P450scc mRNA Levels Research Articles

Differential effects of tumor necrosis factor-alpha and interleukin-1 on 3 beta-hydroxysteroid dehydrogenase/delta 5-->delta 4 isomerase expression in mouse Leydig cells.

Immune-endocrine interactions are important to the regulation of Leydig cell steroidogenesis. We have shown previously that both tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1-beta) inhibit 8-bromo-cAMP-(8-Br-cAMP)-stimulated steroidogenesis in mouse Leydig cells. TNF and IL-1 both inhibit cAMP-stimulated testosterone production as well as mRNA and protein levels of cholesterol side chain cleavage enzyme (P450scc) and 17 alpha-hydroxylase/C17,20 lyase (P450c17) in mouse Leydig cells. Neither TNF nor IL-1 affects basal levels of P450scc mRNA and protein. In the present study, we tested the effects of TNF and IL-1 on basal testosterone production and 8-Br-cAMP-stimulated 3 beta-hydroxysteroid dehydrogenase/delta 5-->delta 4 isomerase (3 beta HSD) expression in Leydig cells. Purified and macrophage-depleted Leydig cells were cultured for 5 d with daily changes of media, and then treated with increasing concentrations of recombinant mouse TNF or IL-1 in the presence or absence of 8-Br-cAMP (50 microM) for 24 h. The media were collected for testosterone RIA and RNA and protein were extracted from cells. Basal testosterone production was inhibited by TNF, but not IL-1. Treatment of Leydig cells with 8-Br-cAMP alone caused a marked increase in 3 beta HSD mRNA, and protein levels. Both TNF and IL-1 inhibited cAMP-stimulated 3 beta HSD mRNA and protein levels, but only TNF inhibited basal 3 beta HSD expression. These results demonstrate that TNF and IL-1 have different effects on basal steroidogenesis in Leydig cells and suggest that TNF-mediated inhibition of basal testosterone production may be owing to the inhibition of basal 3 beta-HSD expression in Leydig cells.

Regulation of cytochrome P450 cholesterol side-chain cleavage, 3 β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase type 1 and estradiol-17 β-hydroxysteroid dehydrogenase mRNA levels by calcium in human choriocarcinoma JEG-3 cells

In human placenta the cytochrome P450 side-chain cleavage (P450scc) and 3 β-hydroxysteroid dehydrogenase type 1 (3 β-HSD-1) convert cholesterol and pregnenolone producing progesterone, whereas 17 β-hydroxysteroid dehydrogenase type 1 (17 β-HSD-1) mediates the interconversion of estrone and estradiol. We have examined the effects of calcium on phorbol ester- and cAMP-induced P450scc, 3 β-HSD-1 and 17 β-HSD-1 mRNAs in human JEG-3 cells. A23187 increased in a dose-dependent fashion in the 1.3 kb 17 β-HSD-1 mRNA whereas a weaker increase followed by a gradual depletion effect of A23187 was observed on 3 β-HSD-1 mRNA. No significant effect of A23187 on P450scc mRNA was observed. Using 0.50 μM of A23187 the induction of 3 β-HSD-1 and 17 β-HSD-1 mRNAs was maximum within about 6 h whereas P450scc mRNA levels stayed unaffected throughout the time-course period. The action of A23187 was synergistic on cAMP-stimulated 17 β-HSD-1 mRNA levels, while in a dose-dependent manner A23187 progressively depleted 3 β-HSD-1 and P450scc mRNA abundance probably by activation of a calcium-/calmodulin-dependent phosphodiesterase. On the phorbol 12-myristate, 13-acetate (PMA)-stimulated 3 β-HSD-1, 17 β-HSD-1 and P450scc mRNA levels only the lowest concentration of A23187 potentialized the PMA effect on the 17 β-HSD-1 mRNA levels. Using thapsigargin (TG), a cell-permeable sesquiterpene lactone that releases calcium by inhibiting sarco/endoplasmic reticular calcium-ATPase, our data indicated the presence in JEG-3 cells of TG-sensitive and TG-insensitive calcium-ATPases regulating 3 β-HSD-1 and 17 β-HSD-1 mRNA levels. These results emphasized the complexity of calcium contribution with the protein kinase A and C pathways in the regulation of P450scc, 3 β-HSD-1 and 17 β-HSD-1 mRNA levels. In addition, the different sensitivity of these genes to calcium suggest they could be activated by different subclasses of PKCs.

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.

Estrogen regulation of placental P-450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid levels and activity during baboon pregnancy.

The present study was conducted to determine whether estrogen regulates the P-450 cholesterol side-chain cleavage (P-450scc) enzyme component of the progesterone biosynthetic pathway in the placenta during the second half of baboon pregnancy. Placental estrogen formation was suppressed by removing the fetus, i.e. fetectomy, and thus fetal adrenal C19-steroid estrogen precursors, on day 100 of baboon gestation (term = 184 days). P-450scc activity and messenger ribonucleic acid (mRNA) levels were then determined in placentas obtained on day 160 after fetectomy alone and after fetectomy and s.c. administration of the estrogen precursor and rostenedione or estradiol benzoate. Placentas were maintained in situ after fetectomy, and placental villi were comprised of syncytiotrophoblasts that seemed morphologically normal, based on their histology and immunocytochemical expression of pregnancy-specific-beta 1-glycoprotein. In untreated baboons, peripheral serum estradiol increased with advancing gestation, and mean (+/-SE) concentrations were 1.22 +/- 0.05 ng/ml on days 101-160 of gestation. After fetectomy serum estradiol concentrations decreased to 24% (P < 0.01) of normal. Androstenedione or estradiol administration after fetectomy increased serum estradiol levels to values that were 57% (P < 0.01) of, or 90% (P < 0.001) greater than intact controls, respectively,. Placental P-450scc specific activity, determined on a mitochondrial-enriched fraction of villous tissue, was 281.1 +/- 15.0 pmol pregnenolone plus progesterone formed per mg mitochondrial protein in untreated control baboons. Fetectomy resulted in a 52% decrease (P < 0.001) in placental P-450scc activity. Administration of androstenedione or estradiol after fetectomy increased P-450scc activity to values that were not significantly different from control. P-450scc mRNA levels were quantified by competitive RT-PCR. P-450scc mRNA levels in placental villous tissue of fetectomized baboons was 38% lower (P < 0.01) than that in the intact controls (110.9 +/- 5.9 attomoles/microgram RNA). The administration of androstenedione after fetectomy restored P-450scc mRNA to a level that was not different from the untreated controls. The results of this study show that there was close association between the levels of estrogen and the specific activity of and the mRNA levels for placental P-450scc in the second half of baboon pregnancy. Therefore, we propose that the P-450scc enzyme that catalyzes the conversion of substrate cholesterol to pregnenolone is regulated, for the most part, by estrogen in the primate placenta.

Estrogen stimulation of P450 cholesterol side-chain cleavage activity in cultures of human placental syncytiotrophoblasts.

The present study determined whether estrogen has a role in regulating the P450 cholesterol side-chain cleavage enzyme (P450scc) and/or de novo/deesterification cholesterol pathways involved in progesterone biosynthesis within human syncytiotrophoblasts. Human placental syncytiotrophoblasts were cultured for 48 h with estradiol, and P450scc activity was determined by the formation of progesterone from 25-hydroxycholesterol. Estradiol at 10(-7) or 10(-6) M and 25-hydroxycholesterol increased mean (+/- SE) progesterone production by syncytiotrophoblasts (ng/0.5 x 10(6) cells) to a value (19.2 +/- 1.1) that was 104% (p < 0.001) higher than that of the untreated controls (9.4 +/- 0.8) and 52% higher (p < 0.001) than with 25-hydroxycholesterol alone (12.6 +/- 0.9). The stimulation of progesterone secretion apparently was not the result of a change in progesterone metabolism to its principal metabolite, because 20alpha-hydroxypregn-4-en-3-one represented a minor secretory component (0.7-1.7 ng/0.5 x 10(6) cells) under these conditions, and levels were not substantially altered by estrogen. In contrast to the stimulatory effect of estradiol on P450scc activity, estrogen did not alter either the P450scc mRNA levels or the activities of 3-hydroxy-3-methylglutaryl coenzyme-A reductase and cholesterol ester hydrolase-rate-limiting enzymes for the de novo and deesterification pathways, respectively, for cholesterol formation in syncytiotrophoblasts in culture. Collectively, these results indicate that estrogen regulates the P450scc component of the progesterone biosynthetic pathway, which we suggest signals functional/biochemical differentiation of syncytiotrophoblasts during primate pregnancy.

Steroidogenic factor-1 expression is transiently repressed and c-myc expression and deoxyribonucleic acid synthesis are induced in rat granulosa cells during the periovulatory period.

The expression of steroidogenic factor 1 (SF-1), cytochrome P450 aromatase (P450arom), and cytochrome P450 cholesterol side-chain cleavage (P450scc) was examined during the periovulatory period. Immature rats were injected with eCG to induce development of ovarian follicles to the preovulatory stage. At 48 h after the eCG injection, the LH surge was simulated by an injection of an ovulatory dose of hCG, and RNA was isolated at 0, 2, 4, 6, 8, and 24 h after hCG injection. The mRNA levels for SF-1, P450arom, and P450scc were relatively high in total ovarian RNA samples from eCG-treated rats. Levels of SF-1 and P450arom mRNA decreased within 2 h after injection of hCG. The SF-1 mRNA levels gradually increased from 4 to 24 h. Aromatase levels remained undetectable until 24 h after hCG. P450scc mRNA levels increased throughout the 24-h period after hCG. Levels of SF-1 and P450arom, but not P450scc, mRNA were also reduced in RNA samples from isolated granulosa cells at 4 h after hCG treatment relative to those from eCG-treated rats. In situ hybridization analysis also revealed that hCG uniformly suppressed SF-1 mRNA levels an all granulosa cells compared to those of eCG-treated controls. The relationship of SF-1 expression to immediate/early gene expression and cell cycle traverse was also examined. C-myc mRNA levels were induced by up to 10-fold at 4 h after hCG injection. Similarly, DNA synthesis, as measured by the percentage of granulosa cells that incorporated 5'-bromodeoxyuridine, was increased from 16 +/- 4% in eCG-treated rats to 61 +/- 7% at 4 h after hCG treatment (p < 0.05). This study provides the novel finding that SF-1 expression is transiently repressed to very low levels in response to the LH surge. Further, these studies suggest that granulosa cells traverse the cell cycle before becoming terminally differentiated luteal cells.

Prostaglandin F2α reduces steroidogenic acute regulatory (StAR) protein messenger ribonucleic acid expression in the rat ovary

Steroid biosynthesis begins with the enzymatic conversion of cholesterol to pregnenolone. This reaction is catalyzed by the cytochrome P450 side-chain cleavage enzyme (P450scc), which is located on the matrix side of the inner mitochondrial membrane. Although the rate-limiting enzymatic step in steroidogenesis is the conversion of cholesterol to pregnenolone by the side-chain cleavage enzyme, the true rate-limiting step in this process is the delivery of cholesterol to the inner mitochondrial membrane. Steroidogenic acute regulatory (StAR) protein is thought to mediate the rapid increase in steroid hormone biosynthesis in response to tropic hormones by facilitating cholesterol transport to the inner mitochondrial membrane. Cholesterol transport across the inner mitochondrial membrane has also been implicated as the target for prostaglandin F2α's (PGF2α) antisteroidogenic activity. Since cholesterol delivery to the P450scc is a rapidly regulated step in steroidogenesis, StAR mRNA levels were examined after the administration of a luteolytic injection of PGF2α. The results of this investigation revealed that both major StAR RNA transcripts were decreased in the ovary, 10 d after ovulation, following PGF2α administration. Serum progesterone levels were decreased following PGF2α administration in parallel with the decreased expression of StAR. Following PGF2α treatment, ovarian StAR transcripts at 3.4 and 1.6 kb were reduced 4-fold (p<0.01) and 2.5-fold (p<0.025), respectively, after 4 h. Ovarian P450scc mRNA levels were also reduced (70%) 4 h after PGF2α injection. Time course experiments following PGF2α administration showed a significant decrease in StAR expression as early as 30 min (p<0.02) following injection. In contrast to StAR's expression after PGF2α administration, StAR mRNA levels were elevated in response to human chorionic gonadotropin (hCG) 3 h postinjection. Administration of PGF2α followed by hCG injection effectively blocked induction of StAR expression. StAR mRNA levels were reduced 1.5-fold relative to control animals and 3.5-fold relative to the hCG-treated animals (p<0.05). The levels of serum progesterone paralleled the change in ovarian StAR mRNA in all experiments. This study provides the first evidence that StAR mRNA expression is mediated by prostaglandins in the rat ovary further supporting its important role in the regulation of steroid hormone biosynthesis.

Hormonal regulation of steroidogenic acute regulatory (StAR) protein messenger ribonucleic acid expression in the rat ovary

Steroidogenic acute regulatory (StAR) protein is thought to mediate the rapid increase in steroid hormone biosynthesis in response to tropic hormones by facilitating cholesterol transport to the inner mitochondrial membrane where the P450 side-chain cleavage enzyme (P450scc) is located. Since cholesterol delivery is the regulated step in steroidogenesis and is dependent onde novo protein synthesis, StAR mRNA levels were examined in response to the tropic hormones, pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The results of this investigation revealed that major StAR mRNA transcripts of 3.4 and 1.6 kb and a less abundant transcript of 1.2 kb were detected in the adrenal, ovary, and testis. Within the ovary, StAR mRNA levels were regulated by PMSG and hCG. The two major transcripts were increased in the immature rat ovary following PMSG administration and in the ovary, 8 d after ovulation, in response to stimulation by hCG. Serum progesterone levels were increased following hCG treatment in parallel with the enhanced expression of StAR. Following PMSG treatment, ovarian StAR transcripts at 3.4 and 1.6 kb were each increased twofold. In the ovary, 8 d following ovulation, basal ovarian StAR mRNA levels were elevated up to sixfold relative to the preovulatory StAR mRNA levels. Even with the enhanced basal level of StAR mRNA within the ovary 8 d postovulation, hCG administration still resulted in a 2.5- and 7-fold increase in the 3.4 and 1.6 kb (p<0.025) transcripts, respectively, and a 58% increase in serum progesterone. In contrast to the dramatic alterations in StAR mRNA expression following hormonal stimulation, P450scc mRNA levels remained unchanged in response to hCG stimulation. The levels of serum progesterone paralleled the change in ovarian StAR mRNA in all experiments. This study provides the first evidence that StAR mRNA expression in the rat ovary is mediated by gonadotropins, further supporting its important role in the regulation of steroid hormone biosynthesis.

Developmental expression of placental trophoblast P-450 cholesterolside-chain cleavage, adrenodoxin and Δ 5-3β-Hydroxysteroid dehydrogenase/isomerase messenger ribonucleic acids during baboon pregnancy

The present study determined whether the elevation in oestrogen, which occurs with advancing baboon pregnancy, is associatedwith a developmental increase in expression of the placental enzymes catalysing progesterone synthesis. The mRNA levels for P-450 cholesterol side-chain cleavage (P-450 scc), adrenodoxin, and Δ 5-3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) were assessed by Northern blot analysis in placental syncytiotrophoblasts isolated from baboons in early (days 58–65), mid- (days 97–113) and late (days 161–175), gestation (term=184 days). Placental villous tissue was dispersed and subjected to 50 per cent Percoll density gradient centrifugation to obtain primarily syncytiotrophoblasts. Mean ( ± s.e.) P-450 scc mRNA level, expressed as a ratio of β-actin in the syncytiotrophoblast-rich fraction, progressively increased with advancing pregnancy to a level in late gestation (1.81 ± 0.28 arbitrary units) that was approximately sixfold ( P<0.01) greater than in early gestation (0.31 ± 0.08) and approximately twofold greater ( P<0.05) than in mid-gestation (0.97 ± 0.24). In contrast adrenodoxin mRNA expression was similar at early (0.97), mid- (1.14 ± 0.12) and late (1.16 ± 0.13) gestation. Syncytiotrophoblast 3β-HSD mRNA levels also remained constant in early (1.69), mid- (1.89 0.41) and late (1.34 0.41) gestation. On the basis of these findings, we propose that villous syncytiotrophoblasts undergo functional/biochemical differentiation, resulting in a coordinated upregulation of specific components of the steroid biosynthetic pathway required for progesterone biosynthesis during the course of primate pregnancy.

The effect of insulin and insulin-like growth factors on the expression of steroidogenic enzymes in a human ovarian thecal-like tumor cell model

To determine the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on steroidogenesis and steroidogenic enzyme expression in a human ovarian thecal-like tumor cell culture model system. Human ovarian thecal-like tumor cells treated with forskolin and insulin IGF-I or IGF-II were evaluated for media accumulation of P and androstenedione (A) as well as 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and cytochrome P450 17 alpha-hydroxylase (P450c17) enzyme activity. Northern analysis of cytochrome P450 side chain cleavage (P450scc), P450c17, and 3 beta HSD messenger RNA (mRNA) also was performed. Basal hormone secretion, enzyme activity, and mRNA levels were not affected by treatment with insulin or the IGFs. Forskolin treatment stimulated steroid production, enzyme activity, and mRNA content. Forskolin-stimulated P secretion was augmented 30% by treatment with insulin and IGFs, whereas 3 beta HSD activity was augmented twofold to threefold. Forskolin stimulated A and P450c17 activity were enhanced by treatment with insulin and the IGFs. In forskolin-treated cells. P450c17 and P450scc mRNA levels were not affected by insulin (100 nM) or IGF (10 nM) treatment; however, 3 beta HSD mRNA levels were augmented by treatment with insulin and IGFs. We observed that forskolin-stimulated human ovarian thecal-like tumor cell steroidogenesis, P450c17, and 3 beta HSD activity, as well as mRNA content for P450scc, 3 beta HSD, and P450c17. Insulin and the IGFs augmented forskolin-stimulated production of P and the expression of 3 beta HSD, with little effect on A production, P450scc, or P450c17 expression.

Regulation of cytochrome P450scc synthesis and activity in the ovine corpus luteum

The rate-limiting step in luteal biosynthesis of progesterone consists of cleavage of the side chain of cholesterol by mitochondrial cytochrome P450 side-chain cleavage enzyme (P450scc) to form pregnenolone. Luteal mRNA encoding P450scc, quantitated on selected days of the 16-day ovine estrous cycle, was similar on days 3 and 6, increased by 2-fold on day 9 (P < 0.05) and remained elevated on day 15. Levels of P450scc mRNA on day 15 of pregnancy were not different from those found on any day of the cycle (P < 0.05). To determine whether levels of mRNA encoding P450scc are hormonally regulated, ewes on day 10 of the estrous cycle were injected with hCG or prostaglandin F2α (PGF2α). P450scc mRNA was not increased for up to 36 h after injection of hCG, nor decreased within 8 h after injection of PGF2α (P < 0.05). An assay for P450scc activity was developed which utilized ovine small and large luteal cells in the presence of 22R-hydroxycholesterol and ovine high density lipoprotein. Enzyme activity was quantitated by measurement of progesterone production. In small luteal cells activation of the protein kinase A (PKA) second-messenger system by treatment with LH resulted in 910% increase in progesterone production without altering activity of P450scc. Activation of the protein kinase C (PKC) second-messenger system with phorbol 12-myristate 13-acetate caused a 51% reduction in progesterone secretion from large luteal cells but did not alter activity of P450scc. These findings suggest that in mature luteal tissue steady state levels of mRNA encoding P450scc, and enzyme activity are independent of acute regulation by activation of PKA or PKC second-messenger systems.

Regulation of corticotropin and steroidogenic enzyme mRNAs in human fetal adrenal cells by corticotropin, angiotensin-II and transforming growth factor β 1

Using cultured human fetal adrenal cells, we have investigated the basal secretion of cortisol and dehydroepiandrosterone sulfate (DHAS) and the effect of corticotropin (ACTH), angiotensin-II (A-II) and transforming growth factorβ 1 (TGFβ 1) on the secretion of these steroids and on the mRNA levels of ACTH receptor (ACTH R), cytochrome P450scc (cholesterol side-chain cleavage), P450 17α (17α-hydroxylase/17–20 lyase) and 3β-HSD (3β-hydroxysteroid dehydrogenase). The basal DHAS/cortisol ratio declined progressively between 12.5 and 21 weeks. ACTH treatment enhanced the secretion of cortisol and to a lesser extent that of DHAS, and increased the steroidogenic response to an acute stimulation with ACTH. These changes were associated with increased mRNA levels of ACTH R and of the steroidogenic enzymes. A-II treatment also increased the secretion of both DHAS and cortisol, but less than ACTH, enhanced the responsiveness to ACTH and increased ACTH R, P450scc and P450 17α mRNA levels. In contrast, TGFβ 1 alone or together with ACTH decreased DHAS secretion, but not cortisol secretion. Moreover, TGFβ 1 had no effect on ACTH R and P450scc mRNA levels, decreased by about 50% the mRNA levels of P450 17α both in the absence or presence of ACTH, but enhanced the stimulatory effects of ACTH on 3β-HSD mRNA. These results, along with those previously reported, suggest that both A-II and TGFβ may play a role in fetal adrenal function. In addition, they show that the effects of both peptides are qualitatively different from, even sometimes opposite to, those previously reported in bovine and ovine adrenal cells.

Induction of rat granulosa cell steroidogenic enzyme activities and their messenger ribonucleic acids by a splenocyte-derived factor

We have previously identified and purified a splenocyte-derived factor (PSF) that stimulates the accumulation of progesterone and 20α-dihydroprogesterone (20α-OH-P) in rat ovarian granulosa cells independently of FSH. In the present study, time course experiments comparing the response to PSF with that to FSH revealed that PSF-stimulated progesterone accumulation was slower than that of FSH, but PSF-stimulated 20α-OH-P accumulation had a time course similar to that of FSH. To determine the basis for the slower progesterone response to PSF, the effect of these two agents on each step of the steroidogenic pathway was assessed. First, to examine whether PSF-stimulated cholesterol mobilization was limiting, cultured granulosa cells were treated with 22( R)-hydroxycholesterol. While both FSH-and PSF-stimulated progesterone and 20α-OH-P accumulation approximately doubled, the overall time courses did not change indicating that cholesterol availability was not the factor limiting the response to PSF. Next, PSF and FSH induction of steroidogenic enzyme activities and messenger RNAs were compared. While FSH-stimulated cytochrome P450 side chain cleavage enzyme (SCC) activity rapidly increased (peaking at 2 days) and then slowly declined, PSF-stimulated SCC activity gradually increased over 5 days to approximately 35% of the maximal activity stimulated by FSH. PSF also induced slower increases in P450scc mRNA levels than did FSH. In addition, PSF stimulated 3β-hydroxysteroid dehydrogenase (3β-HSD) activity more slowly than did FSH, but after 3 days of culture, PSF-stimulated activity was significantly higher than that induced by FSH. A similar pattern was also observed with 3β-HSD mRNA levels. In contrast, FSH stimulated only minimal increases in 20α-hydroxysteroid dehydrogenase (20α-HSD), whereas PSF stimulated larger and more rapid increases in 20α-HSD activity, which were maximal by 3 days. These studies indicate that the slower onset of PSF-stimulated progesterone production is due, at least in part, to slower induction of mRNAs for P450scc and 3β-HSD leading to slower increases in the activities of their respective enzymes. Additionally, the higher 20α-OH-P levels produced by PSF may be due to greater induction of 20α-HSD activity.

Evidence of a steroidogenic enzyme gene dose effect on adrenal gene expression in hereditary rabbit congenital adrenal hyperplasia.

We previously reported the gene deletion encoding cytochrome P-450 cholesterol side-chain cleavage enzyme (P-450SCC, resulting in complete elimination of the adrenal gene expression and causing congenital adrenal hyperplasia in the rabbit. Using the rabbit congenital adrenal hyperplasia model, we investigated the wild type (wt) P-450SCC gene dose effect on gene expression in three P-450SCC genotype animals [wt/wt, wt/mutant (mt), mt/mt] identified by Southern blot analysis. Northern blots using a rabbit P-450SCC cDNA probe revealed no detectable P-450SCC mRNA in individual adrenals of animals with congenital adrenal hyperplasia (mt/mt) and approximately half or slightly less than half the levels of the mRNA in the pooled adrenals of five heterozygous (wt/mt) newborn animals compared with the mRNA levels in the pooled adrenals of five homozygous normal (wt/wt) newborn animals. Identical P-450SCC mRNA levels were found individual adrenals of adult animals with regard to the P-450SCC genotype, although at a higher expression level than in the newborn animals of the same genotype. Control Northern blots using human CPY21-B cDNA and cytoplasmic actin cDNA probes confirmed the accuracy and integrity of RNA. Western immunoblotting using anti-ovine P-450SCC antibody revealed decreased P-450SCC protein in the adrenals of wt/mt animals at approximately half the level of the P-450SCC protein in the adrenals of the wt/wt animals. Baseline and ACTH-stimulated serum corticosterone (B) levels in vivo were similar between the age-matched wt/mt and wt/wt animals, whereas ACTH-stimulated B levels in adult animals were higher than those in the newborn animals irrespective of P-450SCC genotype.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids.

A sensitive assay of multiple mRNAs by reverse transcriptase-polymerase chain reaction was adopted to study the hormonally regulated expression of steroidogenic enzymes in primary rat granulosa cells in culture. As little as 15-60 ng total RNA prepared from cultured cells were reverse transcribed in the presence of pd(T)6, and polymerase chain reaction was conducted in the presence of specific oligonucleotide pairs designed to identify cDNAs of steroidogenic enzymes. In combination with Northern blot analysis of cholesterol side-chain cleavage cytochrome P450 (P450scc) message, it is shown that a novel protein kinase inhibitor, tyrphostin AG18, arrests the FSH-induced accumulation of P450scc mRNA. This inhibition is dose dependent (IC50, 15 microM) and reversible. The addition of 80 microM AG18 to cells containing high levels of P450scc mRNA caused a rapid decline of the cytochrome message (t 1/2, 5 h), similar to the effect of 30 micrograms/ml alpha-amanitin. However, concomitant addition of the two drugs did not accelerate the mRNA degradation process, suggesting that AG18 does not affect message stabilization. Tyrphostin AG18 did not affect mRNA species that are not FSH inducible, such as the ribosomal protein L19, or the constitutively expressed low levels of steroid 5 alpha-reductase mRNA. Moreover, even the extremely high levels of P450scc mRNA in granulosa-lutein cells, being cAMP independent and terminally differentiated a few hours after LH surge, were not affected by the addition of AG18 in culture. In contrast, two additional key and FSH-inducible steroidogenic enzymes, i.e. aromatase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase-I, were inhibited by AG18 at their mRNA levels. These results suggest that an as yet undetermined tyrosine kinase pathway is involved in the cAMP-dependent signal transduction pathway of FSH action, so that the presence of AG18 does not allow FSH induction of gene expression to occur.

Recombinant murine tumor necrosis factor-alpha inhibits cholesterol side-chain cleavage cytochrome P450 and insulin-like growth factor-I gene expression in rat Leydig cells

The purpose of the present study was to evaluate the effects of murine recombinant tumor necrosis factor-α (TNF-α) on rat Leydig cell function. In primary cultures of Leydig cells, we found that in the presence of hCG (10 ng/ml), testosterone levels were markedly elevated, 69.3 ± 3.1 ng/10 6 cells/h (mean ± SE). TNF-α in a concentration of 1 ng/ml markedly inhibited testosterone biosynthesis (a 69% reduction; p < 0.01) and 100 ng/ml of TNF-α almost completely inhibited testosterone formation ( p < 0.001). TNF-α (10 ng/ml) inhibited hCG (0.1, 1 and 10 ng/ml)-induced testosterone formation by 63%, 67% and 61%, respectively. TNF-α (10 ng/ml) also markedly inhibited 8-bromo cAMP-induced testosterone formation from 76 ± 9 ng/10 6 cells/h to 4.9 ng/10 6 cells/h. This indicates that the major effect of TNF-α is at $teps beyond LH receptor site. To further evaluate the site(s) of action of TNF-α, we evaluated its effect on the conversion of precursor steroids to testosterone. We found that the addition of 20-hydroxy-cholesterol could not reverse inhibitory effects of TNF-α on hCG-in- duced testosterone formation. TNF-α had no effect on the conversions of pregnenolone, 17-OH-pregnenolone, DHEA and androstenedione to testosterone. This indicates that the major effect of TNF-α is at the key steroidogenic enzyme, P450scc. We reported previously that human recombinant TNF-α had no effect on hCG-induced testosterone formation but did enhance the inhibitory effects of human recombinant IL-1β. In the present study, we demonstrated that both murine TNF-α and human IL-1β were potent inhibitors of hCG-induced testosterone formation. IL-1β alone in concentrations of 0.1, 1 and 10 ng/ml inhibited testosterone formation by 45%, 62% and 91%, respectively, in the presence of TNF-α (10 ng/ml), IL-1β in a concentration as low as 0.1 ng/ml completely blocked hCG-induced testosterone formation. We next evaluated the effect of TNF-α on P450scc gene expression. There was no constitutively expressed P450scc mRNA in Leydig cells after 24 h in culture. In response to hCG, there was a 33-fold increase in the P450scc mRNA level. Both TNF-α and IL-1β inhibited hCG-induced expression of P450scc mRNA. Finally, the effect of TNF-α on IGF-I gene expression was investigated since IGF-I enhances Leydig cell androgen formation and IGF-I gene is expressed in high levels in Leydig cells. TNF-α inhibited both large (7.4 kb) and small species (0.8-1.2 kb) IGF-I mRNA levels in a dose-dependent manner. In conclusion, murine TNF-α is a potent inhibitor of Leydig cell function. TNF-α inhibited both P450scc and IGF-I mRNA gene expression.

Human cultured adrenal fasciculata-reticularis cells are targets for angiotensin-II: effects on cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17 alpha-hydroxylase, and 3 beta-hydroxysteroid-dehydrogenase messenger ribonucleic acid and proteins and on steroidogenic responsiveness to corticotropin and angiotensin-II.

In the present study we have examined the effects of ACTH and angiotensin-II (A-II) on cultured human adult fasciculata-reticularis-specific functions. When cells were cultured in a chemically defined medium, the mRNA levels of cholesterol side-chain cleavage enzyme (P450scc), 17 alpha-hydroxylase/17,20-lyase (P45017 alpha), and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) progressively declined, so that by day 6 of culture, less than 20% of those observed in freshly isolated cells were present. ACTH and A-II, in a dose- and time-dependent manner, enhanced the mRNA levels of the three enzymes and the protein levels of P45017 alpha and 3 beta HSD, but not protein levels of P450scc. At maximal concentrations, the effects of ACTH on P450scc mRNA levels and P45017 alpha mRNA and protein levels were significantly greater than those induced by A-II, but the effects of both hormones on 3 beta HSD mRNA and protein were similar. At maximal concentrations, the effects of ACTH and A-II were additive only on 3 beta HSD mRNA and protein. The cortisol production of cells pretreated with ACTH or A-II was significantly higher than that of control cells, but the effects of ACTH were greater than those of A-II. Moreover, the acute steroidogenic responses to ACTH or A-II of cells pretreated with either hormone, were significantly higher than those of control cells. In conclusion, the present results demonstrate that human adult adrenal fasciculata-reticularis cells are targets for A-II, because 1) A-II acutely stimulates cortisol secretion and causes a long term increase in P450scc, P45017 alpha, and 3 beta HSD mRNA levels; 2) the steroidogenic responsiveness of A-II-pretreated cells to both ACTH and A-II was increased; and 3) the positive effects of A-II alone or in association with ACTH on steroidogenic enzyme gene expression are opposite those previously reported on bovine and ovine adrenal cells.

Changes in Levels of Messenger Ribonucleic Acid for Cytochrome P450 Side-Chain Cleavage and 3β-Hydroxysteroid Dehydrogenase during Prostaglandin F2α-Induced Luteolysis in Cattle1

Regression of the CL causes a dramatic decrease in plasma progesterone levels. To test the hypothesis that the decrease in progesterone involves the down-regulation of mRNA encoding the steroidogenic enzymes, cytochrome P450 side-chain cleavage (P450scc) and/or 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), levels of plasma progesterone and luteal mRNA for P450scc and 3 beta-HSD were measured and correlated during induced luteolysis. Holstein heifers (n = 25) were injected with 25 mg prostaglandin F2 alpha (PGF2 alpha) on Day 6 or 7 of the estrous cycle (Day 0 = estrus) to induce luteal regression. To determine acute changes in plasma progesterone during luteolysis, jugular blood samples were obtained from 5 heifers hourly for 12 h, beginning immediately before injection of PGF2 alpha, and assayed for progesterone by RIA. A significant decrease in plasma progesterone levels was observed as early as 1 h after the PGF2 alpha injection (3.62 vs. 2.72 ng/ml, p < 0.05). Progesterone levels continued to decline with time through 12 h after administration of PGF2 alpha. The other 20 animals were ovariectomized at 0 (n = 6), 2 (n = 4), 12 (n = 4), or 24 (n = 6) h after PGF2 alpha. Levels of P450scc and 3 beta-HSD mRNA were determined in extracts of total luteal RNA by ribonuclease (RNase) protection assay. By 2 h after PGF2 alpha, 3 beta-HSD mRNA levels had decreased by about 40% as compared with levels at time 0 (p < 0.05), and a further significant decrease occurred between 2 and 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)