Cytochrome P450 Side-chain Cleavage Enzyme Research Articles

Estrogen receptor-beta expression in relation to the expression of luteinizing hormone receptor and cytochrome P450 enzymes in rat ovarian follicles.

Changes in mRNA expression for estrogen receptor (ER beta) in relation to mRNAs for LH receptor (LHr) and cytochrome P450 enzymes were examined in granulosa and theca cells from proestrous rat ovarian follicles. Of the 30 ovaries harvested from 15 adult rats, 24 were processed for in situ hybridization, and the remaining were used for reverse transcription-polymerase chain reaction. Messenger RNAs for ER beta, LHr, cytochrome P450 side-chain cleavage enzyme (P450(scc)), 17 alpha-hydroxylase (P450(c17)), aromatase (P450(arom)), and steroidogenic acute regulatory protein (StAR) were localized in cross sections of ovaries by in situ hybridization and quantified in granulosa and theca cell layers by a computer-image analyzing system. Ovarian follicles were classified as healthy or atretic. Healthy follicles were divided into four size groups: very small (40-100 microm), small (101-275 microm), medium (276-450 microm), and large (451-850 microm). Atretic follicles were divided into medium (276-450 microm) or large follicles (451-850 microm). A low level of ER beta mRNA expression was first detected in granulosa cells of very small healthy follicles, and the expression increased progressively up to medium-sized follicles. The expression of ER beta mRNA was highest (P < 0.01) in medium-sized follicles that was followed by a decrease (P < 0.01) in large follicles. Messenger RNAs for LHr, P450(scc), and P450(arom) were first detected in granulosa cells of medium-sized healthy follicles, while mRNAs for LHr, P450(scc), P450(c17), and StAR were first detected in theca cells associated with very small follicles. The highest expression of LHr, P450(scc), P450(c17), P450(arom), and StAR was seen in granulosa and/or theca cells of large healthy follicles. In atretic follicles, level of gene expression was relatively low in both granulosa and theca cells. In conclusion, stage-specific expression of ER beta mRNA was observed in granulosa cells during follicular development. The increased expression of ER beta and a concomitant initiation of LHr, P450(scc), and P450(arom) expression in granulosa cells of medium follicles may signify a role for estrogen in follicular development. Also, a strong correlation between ER beta mRNA expression in granulosa cells, and the expression of mRNAs for LHr, P450(scc), P450(c17), and StAR in theca cells associated with growing follicles suggests a possible role for estrogen in steroidogenesis.

Steroidogenic factor-1 influences protein-deoxyribonucleic acid interactions within the cyclic adenosine 3,5-monophosphate-responsive regions of the murine steroidogenic acute regulatory protein gene.

De novo synthesis of the steroidogenic acute regulatory protein (StAR) in response to trophic hormonal stimulation of steroidogenic cells is required for the delivery of cholesterol from the mitochondrial outer membrane to the mitochondrial inner membrane and the cytochrome P450 side-chain cleavage enzyme. StAR expression is transcriptionally regulated by cAMP-mediated mechanisms, and we have identified a 45-bp region within the mouse promoter that is important for cAMP responsiveness of the gene. This region, located between -105 and -60 of the start site of transcription, contains a SF-1-binding site, a highly conserved C/EBPbeta-AP-1-nuclear receptor half-site sequences (CAN region), and a GATA-4-binding site. The SF-1 element and CAN region are required for full basal activity, whereas the GATA-4 element may account for 20% of the cAMP response in MA-10 mouse Leydig tumor cells. A cAMP-dependent protein-DNA complex was observed with the CAN region and mutation of a nonconsensus AP-1 site within this region greatly diminished promoter strength. Complex protein-DNA interactions within the cAMP response region (-105/-60) were shown to require the SF-1 element (-95), suggesting that SF-1 is required for protein-DNA interaction at the CAN (-79) region and maximal activity of the promoter.

Mechanisms underlying the steroidogenic synergy of insulin and luteinizing hormone in porcine granulosa cells: joint amplification of pivotal sterol-regulatory genes encoding the low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory (StAR) protein and cytochrome P450 side-chain cleavage (P450scc) enzyme

Growth of ovarian Graafian follicles and cytodifferentiation of granulosa and theca cells are regulated by gonadotropins, sex steroids and peptidyl growth factors. For example insulin and intraovarian insulin-like growth factor type I (IGF-I) may amplify the actions of both follicle stimulating hormone (FSH) and luteinizing hormone (LH) in promoting biochemical luteinization and enhancing steroidogenesis. To explore further the notion of interactions between insulinomimetic peptides and LH and to examine the associated mechanisms, we have established porcine granulosa cells in monolayer culture for 48 h in 3% serum with insulin (1 μg/ml), estradiol (0.5 μg/ml), and follicle stimulating hormone (FSH, 5 ng/ml) to allow cell anchorage, facilitate in vitro cytodifferentiation and confer LH responsiveness. To limit any carry-over effects of serum, granulosa cells were stabilized overnight in serum-free medium. Studies were then initiated to assess the impact of insulin on the dose-responsive actions of LH. A maximally effective concentration of insulin (1 μg/ml) synergistically augmented LH’s dose-dependent ampilification of progesterone and cAMP accumulation; viz. by ≈twofold (progesterone) and ≈2.5-fold (cAMP) above that observed in maximally LH-stimulated cultures ( P<0.001). Mechanistically, insulin significantly enhanced the sensitivity of granulosa cells to LH’s drive of cAMP accumulation [ED 50 for LH 61±14 ng/ml (control) vs. 10±1.0 ng/ml (insulin) ( P<0.01)]. Insulin also augmented the maximal stimulatory effect of LH; i.e. LH efficacy rose from 6.5±0.4 to 17±1.4 (pmole cAMP/μg DNA/48 h; P<0.001). Insulin dose-response analysis showed that insulin alone minimally elevated basal, but significantly heightened LH's stimulation of progesterone and cAMP accumulation at (insulin) concentrations as low as 3–10 ng/ml. The molecular mechanisms underlying insulin and LH’s synergy were assessed by RNase protection assays with (porcine) cRNA probes encoding the low density lipoprotein receptor (LDL-R), Steroidogenic Acute Regulatory Protein (StAR), P450 cholesterol sidechain cleavage enzyme (P450scc) and (as a possible negative control) Sterol Carrier Protein 2 (SCP-2) [data normalized to constitutive 18S rRNA]. Non linear least-squares analysis was applied to confirm or refute an hypothesis of interactive synergy between LH and insulin on gene expression. LH and insulin alone exerted no effect on StAR message accumulation, and LH alone minimally stimulated P450scc and LDL-R mRNA’s accumulation at 48 h. In contrast, insulin in combination with LH augmented StAR mRNA concentrations by ≈5–10-fold and stimulated LDL-R message levels by threefold above the respective maximally LH-driven values ( P<0.01). Maximal P450scc mRNA expression was enhanced twofold by cotreatment with LH and insulin compared with maximal LH-treated cultures. In contrast SCP-2 mRNA accumulation remained unaffected by any treatment. In summary, we have used a serum-free, in vitro differentiated porcine granulosa cell culture system to assess regulatory interactions between the disparate first messengers, LH and insulin. We observe marked LH-insulin steroidogenic synergy after 48 h of joint hormonal stimulation, and further clarify that the mechanism(s) of synergy include augmentation of cAMP production and increased steady-state concentrations of transcripts of key sterol-regulatory genes; namely, LDL-R, StAR, and P450scc, but not SCP-2. Since the encoded products of these genes variously control sterol substrate uptake, delivery to and utilization in mitochondrial steroidogenesis, we speculate that the concerted actions of insulin-like peptides and LH may contribute to steroidogenic differentiation during the later stages of follicular maturation and the granulosa-luteal cell transition.

Pregnenolone, pregnenolone sulfate, and cytochrome P450 side-chain cleavage enzyme in the amphibian brain and their seasonal changes.

To clarify whether the amphibian brain synthesizes de novo neurosteroids, we examined pregnenolone, pregnenolone sulfate ester, and cytochrome P450 side-chain cleavage enzyme (cytochrome P450scc), an enzyme converting cholesterol to pregnenolone, using amphibians. Pregnenolone and its sulfate ester in the brain, gonad, and plasma of Xenopus laevis were measured by a specific pregnenolone RIA. The concentrations of these two steroids in the female brain were significantly larger than those in the ovary and plasma. A similar tendency was evident in the male. In both sexes, pregnenolone and its sulfate ester were concentrated more highly in the cerebellum than in the telencephalon, diencephalon, or midbrain. An immunoreactive protein band of electrophoretic mobility in the proximity of bovine adrenal P450scc was detected in the Xenopus brain as well as the testis by Western blot analysis. Immunohistochemical analysis indicated that Purkinje cells in the Xenopus cerebellum were specifically immunostained with the P450scc antibody. P450scc-like immunoreactive cells were further found in several telencephalic and diencephalic regions, such as the pallium mediale and nucleus preopticus, in the Xenopus brain. A similar localization of P450scc-like immunoreactive cells was evident in Rana nigromaculata, a seasonally breeding amphibian. In the present study, seasonal changes in pregnenolone and its sulfate ester were further examined as a possible physiological change using R. nigromaculata. In both sexes, pregnenolone concentrations in the brain were almost constant during the seasonally breeding cycle. In contrast, the pregnenolone sulfate concentration in the brain was significantly lower in the hibernating quiescent phase and higher in the breeding and postbreeding active phases, independent of the plasma steroid level. These results taken together suggest that the amphibian brain possesses steroidogenic enzyme P450scc and produces pregnenolone and its sulfate ester. Pregnenolone sulfate may function well during the breeding and postbreeding active phases of the year in the seasonal breeder.

Pregnenolone, Pregnenolone Sulfate, and Cytochrome P450 Side-Chain Cleavage Enzyme in the Amphibian Brain and Their Seasonal Changes

To clarify whether the amphibian brain synthesizes de novo neurosteroids, we examined pregnenolone, pregnenolone sulfate ester, and cytochrome P450 side-chain cleavage enzyme (cytochrome P450scc), an enzyme converting cholesterol to pregnenolone, using amphibians. Pregnenolone and its sulfate ester in the brain, gonad, and plasma of Xenopus laevis were measured by a specific pregnenolone RIA. The concentrations of these two steroids in the female brain were significantly larger than those in the ovary and plasma. A similar tendency was evident in the male. In both sexes, pregnenolone and its sulfate ester were concentrated more highly in the cerebellum than in the telencephalon, diencephalon, or midbrain. An immunoreactive protein band of electrophoretic mobility in the proximity of bovine adrenal P450scc was detected in the Xenopus brain as well as the testis by Western blot analysis. Immunohistochemical analysis indicated that Purkinje cells in the Xenopus cerebellum were specifically immunostained with the P450scc antibody. P450scc-like immunoreactive cells were further found in several telencephalic and diencephalic regions, such as the pallium mediale and nucleus preopticus, in the Xenopus brain. A similar localization of P450scc-like immunoreactive cells was evident in Rana nigromaculata, a seasonally breeding amphibian. In the present study, seasonal changes in pregnenolone and its sulfate ester were further examined as a possible physiological change using R. nigromaculata. In both sexes, pregnenolone concentrations in the brain were almost constant during the seasonally breeding cycle. In contrast, the pregnenolone sulfate concentration in the brain was significantly lower in the hibernating quiescent phase and higher in the breeding and postbreeding active phases, independent of the plasma steroid level. These results taken together suggest that the amphibian brain possesses steroidogenic enzyme P450scc and produces pregnenolone and its sulfate ester. Pregnenolone sulfate may function well during the breeding and postbreeding active phases of the year in the seasonal breeder.

Hormonal regulation of messenger ribonucleic acid expression for steroidogenic factor-1, steroidogenic acute regulatory protein, and cytochrome P450 side-chain cleavage in bovine luteal cells.

To examine hormonal regulation of genes pertinent to luteal steroidogenesis, bovine theca and granulosa cells derived from preovulatory follicles were cultured with various combinations of forskolin and insulin. On Day 8 of culture, progesterone production was measured, and mRNA levels of steroidogenic factor-1 (SF-1), cytochrome P450 side-chain cleavage enzyme (P450scc), and steroidogenic acute regulatory protein (StAR) were determined by means of semiquantitative reverse transcription-polymerase chain reaction. Notably, the combination of forskolin plus insulin stimulated progesterone production in luteinized theca cells. This was probably a result of a synergistic interaction between forskolin and insulin, observed on both StAR and P450scc mRNA levels. However, in luteinized granulosa cells (LGC), forskolin and insulin each independently were able to up-regulate the levels of P450scc and StAR mRNA levels, respectively. Moreover, insulin alone was sufficient to maintain the high steady-state levels of StAR mRNA in LGC. Both insulin and insulin-like growth factor I enhanced StAR gene expression in LGC. SF-1 was constitutively expressed in bovine luteal cells; its amounts did not vary between the two luteal cell types or with hormonal treatments. In summary, this study demonstrates a distinct, cell-type specific regulation of StAR and P450scc mRNA in the two bovine luteal cell types.

Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in the rat Purkinje neuron during neonatal life.

Recently, we demonstrated that cytochrome P450 side-chain cleavage enzyme (P450scc) occurs in the rat cerebellar Purkinje cell after differentiation and remains during neonatal development and into adulthood. 3Beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3betaHSD) is also an essential enzyme for progesterone biosynthesis not only in peripheral steroidogenic glands but also in the nervous system. In the present study, therefore, the expression of 3betaHSD in the rat cerebellum was investigated during neonatal development and in the adult. RT-PCR analysis showed that the expression of 3betaHSD messenger RNA (mRNA) in the cerebellum was higher at 7-14 days of age than at other times. Biochemical studies together with HPLC analysis revealed that cerebellar slices at 10 days of age converted pregnenolone to progesterone, suggesting enzymatic activity of 3betaHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3betaHSD. A specific RIA indicated that progesterone concentrations in the cerebellum were higher at 3 and 10 days of age than at 60 days of age. The progesterone level in the cerebellum was significantly higher than that in plasma at 10 days of age. In contrast, the concentrations in both cerebellum and plasma at 3 and 60 days of age were similar. In the present study, the site of 3betaHSD mRNA expression in the cerebellum was further examined in neonatal and adult rats using in situ hybridization. The cerebellar expression of 3betaHSD mRNA was obscure at 3 days of age, whereas intense expression occurred in Purkinje cells and external granule cells throughout the cerebellum at 10 days of age. 3BetaHSD mRNA was also expressed in Purkinje cells and granule cells at 60 days of age, but a restricted expression was observed along the cerebellar meninges. These results suggest that the steroidogenic enzyme 3betaHSD as well as P450scc are expressed at least in the cerebellar Purkinje cell. The expression of 3betaHSD, however, may increase for a limited period around 10 days of age, unlike P450scc.

Expression and Activity of 3 -Hydroxysteroid Dehydrogenase/ 5- 4-Isomerase in the Rat Purkinje Neuron during Neonatal Life

Recently, we demonstrated that cytochrome P450 side-chain cleavage enzyme (P450scc) occurs in the rat cerebellar Purkinje cell after differentiation and remains during neonatal development and into adulthood. 3β-Hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3βHSD) is also an essential enzyme for progesterone biosynthesis not only in peripheral steroidogenic glands but also in the nervous system. In the present study, therefore, the expression of 3βHSD in the rat cerebellum was investigated during neonatal development and in the adult. RT-PCR analysis showed that the expression of 3βHSD messenger RNA (mRNA) in the cerebellum was higher at 7–14 days of age than at other times. Biochemical studies together with HPLC analysis revealed that cerebellar slices at 10 days of age converted pregnenolone to progesterone, suggesting enzymatic activity of 3βHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3βHSD. A specific RIA indicated that progesterone concentrations in the cerebellum were higher at 3 and 10 days of age than at 60 days of age. The progesterone level in the cerebellum was significantly higher than that in plasma at 10 days of age. In contrast, the concentrations in both cerebellum and plasma at 3 and 60 days of age were similar. In the present study, the site of 3βHSD mRNA expression in the cerebellum was further examined in neonatal and adult rats using in situ hybridization. The cerebellar expression of 3βHSD mRNA was obscure at 3 days of age, whereas intense expression occurred in Purkinje cells and external granule cells throughout the cerebellum at 10 days of age. 3βHSD mRNA was also expressed in Purkinje cells and granule cells at 60 days of age, but a restricted expression was observed along the cerebellar meninges. These results suggest that the steroidogenic enzyme 3βHSD as well as P450scc are expressed at least in the cerebellar Purkinje cell. The expression of 3βHSD, however, may increase for a limited period around 10 days of age, unlike P450scc.

Effect of truncated forms of the steroidogenic acute regulatory protein on intramitochondrial cholesterol transfer.

It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type StAR, N-47, or the C-28 constructs. Lysates from the transfected COS-1 cells were then incubated with mitochondria from MA-10 mouse Leydig tumor cells that were preloaded with [3H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [3H]cholesterol content was determined in each membrane fraction. Incubation of MA-10 mitochondria with wild-type StAR containing cell lysate resulted in a significant 34.9% increase in [3H]cholesterol content in contact sites and a significant 32.8% increase in inner mitochondrial membranes. Incubations with cell lysate containing N-47 StAR protein also resulted in a 16.4% increase in [3H]cholesterol in contact sites and a significant 26.1% increase in the inner membrane fraction. In contrast, incubation with the C-28 StAR protein had no effect on cholesterol transfer. The cholesterol-transferring activity of the N-47 truncation, in contrast to that of the C-28 mutant, was corroborated when COS-1 cells were cotransfected with F2 vector (containing cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reductase) and either pCMV empty vector or the complementary DNAs of wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was significantly increased in both wild-type and N-47-transfected cells, whereas that in C-28-transfected cells was similar to the control value. Finally, immunolocalization studies with confocal image and electron microscopy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that wild-type and most of the C-28 StAR protein were imported into the mitochondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol transfer to the inner mitochondrial membrane, that StAR need not enter the mitochondria to produce this transfer, and the importance of the C-terminus of StAR in this process.

Differentiation of the corpus luteum of the mink (Mustela vison): mitogenic and steroidogenic potential of luteal cells from embryonic diapause and postimplantation gestation.

The mink corpus luteum (CL) involutes after ovulation and remains dormant, synthesizing low amounts of progesterone until reactivated to terminate embryonic diapause. We examined the mitotic and steroid synthetic capacity of luteal cells from the diapause and postimplantation phases of mink gestation. Cells from diapause divided in vitro, reaching confluence in 7-8 days. Three phenotypes were distinguishable: a fusiform cell in whorls, a hypertrophied epithelioid cell, and a small epithelioid cell. The first and second cell types divided in vitro after confluence, evidenced by localization of proliferating cell nuclear antigen (PCNA) in their nuclei. The small epithelioid cells were present in cell nests and showed no PCNA activity. Cells derived from reactivated CL did not reach confluence and had no PCNA activity. Progesterone accumulation was enhanced in luteal cells from diapause by LH, FSH, and dibutyryl (Bu2)cAMP, and by LH and (Bu2)cAMP in cells from reactivated CL. In luteal cells from the diapause phase of gestation, LH and (Bu2)cAMP induced increases in mRNA coding for steroidogenic acute regulatory protein, while cytochrome P450 side-chain cleavage enzyme mRNA was increased by prolactin, LH and (Bu2)cAMP. Cellular concentrations of 3beta-hydroxysteroid dehydrogenase-delta5-4-isomerase mRNA were increased by prolactin and (Bu2)cAMP. Thus, luteinization in the mink CL does not engender exit from the cell cycle, as both fusiform and hypertrophied cells from diapause divide in vitro. Reactivation appears to represent terminal differentiation. LH is capable of stimulating steroidogenesis in vitro in luteal cells from diapause, and prolactin and LH appear to have both specific and overlapping stimulatory effects on the CL of this species.

Interferon-gamma inhibits the steroidogenic acute regulatory protein messenger ribonucleic acid expression and protein levels in primary cultures of rat Leydig cells.

Interferon-gamma (IFNgamma) is an immunomodulating cytokine that has profound effects on reproductive function. IFNgamma inhibits steroidogenesis both in vivo and in vitro. The mechanism by which IFNgamma inhibits Leydig cell steroidogenesis remains unclear. In the present study, we evaluated the effect of IFNgamma on the expression and regulation of the steroidogenic acute regulatory protein (StAR) gene in primary cultures of rat Leydig cells. StAR facilitates the efficient production of steroid hormone by regulating the translocation of cholesterol from the outer to the inner mitochondrial membrane, the site of the cytochrome P450 side-chain cleavage (P450scc) enzyme system that converts cholesterol to pregnenolone. IFNgamma inhibited hCG-induced StAR messenger RNA (mRNA) levels in a dose-dependent manner. The addition of IFNgamma in a concentration of 500 U/ml decreased hCG-induced 3.8- and 1.7-kilobase StAR mRNA by 78% and 70%, respectively. IFNgamma also reduced hCG-stimulated P450scc mRNA levels by 69%. The inhibitory effects of IFNgamma on StAR mRNA levels were confirmed by ribonuclease protection assay. As early as 12 h after the addition of IFNgamma, hCG-induced StAR mRNA levels decreased by more than 44%. To evaluate the effects of IFNgamma on StAR protein levels, Western blot analyses were performed. hCG in a concentration of 10 ng/ml increased StAR protein by 5.6-fold. Treatment of Leydig cells with IFNgamma (500 U/ml) decreased hCG-induced StAR protein by 44%. In contrast, interleukin-1 and murine tumor necrosis factor-alpha reduced hCG-induced P450scc mRNA expression without inhibiting StAR mRNA or protein levels. In conclusion, IFNgamma inhibits Leydig cell steroidogenesis by down-regulating StAR gene expression and protein production.

Detection of steroidogenic acute regulatory protein (stAR) in mitochondria of cultured rat Sertoli cells incubated with follicle-stimulating hormone.

Previous work established biochemical homology between proteins named SCc1 and SCc2 in FSH-treated Sertoli cell cultures and a family of 30-kDa proteins, collectively referred to as Steroidogenic Acute Regulatory protein (StAR), in hormone-treated adrenal and gonadal cells. The purpose here was to establish the presence of StAR in FSH-treated Sertoli cell cultures using a StAR-specific antiserum. Immunofluorescence microscopy and immunoblot analysis were used to detect StAR in Sertoli cell cultures and in R2C rat Leydig tumor cell cultures that served as a positive control for StAR expression. FSH dramatically enhanced the appearance of StAR in mitochondria of Sertoli cells, whereas R2C cells expressed StAR constitutively. It has been proposed that StAR participates in steroidogenesis by facilitating the conversion of cholesterol to pregnenolone by the cytochrome P450 side-chain cleavage enzyme. FSH-treated Sertoli cells were shown here to lack detectable levels of side-chain cleavage enzyme protein and RNA, while R2C cells expressed both. We conclude that the appearance of StAR in Sertoli cell mitochondria is regulated by FSH, but that its function in Sertoli cells is not associated with the conversion of cholesterol to pregnenolone.

Structure and function of the peripheral-type benzodiazepine receptor in steroidogenic cells.

Steroidogenesis depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane cytochrome P-450 side-chain cleavage enzyme. Using steroidogenic cell submitochondrial fractions, mitochondrial preparations, various cell models, and animal models and with the help of pharmacological, biochemical, morphological, and molecular approaches, we provide evidence that the peripheral-type benzodiazepine receptor mediates the intramitochondrial cholesterol transport and the subsequent adrenal, gonadal, placental, and brain steroid biosynthesis.

Specific effect of arachidonic acid on 17β-hydroxysteroid dehydrogenase in rat Leydig cells

It is well known that arachidonic acid (AA) acts as an intratesticular factor regulating luteinizing hormone-mediated testicular steroidogenesis. The present studies were conducted to determine the effect of AA on steroidogenic enzymes in rat Leydig cells. Exogenously added AA significantly inhibited 22( R)-hydroxy-cholesterol-stimulated testosterone production, which is a clear indication that AA is acting at some point after cholesterol transport to the inner mitochondrial membrane. AA failed to block the conversion of 22( R)-hydroxycholesterol to pregnenolone, indicating that the cytochrome P-450 side-chain cleavage enzyme complex is not the site of inhibition. The present results demonstrate that only 17β-hydroxysteroid dehydrogenase seems to be involved in the AA action, since nearly 60% inhibition of testosterone production was found when the cells were incubated with androstenedione. Furthermore, no effect of AA was found when androstenediol was used as substrate in the testosterone synthesis, which indicates that 3β-hydroxysteroid dehydrogenase is not affected by AA. The conversion of AA to its metabolites is not required for its action on 17β-hydroxysteroid dehydrogenase and the activation of protein kinase C is not involved in the inhibitory effect.

Cytochrome P450 side-chain cleavage enzyme in the cerebellar Purkinje neuron and its neonatal change in rats.

Neurosteroids are de novo synthesized in the nervous system through mechanisms at least partly independent of peripheral steroidogenic glands. However, the concept of neurosteroidogenesis in neurons is not clear in mammalian brains. The present study identified the presence of cytochrome P450scc in the rat Purkinje cell, a typical cerebellar neuron. Immunohistochemical analysis with the antibody against the purified bovine adrenal P450scc showed an immunoreaction restricted to somata and dendrites of the Purkinje cells in adult cerebella. Preadsorbing the antibody with P450scc resulted in a complete absence of the immunoreaction. The antibody against inositol triphosphate receptor, a marker of the Purkinje cell, recognized P450scc-immunoreactive cerebellar cells that showed no immunoreaction with glial fibrillary acidic protein, a specific marker of glial cells. Expression of the P450scc-like protein in the cerebellum was verified by Western blot analysis, and cerebellar P450scc messenger RNA, by RT-PCR analysis in adulthood. On the other hand, P450scc-immunoreactive cells were found to scatter throughout the cerebellum at 0 day of age, before the differentiation of the first Purkinje cells, while the site of expression of this protein was localized only in somata of Purkinje cells at 3 days of age. Immunoreactive dendrites of the Purkinje cell spread into the molecular layer during neonatal development concurrently with its maturation. The intensity of the immunoreaction did not change during neonatal life. Expression of the cerebellar P450scc messenger RNA was also detected after birth, and the level was almost constant during neonatal life. A specific RIA indicated that the pregnenolone concentration was unexpectedly high at 0 day and decreased until 7 days. The total amount of pregnenolone in the cerebellum was almost constant from 0-7 days and increased during 7-21 days concurrently with the cerebellar development. In contrast, the pregnenolone sulfate ester level was low and did not significantly change among the developmental stages. These results suggest that steroidogenic enzyme P450scc appears in the rat Purkinje cell immediately after its differentiation. The expression of this enzyme may remain during neonatal development and in adulthood.

Cytochrome P450 Side-Chain Cleavage Enzyme in the Cerebellar Purkinje Neuron and Its Neonatal Change in Rats

Neurosteroids are de novo synthesized in the nervous system through mechanisms at least partly independent of peripheral steroidogenic glands. However, the concept of neurosteroidogenesis in neurons is not clear in mammalian brains. The present study identified the presence of cytochrome P450scc in the rat Purkinje cell, a typical cerebellar neuron. Immunohistochemical analysis with the antibody against the purified bovine adrenal P450scc showed an immunoreaction restricted to somata and dendrites of the Purkinje cells in adult cerebella. Preadsorbing the antibody with P450scc resulted in a complete absence of the immunoreaction. The antibody against inositol triphosphate receptor, a marker of the Purkinje cell, recognized P450scc-immunoreactive cerebellar cells that showed no immunoreaction with glial fibrillary acidic protein, a specific marker of glial cells. Expression of the P450scc-like protein in the cerebellum was verified by Western blot analysis, and cerebellar P450scc messenger RNA, by RT-PCR analysis in adulthood. On the other hand, P450scc-immunoreactive cells were found to scatter throughout the cerebellum at 0 day of age, before the differentiation of the first Purkinje cells, while the site of expression of this protein was localized only in somata of Purkinje cells at 3 days of age. Immunoreactive dendrites of the Purkinje cell spread into the molecular layer during neonatal development concurrently with its maturation. The intensity of the immunoreaction did not change during neonatal life. Expression of the cerebellar P450scc messenger RNA was also detected after birth, and the level was almost constant during neonatal life. A specific RIA indicated that the pregnenolone concentration was unexpectedly high at 0 day and decreased until 7 days. The total amount of pregnenolone in the cerebellum was almost constant from 0–7 days and increased during 7–21 days concurrently with the cerebellar development. In contrast, the pregnenolone sulfate ester level was low and did not significantly change among the developmental stages. These results suggest that steroidogenic enzyme P450scc appears in the rat Purkinje cell immediately after its differentiation. The expression of this enzyme may remain during neonatal development and in adulthood.

Role of steroidogenic-factor 1 in basal and 3',5'-cyclic adenosine monophosphate-mediated regulation of cytochrome P450 side-chain cleavage enzyme in the mouse.

The orphan receptor steroidogenic factor-1 (SF-1) plays a major role in adrenal and gonadal development and sexual differentiation, and it has been shown to interact with shared promotor elements to increase the expression of cytochrome P450 side-chain cleavage (P450scc) and other steroid hydroxylases in vitro. We took a step-wise approach to define the role of SF-1 in regulation of hormone-induced steroidogenesis. In a mouse Leydig cell line (MA-10) we show that hCG and forskolin are effective inducers of progesterone production and P450scc expression. In contrast, endogenous SF-1 expression was not increased by either hCG or forskolin. Similarly, these agents did not enhance the activity of SF-1 promoter transfected into MA-10 cells. The transcriptional activity of SF-1, measured by induction of an SF-1 synthetic reporter, was only minimally increased by forskolin. Within the context of the rat P450SCC promoter, mutation of the two SF-1-binding sites caused a dramatic decrease in constitutive activity of this promoter, but the degree of induction by 8-bromo-cAMP was only reduced from 7.9-fold to 5.9-fold. We conclude that SF-1 is required for the constitutive activity of P450scc, but that it does not play a direct role in the early induction of steroidogenesis by hCG or forskolin in MA-10 cells.

Corticotropin-releasing hormone stimulates the expression of the steroidogenic acute regulatory protein in MA-10 mouse cells.

We have previously demonstrated that corticotropin-releasing hormone (CRH) treatment of MA-10 mouse Leydig tumor cells results in a dose-dependent stimulation of progesterone production. In view of this observation we wished to determine the effects of CRH on the synthesis of the steroidogenic acute regulatory (StAR) protein in these cells. StAR is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage enzyme. Treatment of MA-10 cells with the cAMP analogue dibutyryl cAMP (dbcAMP) resulted in a dose- and time-dependent increase in the levels of StAR protein that reached a maximum at 800 microM dbcAMP and within a time period of 6 h. Further, treatment of MA-10 cells with CRH also resulted in a dose-dependent increase in the synthesis of the StAR protein with a maximal response observed at 1 microM. Slightly different from that observed with dbcAMP, the maximal response to 1 microM CRH was seen at 4 h following stimulation. These results indicate that the observed increase in steroid production in response to CRH in MA-10 Leydig tumor cells is similar to that previously seen with trophic hormone stimulation acting through the cAMP second messenger pathway, and that it occurs as a result of an increase in the synthesis of the StAR protein.

Coordinate developmental expression of genes regulating sterol economy and cholesterol side-chain cleavage in the porcine ovary.

To investigate the coordinate developmental expression of low-density lipoprotein (LDL) receptor, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, sterol carrier protein 2 (SCP2), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450scc) enzyme messages throughout the pig estrous cycle, RNase protection analysis was performed using homologous (partially cloned) porcine sequences. Total RNA was isolated from ovarian tissues from unstimulated prepubertal gilts and gilts stimulated with eCG (Day -3) and hCG (Day 0) to induce follicular growth and ovulation. Specific transcripts (relative to 18S rRNA) were quantified in immature ovaries, preovulatory follicles (> or = 5 mm), corpora lutea (CL), and corpora albicantia. As an index of steroidogenesis, tissue progesterone content (per microgram protein) was low in the unstimulated ovary and preovulatory follicles, and it began to increase 4 days post-hCG, peaked at 12 days, and returned to preovulatory concentrations by 20 days post-hCG. HMG-CoA reductase mRNA was expressed at low levels and did not change significantly throughout the estrous cycle. The amount of LDL receptor mRNA increased approximately 6-fold after eCG stimulation and was expressed at similar concentrations in both preovulatory follicles and functional CL. Expression of SCP2 mRNA did not differ among the four tissue types but tended to be highest in midcycle (Day 12) CL compared other stages of CL (p = 0.007). StAR mRNA expression was minimal in unstimulated ovaries, was higher in preovulatory follicles (p = 0.014), and then rose again in CL (p = 0.009 compared with unstimulated ovary). P450scc mRNA concentrations were low in unstimulated ovaries, increased in preovulatory follicles (p = 0.044), and increased further in CL (p = 0.001 compared with preovulatory follicles). P450scc and StAR mRNA levels correlated with progesterone levels (r = +0.37, p = 0.025, and r = +0.71, p < 0.001, respectively). The expression of LDL receptor, StAR, and P450scc messages showed a dramatic decline by Day 20 post-hCG (p = 0.002, p = 0.003, p = 0.006, respectively, compared with CL) corresponding with functional regression of the CL. In summary, P450scc and StAR message expression are coordinately amplified during the pig follicular and luteal phase, whereas LDL receptor message after an initial increase is expressed at constitutively high levels, thus indicating a differential regulation of ovarian sterol-metabolizing genes during the steroidogenic life of the follicle and CL.

II. Adrenal cortex and steroid 21-hydroxylase autoantibodies in children with organ-specific autoimmune diseases: markers of high progression to clinical Addison's disease.

Adrenal cortex autoantibodies (ACA) were measured by immunofluorescence in 808 children with organ-specific autoimmune diseases without adrenal insufficiency. ACA were found in 14 children (1.7%), mostly in hypoparathyroidism (48%). Ten ACA-positive and 12 ACA-negative children were followed up for a maximum of 10 yr by evaluation of adrenocortical function (ACTH test) and autoantibody status. In all patients steroid-producing cell autoantibodies were assessed by immunofluorescence and autoantibodies to steroid 21-hydroxylase, 17 alpha-hydroxylase, and cytochrome P450 side-chain cleavage enzyme by immunoprecipitation assay. All 10 ACA-positive patients were positive for 21-hydroxylase autoantibodies. Six were positive for steroid-producing cell autoantibodies and 5 also for autoantibodies to 17 alpha-hydroxylase and/or P450 side-chain cleavage enzyme. Overt Addison's disease developed in 9 (90%) ACA/21-OH-antibody-positive children after 3-121 months, and 1 remaining child had subclinical hypoadrenalism. By contrast, all ACA/21-OH antibody-negative children maintained normal adrenal function. Adrenal failure was not related to ACA titres, sex, adrenal function, type of preexisting autoimmune disorder, or human leucocyte antigens D-related status. In conclusion, in children with autoimmune endocrine diseases, ACA/21-hydroxylase autoantibodies are important predictive markers for the development of Addison's disease.