in significantly higher amounts than either normal or cholesterol-poor mitochondria after removal of the aminoglutethimide block. However, addition of (Bu)2cAMP or protein kinase to Ca+2-treated cholesterol-rich mitochondria failed to additionally stimulate pregnenolone synthesis. We conclude from these observations that the mitochondrial membrane normally excludes protein kinase and (Bu)2cAMP from any stimulatory action on cholesterol side-chain cleavage. Disruption of the mitochondrial membrane by high Ca+2 concentrations eliminates this barrier and permits (Bu)2cAMP and protein kinase stimulation of the CSCC enzyme system. The mechanism of stimulation is not clear. It could involve direct action on the CSCC enzyme. Alternatively, an increase in either intramitochondrial transport or binding of cholesterol substrate to the CSCC enzyme could be facilitated by protein kinase action. Direct stimulation of the enzyme by protein kinase seems less likely, since increased enzyme activity was not observed in the presence of high concentrations of intramitochondrial cholesterol substrate.     

Regulation of cholesterol metabolism in rat adrenal mitochondria.

Addition of cholesterol to rat adrenal mitochondria resulted in a stimulation of pregnenolone synthesis. The slow step of the mitochondrial cholesterol side-chain cleavage reaction could be the interaction of the sterol with cytochrome P-450. The rate of cholesterol binding to this enzyme as observed spectroscopically correlated with the equilibration period (20 min) of the mitochondria and exogenous cholesterol required for maximal rates of pregnenolone synthesis. It is suggested that translocation of cholesterol between different sterol pools occurs within the mitochondria. Potential intracellular effectors that could be of importance in the movement or regulation of mitochondrial cholesterol include bivalent metallic ions, prostaglandins, cyclic nucleotides, polyamines and polylysine. Of the effectors studied, only calcium ions and polylysine markedly stimulated pregnenolone synthesis. These effectors might stimulate steroidogenesis by lateral displacement of cholesterol in the mitochondrial membrane into a compartment easily accessible to the cholesterol side-chain cleavage enzyme complex.     

Effects of 8-bromo-cAMP on expression of endocrine functions by cultured human trophoblast cells. Regulation of specific mRNAs

There is little information on the molecular events underlying the effects of cAMP on human chorionic gonadotropin (hCG) and particularly steroidal hormone production in normal trophoblasts. We examined the effects of 8-bromo-cAMP on mRNAs encoding two components of the cholesterol side-chain cleavage system, cytochrome P-450scc and adrenodoxin, and the alpha- and beta-subunits of hCG in cultured cytotrophoblasts. cAMP caused an increase in all of these mRNAs within 24 h, whereas actin mRNA declined. alpha-hCG mRNA increased first, followed by adrenodoxin, beta-hCG and cytochrome P-450scc mRNAs. The effects of 8-bromo-cAMP on alpha- and beta-hCG, adrenodoxin, and cytochrome P-450scc mRNAs, in cytotrophoblasts and JEG-3 choriocarcinoma cells, required the catalytic unit of protein kinases since H-7, a kinase inhibitor, blocked the increase in the mRNAs and prevented the stimulation of hCG and progesterone secretion. 8-Bromo-cAMP promoted a rapid increase in alpha-hCG mRNA in cytotrophoblasts in the presence of cycloheximide, an inhibitor of protein synthesis. In cytotrophoblasts, cycloheximide reduced basal and 8-bromo-cAMP-stimulated adrenodoxin mRNA abundance. In contrast, basal and cAMP-stimulated adrenodoxin mRNA was augmented by cycloheximide in JEG-3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biosynthesis of cholesterol side-chain cleavage cytochrome P-450 in the rabbit corpus luteum depends upon estrogen

To gain a better understanding of the luteotropic action of estrogen, we have investigated the effect of estrogen on the synthesis of the enzyme, cholesterol side-chain cleavage cytochrome P-450 (P-450scc) in the rabbit corpus luteum. Using an established protocol, rabbits were treated with estradiol, and the estradiol was then withdrawn on day 9 of pseudopregnancy, which caused an 88% fall in serum progesterone within 48 h. In other rabbits, estradiol was replaced at 48 h which stimulated a 6.6-fold increase in serum progesterone concentration within the next 24 h. Luteal tissues were incubated with [35S]methionine and homogenized, and a mitochondrial fraction lysate was obtained. Equal trichloroacetic acid-precipitable radioactivity was taken for immunoprecipitation using a well-characterized polyclonal antiserum against bovine adrenal P-450scc. The immunoisolated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radioactivity was visualized by autofluorography. The results indicate that the rate of synthesis of P-450scc in 48 h-estradiol withdrawn animals was markedly reduced, and by 72 h of withdrawal was barely detectable. When estradiol was reintroduced, the synthesis of P-450scc was increased. Despite the prominent changes in P-450scc synthesis, immunoblotting revealed only a minimal (approximately 30%) decrease in relative P-450scc content by 72 h after estradiol withdrawal. Analyses of DNA and protein contents of luteal tissues revealed an increase in DNA per mg luteal tissue, a decline in total tissue protein/DNA ratio, but no change in mitochondrial fraction protein/DNA ratio after estrogen withdrawal. The results indicate that de novo synthesis of P-450scc in the corpus luteum is sensitive to estrogen; however, the estrogen-sensitive rate-limiting step(s) for steroidogenesis are at other sites in the steroid biosynthetic pathway.     

Simultaneous transfection of COS-1 cells with mitochondrial and microsomal steroid hydroxylases: incorporation of a steroidogenic pathway into nonsteroidogenic cells.

Transfected, nonsteroidogenic COS-1 cells derived from monkey kidney are found to be capable of supporting the initial and rate-limiting step common to all steroidogenic pathways, the side-chain cleavage of cholesterol to produce pregnenolone. Endogenous COS-1 kidney cell renodoxin reductase and renodoxin are able to sustain low levels of this activity catalyzed by bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) whose synthesis is directed by a transfected plasmid containing P450scc cDNA. Double transfection with both P450scc and adrenodoxin plasmids leads to greater pregnenolone production and indicates that adrenodoxin plays a role as a substrate for this reaction or that bovine adrenodoxin serves as a better electron donor than the endogenous iron-sulfur protein renodoxin. Also it is found that both the bovine adrenodoxin and P450scc precursor proteins are proteolytically processed upon their uptake by COS-1 cell mitochondria to forms having the same electrophoretic mobility as mature bovine adrenodoxin and P450scc. Following triple transfection of COS-1 cells with P450scc, adrenodoxin, and 17 alpha-hydroxylase cytochrome P450 plasmids, pregnenolone produced in mitochondria by the side-chain cleavage reaction can be further metabolized in the endoplasmic reticulum to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Although this functional steroidogenic pathway can be incorporated into this nonsteroidogenic cell type, it is found to be nonresponsive to cAMP, a potent activator of steroid hormone biosynthesis in adrenal cortex, testis, and ovary. Thus the cellular mechanisms necessary to support both microsomal and mitochondrial steroid hydroxylase activities appear not to be tissue specific, whereas the acute cAMP-dependent regulation of steroidogenesis is not present in transformed kidney (COS-1) cells.     

Polypeptide activators of cholesterol side-chain cleavage

A rate-determining step in the cAMP-dependent action of ACTH on adrenal steroid biosynthesis is the interaction of cholesterol substrate with the cholesterol side-chain cleavage cytochrome P-450 in the mitochondrion. This interaction is rapidly and reversibly sensitive to inhibitors of protein synthesis. For this reason a hormone-dependent, labile protein activator of cholesterol side-chain cleavage has long been postulated as an obligatory intermediate in the tropic regulation of this reaction. Applying recent advances in liquid chromatography, two-dimensional gel electrophoresis, and enzyme reconstitution into liposomes, several laboratories have now reported the isolation and partial characterization of polypeptide candidates for the status of "labile protein."     

Regulation of cholesterol side-chain cleavage and 17 alpha-hydroxylase/lyase activities in proliferating human theca interna cells in long term monolayer culture

In this report we describe the development and characterization of a long term culture system to study regulation of the expression of 17 alpha-hydroxylase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase in human theca interna cells. Conditions have been established for the dispersal, growth, freezing, and storage of functional human theca interna cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Theca interna cells grown under these conditions have a doubling rate of 28-32 h and are morphologically distinct from human granulosa cells grown under the same conditions. Theca interna cells were grown, passed for successive passages, and transferred into serum-free medium containing forskolin, hCG, LH, or cAMP analogs. There was a time- and dose-dependent increase in 17 alpha-hydroxylase activity and progesterone synthesis from endogenous precursors. Added pregnenolone was converted to 17 alpha-hydroxypregnenolone, which was further converted primarily to dehydroepiandrosterone and, to a much lesser extent, androstenedione. Progesterone was converted to 17 alpha-hydroxyprogesterone and 16 alpha-hydroxyprogesterone. In studies using 17 alpha-hydroxyprogesterone as substrate, no metabolism to androstenedione or any other product was detectable. Similarly, 4-pregnen-20 alpha-ol-one (20 alpha-dihydroprogesterone) was not metabolized to any detectable products. Northern analysis performed on total RNA obtained from forskolin-stimulated theca interna cultures verified that the increase in 17 alpha-hydroxylase activity was associated with a corresponding increase in levels of mRNA specific for 17 alpha-hydroxylase cytochrome P-450. Message levels for cholesterol side-chain cleavage P-450 were similarly increased in cells treated with forskolin. No detectable mRNA encoding aromatase cytochrome P-450 was discerned. This procedure for the preparation and study of proliferating human theca internal cells provides an opportunity to study regulation of the expression of steroidogenic enzymes and other cellular processes unique to human ovarian cells.     

Regulation of the cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin mRNAs in cultured choriocarcinoma cells.

Cytochrome P-450scc (P-450scc) catalyzes the cholesterol side-chain cleavage reaction, a rate-limiting enzymatic step for progesterone synthesis in trophoblastic and other steroidogenic cells. Adrenodoxin is the iron/sulfur protein donating electrons to P-450scc during this reaction. We examined the effects of cholera toxin (CT), an activator of adenylate cyclase, and 12-O-tetradecanoylphorbol acetate (TPA), a phorbol ester protein kinase C activator, on the levels of mRNAs encoding P-450scc and adrenodoxin in JEG-3 choriocarcinoma cells. CT induced in a concentration- and time-dependent manner P-450scc and adrenodoxin mRNA levels to 8-fold and 1.5-fold above that of control, respectively. TPA also increased P-450scc and adrenodoxin mRNA levels about 3-fold and 1.5-fold above that of control, respectively. Epidermal growth factor (EGF) was found to weakly induce P-450scc mRNA accumulation with a maximal 20% stimulation above basal levels. The effects of CT and TPA were apparently additive on both mRNAs. The protein synthesis inhibitor cycloheximide diminished basal, CT-, TPA-, and EGF-stimulated P-450scc mRNA accumulation whereas the opposite was observed for the adrenodoxin mRNA. Insulin-like growth factor I (IGF-I) appeared to have no effect on either mRNA. These data indicate that: (1) the accumulation of P-450scc and adrenodoxin mRNAs is mainly controlled by the cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway but their stimulation by TPA- and EGF-induced signals may also play a weaker synergistic role; (2) the protein synthesis inhibitor cycloheximide inhibits basal, CT-, TPA- and EGF-stimulated P-450scc mRNA levels while it increases the expression of adrenodoxin mRNA suggesting that in the malignant trophoblasts these two enzyme mRNAs are differentially controlled.     

Cytochromes P-450scc, P-450(17)alpha, adrenodoxin, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase in bovine follicles and corpora lutea. Changes in specific contents during the ovarian cycle

To investigate the basis for the pattern of ovarian steroid production during the bovine estrous cycle, the tissue concentrations of major steroidogenic enzymes, 17 alpha-hydroxylase cytochrome P-450 and cholesterol side-chain cleavage cytochrome P-450 (cytochrome P-450scc), and their respective electron donors, NADPH-cytochrome P-450 reductase and adrenodoxin, were estimated and compared with those of the nonsteroidogenic enzymes, cytochrome c oxidase and F1-ATPase. The levels of these enzymes were estimated in medium sized (9-11 mm) and large (14-18 mm) follicles after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid late-mid, and late stages of the luteal phase (n = 5 per group). The specific contents of all enzymes and electron donors were determined by immunoblot analysis, except for cytochrome c oxidase, which was quantified by determination of specific activity. The specific (per microgram of tissue homogenate protein) and total (per follicle or corpus luteum) tissue contents of 17 alpha-hydroxylase cytochrome P-450 increased 0.5- and 5-fold respectively from medium sized to large follicles, but then decreased to undetectable levels in corpora lutea in the early luteal phase, and remained undetectable throughout the luteal phase. In contrast, the specific content of NADPH-cytochrome P-450 reductase was similar between follicles and corpora lutea. The specific contents of cytochrome P-450scc, adrenodoxin and cytochrome c oxidase in follicles were similar to those of corpora lutea of the early luteal phase. However, by the early-mid luteal phase the specific contents of luteal cytochrome P-450scc (490 +/- 46 vs. 5709 +/- 982 cpm/micrograms protein) and adrenodoxin (44 +/- 15 vs. 705 +/- 229 cpm/micrograms protein) were increased, by 12- and 15-fold, respectively (P less than 0.05). In contrast, cytochrome c oxidase activity (29.1 +/- 10.1 vs. 108.6 +/- 20.7 nmol/mg tissue protein X min) and the specific content of F1-ATPase increased only 3- to 4-fold reflective of an increase in numbers of mitochondria. The levels of these enzymes remained elevated until the late luteal phase when they declined markedly. It is concluded that the induction of synthesis of P-450scc and adrenodoxin after ovulation is specific and does not merely reflect biogenesis of mitochondria during luteinization. Moreover the changes in the types of steroids produced by the ovarian compartments throughout the estrous cycle are a reflection of changes in the tissue content of steroidogenic enzymes.     

Microanalysis of hormone responsive ovarian interstitial gland cells in miniature culture

Small cell aggregates of functional interstitial tissue were isolated from ovaries of 21- to 24-day-old rats, and their biochemical properties were studied in miniature cultures of 500-4000 cells. The isolated interstitial cells expressed high amounts of the mitochondrial cholesterol side-chain cleavage cytochrome P-450, visualized by immunofluorescent staining. Freshly isolated cells also expressed high activities of 17 alpha-hydroxylase and 17:20-lyase, which were assayed by TLC analysis of [3H]progesterone metabolites. The TLC technique revealed the immediate conversion of progesterone to 5 alpha-reduced progestins. Consequently, no aromatizable androgens were produced but, accumulation of androsterone resulted instead from the direct action of 17 alpha-hydroxylase on 3 alpha-hydroxy-5 alpha-pregnane-20-one (pregnanolone). Both the immunoreactive levels of cholesterol side-chain cleavage cytochrome P-450 and the rate of the 17:20-lyase activity declined rapidly during culture. However, addition of LH to the medium restored both enzymes, indicating the presence of functional LH receptors. The latter were also demonstrable by their ability to evoke cAMP formation in response to LH, but not to FSH. The activity of 5 alpha-reductase in the interstitial cells was much higher (3-fold) than its activity in the granulosa cells. Unlike 17:20 lyase, the activity of 5 alpha-reductase did not decay in culture, nor was it affected by LH. We thus established a novel and sensitive experimental method to isolate and study a minute population of ovarian cells which, unlike the follicular granulosa and theca cells, are enigmatically differentiated at the early stages of ovarian development.     

Estradiol regulation of sterol carrier protein-2 independent of cytochrome P450 side-chain cleavage expression in the rat corpus luteum

A major action of estradiol in the corpus luteum of the pregnant rat is to increase the supply of cholesterol substrate for progesterone production by stimulating both cholesterol synthesis and uptake. To determine whether this steroid also affects cholesterol metabolism and transport, estradiol's action on the expression of cytochrome P450 side-chain cleavage enzyme (P450scc) and the cholesterol transport protein, sterol carrier protein-2 (SCP2), was examined. Mitochondria isolated from corpora lutea of estradiol-treated rats secreted significantly more progestagen than mitochondria of control corpora lutea. Several findings indicate that estradiol enhances cholesterol transport and availability to the P450scc rather than affects the expression of this enzyme: 1) the difference in mitochondrial progestagen synthesis induced by estradiol was obliterated by the presence of 25-hydroxycholesterol; 2) immunoblotting of P450scc indicated no stimulatory effect of estradiol on the amount of enzyme; and 3) levels of P450scc mRNA were not increased by estradiol. Whereas estradiol had no stimulatory effect on P450scc it caused a mark (3-fold) increase in the mitochondrial content of SCP2. Thus, the increase in luteal progestagen synthesis stimulated by estradiol appears to be associated with an increase in mitochondrial SCP2 and is independent of luteal P450 content or message.     

Cell-specific expression of immunoreactive cholesterol side-chain cleavage cytochrome P-450 during follicular development in the rat ovary

Using a specific antiserum against rat cholesterol side-chain cleavage cytochrome P-450 (P-450scc), we examined the expression of this key steroidogenic enzyme during follicular development in PMSG-treated immature rats. The accumulation of the enzyme was monitored in ovary homogenates by quantitative immunodot blot assay, while expression of P-450scc in various cell types was visualized concomitantly by immunofluorescent staining of ovarian cryosections. Before PMSG treatment, no labeling of P-450scc could be observed in follicular granulosa cells. In contrast, steroidogenic cytochrome was markedly expressed in interstitial cells, part of theca interna cells, and hypertrophied theca of atretic follicles. As a result of PMSG treatment, the interstitial thecal cells promptly enriched their P-450scc content within 24 h, whereas the granulosa cells acquired the enzyme at a later time, between 30 and 48 h after hormone administration. After ovulation, many corpora lutea filled most of the ovarian volume, and the ovarian content of P-450scc was 47 times higher than that in control ovaries of untreated rats. In granulosa cell population of a single preovulatory follicle, a downward gradient of P-450scc expression was observed, starting high in the cells abutting the basal lamina and decreasing toward the cells lining the antrum. Cumulus cells failed to express P-450scc. Referring to the basal lamina, theca interna cells exhibited a reverse gradient of P-450scc expression, starting high in peripheral cells close to the theca externa layer and decreasing in cells located near the follicular basement membrane. Immunofluorescent labeling revealed a major difference between P-450scc expression in thecal cells compared to that in granulosa cells. While expression of P-450scc in granulosa cells was restricted exclusively to cells within preovulatory follicles, P-450scc labeling was observed throughout the ovary in thecal and interstitial cells associated with follicles at any phase of follicular maturation. Therefore, it may be proposed that the thecal and interstitial cells represent an all ovarian network which expresses its steroidogenic capacity at early stages of follicular maturation and thereby is able to supply androgens necessary for the follicular development.     

Developmental and hormonal regulation of cholesterol side chain cleavage cytochrome P-450 in the fetal rabbit testis

Male sexual differentiation is dependent upon the induction of testosterone synthesis by the fetal testis at a critical phase of development. In the rabbit, testosterone synthesis by the fetal testis is initiated after 17.5-18 days of gestation, reaches peak values by day 21 and subsequently declines. In the present study, we analyzed the specific activity and concentration of immunoreactive cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) in the fetal rabbit testis during development to assess its possible role as a key regulatory enzyme in fetal testicular steroidogenesis. The effects of human chorionic gonadotropin (hCG) and dibutyryl cyclic AMP on the specific activity and synthesis of cytochrome P-450scc in fetal rabbit testes in vitro also were evaluated. We observed that changes in cholesterol side chain cleavage activity paralleled the induction of testosterone synthesis; the specific activity of this enzyme which was approximately equal to 0.25 pmol min-1 mg-1 protein in testes from 19-day fetal rabbits was increased approximately equal to 10-fold in testes of 21-day fetuses and thereafter declined dramatically. Immunoreactive cytochrome P-450scc, which was first detectable in gonads of 19-day fetal rabbits, was induced markedly in 21-day fetal testes, reached maximum levels on day 24 and declined slightly thereafter. Incubation of testes from 19- and 21-day gestational age fetal rabbits with hCG or dibutyryl cyclic AMP for 24 h resulted in an induction of testosterone synthesis, cholesterol side chain cleavage activity and synthesis of cytochrome P-450scc. These findings are suggestive that androgen synthesis by the fetal Leydig cell is mediated by an induction of the synthesis and specific activity of cytochrome P-450scc. In addition, these data support the hypothesis that the developmental changes in the synthesis of cytochrome P-450scc are regulated by fetal gonadotropin and are mediated by cyclic AMP.     

Proximity of the substrate binding site and the heme-iron catalytic site in cytochrome P-450scc.

As an approach to "mapping" the active site of the cytochrome P-450 that catalyzes cholesterol side-chain cleavage, designated cytochrome P-450scc, we have synthesized steroid derivatives with the potential to interact with both the substrate binding site and the heme-iron catalytic site of the enzyme. The effects of these substrate analogs were studied with cytochrome P-450scc purified from bovine adrenal cortex. One derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, was found to be a potent inhibitor of pregnenolone formation in a reconstituted enzyme system, and a kinetic analysis of the inhibition showed that binding of the derivative is competitive with respect to cholesterol. The spectral properties of a stable complex formed between the steroidal amine and the purified cytochrome suggest that the 22-amine group coordinates directly to the heme-iron. A model for the structure of this inhibitor-enzyme complex is proposed in which the 5-androstene ring system of the steroid occupies the substrate binding site, and the amine group of the side chain occupies an axial coordination position of the Fe(III) center. This places limits on the distance between these two domains in the enzyme and offers support for proposed mechanisms of cytochrome P-450-catalyzed oxygen-insertion reactions in which an iron-bound oxidant directly attacks the substrate.     

2-hydroxyestradiol enhanced progesterone production by porcine granulosa cells: dependence on de novo cholesterol 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.     

Glucocorticoid and cyclic adenosine 3'5'-monophosphate-mediated induction of cholesterol side-chain cleavage cytochrome P450 (P450scc) in MA-10 tumor Leydig cells. Increases in mRNA are cycloheximide sensitive

The regulation of cholesterol side-chain cleavage enzyme (P450scc) was investigated in MA-10 tumor Leydig cells. We recently demonstrated that the constitutive and cAMP-stimulated expression of P450scc in normal mouse Leydig cells is negatively regulated by glucocorticoids. We now report that glucocorticoids have the opposite effect in MA-10 cells causing a 1.7-fold increase in the rate of P450scc synthesis and a 2.1-fold increase in the amount of P450scc mRNA. Treatment of MA-10 cells with 10 microM 8-bromo-cAMP (8-Br-cAMP) (cAMP) resulted in a 1.7-fold increase in P450scc synthesis and a 3-fold increase in P450scc mRNA. Combined treatment with dexamethasone and cAMP resulted in additive increases in synthesis (2.8-fold) and mRNA (5.3-fold). Increases in de novo synthesis and mRNA levels were reflected by modest increases in the amount of immunoreactive P450scc enzyme protein. Dexamethasone-mediated stimulation in synthesis and accumulation of P450scc mRNA were blocked by the antiglucocorticoid RU-486. Cycloheximide blocked both cAMP- and dexamethasone-induced increases but had no effect on constitutive levels of P450scc mRNA. Treatment of MA-10 cells with 10 microM 8-Br-cAMP had no effect on cell morphology and stimulated progesterone accumulation to a minor degree. Treatment of MA-10 cells with 1 mM 8-Br-cAMP resulted in cell rounding and loss of cells from culture dishes. The results of this study demonstrate that: 1) dexamethasone increases P450scc de novo synthesis and mRNA levels in MA-10 tumor Leydig cells, opposite to the effect in normal Leydig cells; 2) dexamethasone- and cAMP-stimulated increases occur via distinct mechanisms; 3) and synthesis of protein factor(s) is required to mediate the action of both dexamethasone and cAMP.