Cortisol induces follicle regression, while FSH prevents cortisol-induced follicle regression in pigs.

During follicular development, a few dominant follicles develop to large antral dominant follicles, whereas the remaining follicles undergo atretic degeneration. Because vascularization on the follicular surface is a morphological feature of dominant follicles, we previously classified these follicles as vascularized follicles (VFs) and non-VFs (NVFs). In NVFs, progesterone producing genes were expressed similarly to that in VFs; however, the progesterone concentration in follicular fluid was low in large NVFs. Therefore, we estimated that progesterone is converted to cortisol, which induces the loss of follicular functions. In this study, we comparative analyzed the expression of genes for progesterone converting enzymes (Cytochrome (CYP)11B1, CYP21A2, Hydroxysteroid (HSD)11B2) and cortisol receptor (NR3C1) in VF and NVF granulosa cells. In NVFs, expression of cortisol producing genes (CYP11B1 and CYP21A2) was higher than in VFs. Expression of the gene for the cortisol metabolizing enzyme HSD11B2 in NVFs was significantly lower than in VFs. In NVFs, accompanied by increasing cortisol concentration in follicular fluid, apoptosis of granulosa and cumulus cells was observed. Cultivation with FSH and metyrapone (a CYP11B1 inhibitor) of NVF cumulus-oocyte complexes inhibited apoptosis of cumulus cells and induced cumulus cell proliferation and oocyte maturation. Cortisol-induced CYP11B1 and CYP21A2 expression, whereas FSH-induced HSD11B2 mRNA expression in VF granulosa cells in the presence of cortisol. Furthermore, an addition of 18ß-glycyrrhetinic acid (18-GA; a HSD17B2 inhibitor) to cortisol and FSH-containing medium increased apoptosis of VF granulosa cells. These results suggested that cortisol is a stimulatory factor that induces follicular atresia; furthermore, inhibition of cortisol production by FSH might increase the number of healthy preovulatory follicles in pigs.

Preeclampsia induced by cadmium in rats is related to abnormal local glucocorticoid synthesis in placenta.

BACKGROUND: Cadmium (Cd) is a major environmental pollutant that causes multiple adverse health effects in humans and animals. In this study, we investigated Cd-mediated toxic effects in rats during pregnancy and endocrine intervention in the placenta. METHODS: We exposed pregnant rats to intraperitoneal Cd (CdCl2) at various doses (0, 0.25, and 0.5 mg/kg BW/day) from days 5 to 19 of pregnancy and evaluated the maternal-placental-fetal parameters linked to preeclampsia. We measured the corticosterone level in rat serum and placental tissue by sensitive ELISA and also analyzed the expression of glucocorticoid synthesis enzymes in the placenta. RESULTS: Key features of preeclampsia (PE), including hypertension, proteinuria, glomerular endotheliosis, placental abnormalities and small fetal size, appeared in pregnant rats after injection with 0.5 mg/kg BW/day Cd. The placental corticosterone production and maternal and fetal plasma corticosterone levels were increased in rats treated with 0.5 mg/kg BW/day Cd (P <0.01). The expression of 21-hydroxylase (CYP21) and 11beta-hydroxylase (CYP11B1), enzymes essential for corticosteroid synthesis, were increased in Cd-exposed placenta (P <0.01). 11beta-hydroxysteroid dehydrogenase (11beta-HSD2), a dominant negative regulator of local glucocorticoid levels, was decreased in Cd-exposed placenta (P <0.01). CONCLUSIONS: Our study demonstrates for the first time that changes in placental glucocorticoid synthesis induced by Cd exposure during pregnancy could contribute to preeclamptic conditions in rats.

Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice.

We have used a simple binomial model of stochastic transgene inactivation at the level of the chromosome or transgene, rather than the cellular level, for the analysis of two mouse transgenic lines that show variegated patterns of expression. This predicts the percentages of cells that express one, both or neither alleles of the transgene in homozygotes from the observed percentages of cells, which express the transgene in hemizygotes. It adequately explained the relationship between the numbers of cells expressing the transgene in hemizygous and homozygous mosaic 21OH/LacZ mouse adrenals and mosaic BLG/7 mouse mammary glands. The binomial model also predicted that a small proportion of cells in mosaic mammary glands of BLG/7 homozygotes would express both BLG/7 alleles but published data indicated that all cells expressing the transgene showed monoallelic expression. Although it didn't fit all of the BLG/7 data as precisely as a more complex model, which used several ad hoc assumptions to explain these results, the simple binomial model was able to explain the relationship in observed transgene expression frequencies between hemizygous and homozygous mosaic tissues for both 21OH/LacZ and BLG/7 mice. It may prove to be a useful general model for analysing other transgenic animals showing mosaic transgene expression.

Leptin alters adrenal responsiveness by decreasing expression of ACTH-R, StAR, and P450c21 in hypoxemic fetal sheep.

The late gestation increase in adrenal responsiveness to adrenocorticotropin (ACTH) is dependent upon the upregulation of the ACTH receptor (ACTH-R), steroidogenic acute regulatory protein (StAR), and steroidogenic enzymes in the fetal adrenal. Long-term hypoxia decreases the expression of these and adrenal responsiveness to ACTH in vivo. Leptin, an adipocyte-derived hormone which attenuates the peripartum increase in fetal plasma cortisol is elevated in hypoxic fetuses. Therefore, we hypothesized that increases in plasma leptin will inhibit the expression of the ACTH-R, StAR, and steroidogenic enzymes and attenuate adrenal responsiveness in hypoxic fetuses. Spontaneously hypoxemic fetal sheep (132 days of gestation, PO(2) ≈ 15 mm Hg) were infused with recombinant human leptin (n = 8) or saline (n = 7) for 96 hours. An ACTH challenge was performed at 72 hours of infusion to assess adrenal responsiveness. Plasma cortisol and ACTH were measured daily and adrenals were collected after 96 hours infusion for messenger RNA (mRNA) and protein expression measurement. Plasma cortisol concentrations were lower in leptin- compared with saline-infused fetuses (14.8 ± 3.2 vs 42.3 ± 9.6 ng/mL, P < .05), as was the cortisol:ACTH ratio (0.9 ± 0.074 vs 46 ± 1.49, P < .05). Increases in cortisol concentrations were blunted in the leptin-treated group after ACTH(1-24) challenge (F = 12.2, P < .0001). Adrenal ACTH-R, StAR, and P450c21 expression levels were reduced in leptin-treated fetuses (P < .05), whereas the expression of Ob-Ra and Ob-Rb leptin receptor isoforms remained unchanged. Our results indicate that leptin blunts adrenal responsiveness in the late gestation hypoxemic fetus, and this effect appears mediated by decreased adrenal ACTH-R, StAR, and P450c21 expression.

Cytochrome P450 dysregulations in thioacetamide-induced liver cirrhosis in rats and the counteracting effects of hepatoprotective agents.

Dysregulations of cytochromes P450 (P450s) under liver injury have been extensively studied. However, little is known about the possible reversing effects of hepatoprotective agents, the understanding of which is of great importance in guiding clinical dosage adjustment for patients with liver injury. This study aims to investigate the dysregulation patterns of major P450s in thioacetamide (TAA)-induced liver cirrhosis in rats and the potential counteracting effects of hepatoprotective agents schisandra lignans extract (SLE) and dimethyl diphenyl bicarboxylate (DDB). TAA intoxications for 6 weeks induced apparent liver injury and dramatically reduced the hepatic protein expressions of CYP1A2, CYP2C6, CYP2E1, and CYP3A2 to 18, 71, 30, and 21% of that in the normal control, respectively. Both SLE and DDB treatments could significantly reverse the TAA-induced loss of P450 protein levels, which may be ascribed to their hepatoprotective effects and direct P450-inducing effects that have been confirmed in healthy rats. However, the recovery of enzyme activities of most P450s by SLE and DDB treatment was less evident than that for the protein expression levels. TAA exhibited NADPH-, time-, and concentration-dependent inactivating effects on all of the four major P450 isozymes; both DDB and GSH showed little effects on counteracting such an inactivation efficacy. These findings provided a good explanation on the disproportional effects of hepatoprotective agents in recovering the protein levels and enzyme activities of TAA-induced dysregulated P450s.

[Unique steroid 21-hydroxylase gene CYP21A2 polymorphism in patients with hyperandrogenism signs].

Hyperandrogenism is a medical condition characterized by excessive production of male sex hormones (androgens) in woman organism. One of the major causes of hyperandrogenism is the autosomal-recessive disorder--congenital adrenal hyperplasia (CAH). The mutational defects in the steroid 21-hydroxylase CYP21A2 gene causing steroid 21-hydroxylase deficiency account for over 90% of CAH cases. Our paper describes the sequencing results of entire CYP21A2 gene from 15 patients with hyperandrogenism signs, which had not nine most prevalent mutations associated with nonclassic CAH as it was previously established. 26 polymorphisms were found by sequencing among which 25 were known previously and 23 of them are referred to "normal" gene variants which do not associated with CAH. At the same time the gene of every patient had unique its own distinctive combination of polymorphisms. New SNP represents synonymous substitution C --> T in 3' part of exon 8. All detected SNPs are not regularly distributed but are clustered along the gene. Notably, they were found in the neighborhood of initiation and termination codons and near the intron-exon boundaries of introns 2, 6 and 8. We hypothesize that "normal" clinically insignificant per se SNPs in unique combinations may influence spatial structure of CYP21A2 mRNA or its pre-mRNA splicing efficiency and decrease gene expression level. This assumption may explain the mechanism of pathological phenotype development in our patients.

Fenofibrate-induced decrease of expression of CYP2C11 and CYP2C6 in rat.

This short communication is aimed to investigate whether the widely used hypolipidemic drug fenofibrate affects CYP2C11 and CYP2C6 in rats, both counterparts of human CYP2C9, known to metabolise many drugs including S-warfarin and largely used non-steroidal antiinflammatory drugs such as ibuprofen, diclofenac and others. The effects of fenofibrate on the expression of rat liver CYP2C11 and CYP2C6 were studied in both healthy Wistar rats and hereditary hypertriglyceridemic rats. Both strains of rats were fed on diet containing fenofibrate (0.1% w/w) for 20 days. Fenofibrate highly significantly suppressed the expression of mRNA of CYP2C11 and less that of CYP2C6 in liver microsomes of both rat strains; this effect was associated with a corresponding decrease in protein levels. The results indicate that the combination of fenofibrate with drugs metabolised by CYP2C9 in humans should be taken with caution as it may lead, for example, to the potentiation of warfarin effects. This type of drug interaction has been observed previously and the results presented here could contribute to the explanation of their mechanism.

Monogenic and polygenic models detected in steroid 21-hydroxylase deficiency-related paediatric hyperandrogenism.

AIMS: Hyperandrogenism, although mostly due to polygenic interactions, is monogenic for some enzymatic adrenal deficiencies. This study evaluates mono- and biallelic 21-hydroxylase deficiency (21OHD)-related hyperandrogenism in pediatric patients. Sensitizing and protective polymorphisms were investigated in carriers and cryptic forms of 21OHD. METHODS: The study involved a monogenic analysis of CYP21A2 in patients (375 nonclassical 21OHD [NC21OHD] children; 306 hyperandrogenic 21OHD carriers, n = 306) and a polygenic association study (CAPN10-UCSNP44, PON1-108, TNFR2-M196R, IGF2-ApaI and IRS1-G972R polymorphisms) of 170 hyperandrogenic carriers plus 277 family members (control groups). The metabolic marker 17OH progesterone defined the degree of deficiency; clinical expressivity was determined by pediatric endocrinologists. RESULTS: The group of 21OHD carriers manifesting hyperandrogenism was enriched in the CAPN-UCSNP44 rare variant in homozygosity (4.9 vs. 0.4%, NCBI data for the general population; p = 0.004). In our patients and controls, contrasting distributions were observed for this and another polymorphism, TNFR2-196R. In a recessive model, their rare variants were more frequently detected among the forms with high (p = 0.048) and low (p = 0.034) expressivity respectively. CONCLUSIONS: 21OHD-related pediatric hyperandrogenism follows monogenic and polygenic models. The opposite behaviors in terms of clinical expressivity detected for CAPN-UCSNP44 and TNFR2-M196R rare variants suggest these variants to be sensitizing and protective factors respectively in adrenal hyperandrogenism.

Functional characterization of three CYP21A2 sequence variants (p.A265V, p.W302S, p.D322G) employing a yeast co-expression system.

Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase (CYP21A2) deficiency is the commonest inborn error in steroid hormone biosynthesis. Functional in vitro assessment of mutant activity generally correlates well with clinical phenotype and therefore has contributed greatly to phenotype prediction in this CAH variant. Three CYP21A2 sequence variants (g.1641C>T, p.A265V; g.1752G>C, p.W302S; and g.2012A>G, p.D322G) identified in patients with non-classic and simple virilizing CAH were characterized using a yeast co-expression system and a computational three-dimensional CYP21A2 model. Computational analysis of the mutants in the three-dimensional structural model predicted no relevant effect of p.A265V, while p.W302S and p.D322G were predicted to impact significantly on enzyme function. Consistent with these findings, in vitro mutant analysis revealed enzyme activity similar to wild-type for p.A265V, whereas p.W302S and p.D322G exerted activities compatible with simple virilizing and non-classical CAH, respectively. The results indicate that p.A265V is an allelic variant rather than a disease-causing amino acid change, whilst p.W302S and p.D322G could be confirmed as functionally relevant mutations. These findings emphasize the value of in vitro functional analysis of sequence variations in predicting genotype-phenotype correlations and disease severity.

Age-specific changes in sex steroid biosynthesis and sex development.

Normal male sex development requires the SRY gene on the Y chromosome, the regression of Müllerian structures via anti-Müllerian hormone (AMH) signalling, the development of the Wolffian duct system into normal male internal genital structures consequent to testosterone secretion by the testicular Leydig cells, and finally, sufficient activation of testosterone to dihydrotestosterone by 5alpha-reductase. All these events take place during weeks 8-12 of gestation, a narrow window of sexual differentiation. Recent studies in human fetal development have demonstrated the early fetal expression of the adrenocorticotrophic hormone (ACTH) receptor and all steroidogenic components necessary for the biosynthesis of cortisol. These findings provide compelling evidence for the assumed pathogenesis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, diminished feedback to the pituitary due to glucocorticoid deficiency, subsequent ACTH excess, and up-regulation of adrenal androgen production with subsequent virilization. Another CAH variant, P450 oxidoreductase deficiency, manifests with 46,XX disorder of sex development (DSD), i.e., virilized female genitalia, despite concurrently low circulating androgens. This CAH variant illustrates the existence of an alternative pathway toward the biosynthesis of active androgens in humans which is active in human fetal life only. Thus CAH teaches important lessons from nature, providing privileged insights into the window of human sexual differentiation, and particularly highlighting the importance of steroidogenesis in the process of human sexual differentiation.

[Expression of the human steroid 21-hydroxylase gene and its mutant variant C169R in insect cells and functional analysis of expression products].

Steroid 21-hydroxylase is a key enzyme of glucocorticoid and mineralocorticoid biosynthesis in the adrenal gland that belongs to the family of microsomal cytochrome P450. The steroid 21-hydroxylase deficiency is the most frequent cause of the congenital adrenal hyperplasia. The human steroid 21-hydroxylase (CYP21 A) and its mutant variant (C 169R) found previously in patient with the classical congenital adrenal hyperplasia were synthesized for the first time in the insect cell lines Sf9 and Hi5 infected by recombinant baculoviruses. Under optimal conditions the level of CYP21A2 production in insect cells achieves 28% of the total microsomal protein. C169R mutation does not effect the synthesis of CYP21 A2 in insect cells and does not prevent the incorporation of the enzyme into the membranes of endoplasmic reticulum. Functional analysis of the mutant enzyme in vitro suggested the virtually complete lack of catalytic activity towards two substrates - progesterone and 17-hydroxyprogesterone.

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450-dependent biotransformation in fission yeast.

Cytochrome P450s are haem-containing monooxygenases that catalyse a variety of oxidations utilizing a large substrate spectrum and are therefore of interest for biotechnological applications. We expressed human CYP21 in fission yeast Schizosaccharomyces pombe as a eukaryotic model for P450-dependent whole-cell biotransformation. The resulting strain displayed strong steroid hydroxylase activity that was accompanied by contrary effects on respiration and non-respiratory oxygen consumption, which combined to a significant decline in total oxygen consumption of the cells. While production of ROS (reactive oxygen species) decreased, the TCA cycle activity increased, as was shown by metabolic flux (METAFoR) analysis. Pentose phosphate pathway (PPP) activity was found to be negligible, regardless of growth phase, CYP21 expression or biocatalytic activity, indicating that NADPH levels in Sz. pombe are sufficiently high to support an exogenous P450 without adaptations of central carbon metabolism. We conclude from these data that neither oxygen supply nor NADPH availability are limiting factors in P450-dependent biocatalysis in Sz. pombe.

Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli.

Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Escherichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2micromol/L culture (>60mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E. coli yielding sufficient quantities for both biochemical and biophysical studies.

Sex difference in the principal cytochrome P-450 for tributyltin metabolism in rats.

Tributyltin is metabolized by cytochrome P-450 (CYP) system enzymes, and its metabolic fate may contribute to the toxicity of the chemical. In the present study, it is examined whether sex differences in the metabolism of tributyltin exist in rats. In addition, the in vivo and in vitro metabolism of tributyltin was investigated using rat hepatic CYP systems to confirm the principal CYP involved. A significant sex difference in metabolism occurred both in vivo and in vitro, suggesting that one of the CYPs responsible for tributyltin metabolism in rats is male specific or predominant at least. Eight cDNA-expressed rat CYPs, including typical phenobarbital (PB)-inducible forms and members of the CYP2C subfamily, were tested to determine their capability for tributyltin metabolism. Among the enzymes studied, a statistically significant dealkylation of tributyltin was mediated by CYP2C6 and 2C11. Furthermore, the sex difference in metabolism disappeared in vitro after anti-rat CYP2C11 antibody pretreatment because CYP2C11 is a major male-specific form in rats. These results indicate that CYP2C6 is the principal CYP for tributyltin metabolism in female rats, whereas CYP2C11 as well as 2C6 is involved in tributyltin metabolism in male rats, and it is suggested that CYP2C11 is responsible for the significant sex difference in the metabolism of tributyltin observed in rats.

Expression of cytochrome P450c17 and other steroid-converting enzymes in the rat kidney throughout the life-span.

We have investigated the metabolism of [14C]-labelled progesterone (P4) and dehydroepiandrosterone (DHEA) by kidney tissues of newborn and 7-, 15-, 30-, 60- and 365-day-old rats of both sexes. The following enzymes were revealed at all ages by radiochemical identification of the corresponding products: 5alpha-reductase, cytochromes P450c17 and P450c21, 3beta-hydroxysteroid dehydrogenase (HSD)/delta5-delta4 isomerase, and 17beta- and 20alpha-HSDs, catalyzing reductive reactions. The major P4 metabolites were 5alpha-reduced C21 steroids, whose formation was almost completely suppressed by the 5alpha-reductase 4-azasteroid inhibitor, PNU 156765. Androstenedione and testosterone were also formed via 17alpha-hydroxyprogesterone, together with 11-deoxycorticosterone and 20alpha-dihydroprogesterone. DHEA was mainly converted to androst-5-ene-3beta,17beta-diol, with smaller amounts of the above androgens. Cytochrome P450c17 mRNA and protein were demonstrated by Northern blotting and Western blotting analyses, respectively. P450c17 mRNA, assessed by Northern blotting, protein and catalytic activity all peaked in the kidney samples at 15 days of life and declined thereafter. Cytochrome P450arom was below the level of detection of semi-quantitative RT-PCR. Since the rat kidney has been previously shown to contain cytochrome P450scc as well as androgen and estrogen receptors, it is suggested that it is capable of autonomous hormonal steroidogenesis and that renal steroids, or nephrosteroids, may act locally, in a paracrine or autocrine fashion.

Identification of principal cytochrome P-450 in triphenyltin metabolism in rats.

The in vivo and in vitro metabolism of triphenyltin using rat hepatic cytochrome P-450 (CYP) systems was investigated to confirm the specific CYP that is closely related to triphenyltin metabolism. No significant sex differences occurred between the in vivo and in vitro metabolic patterns of the chemical, indicating that the principal CYP for triphenyltin metabolism in rats is not a sex-specific form of CYP. In addition, seven types of complementary DNA (cDNA)-expressed rat CYPs, typical phenobarbital (PB)-inducible forms and the CYP2C subfamily were tested to determine the activity of triphenyltin metabolism. Among the CYP isoforms studied, although CYP2B1 had a small metabolic capacity, a marked dearylation of the chemical was induced by CYP2C6. Furthermore, anti-rat CYP2C6 antibodies and cimetidine, a selective CYP2C6 inhibitor, inhibited triphenyltin dearylation activity in the hepatic microsomes of rats. Taken together, these findings suggest that CYP2C6 is the principal CYP for the triphenyltin metabolism in rats.

Interpulse growth hormone secretion in the episodic plasma profile causes the sex reversal of cytochrome P450s in senescent male rats.

Humans as well as other mammals experience an aging-related decline in drug metabolism as well as a diminution in growth hormone secretion. In the case of rats, these events are more pronounced in senescent males, whose expression of male-specific isoforms of cytochrome P450, the major drug-metabolizing enzymes and constituting approximately 60-70% of the total cytochrome P450 in male rat liver, is completely suppressed, whereas female-dependent isoforms are remarkably induced to female-like levels. Overlooked in these independently reported studies is the fact that "signals" inherent in the masculine episodic and female continuous growth hormone profiles regulate expression and/or suppression of the dozen or so sex-dependent cytochrome P450 isoforms in rat liver. Whereas previous studies identified profound reductions in the pulse amplitudes of the masculine growth hormone profile as the cause for the diminished hormone secretion during aging, pulse heights are not recognized by the cytochromes as regulatory signals. Instead, we have shown that just a nominal secretion of growth hormone during the usual growth hormone-devoid interpulse period in the masculine episodic profile can explain the complete repression of male-specific CYP2C11, CYP3A2, and CYP2A2 and induction of female-dependent CYP2C12, CYP2C6, and CYP2A1 observed in senescent male rats.

Steroid 21-hydroxylase expression in cultured rat astrocytes.

Over the past decade or so it has become widely recognised that the brain is a significant steroidogenic organ. Many publications have highlighted the ability of the brain to synthesise and interconvert a large number of steroid products including cholesterol, progesterone and testosterone. In this study, in vitro experiments were performed to determine if 21-hydroxylation of steroids is undertaken by rat brain astrocytes in culture. This is a common reaction that occurs in the adrenal gland and other organs in mammals, catalysing the conversion of pregn-4-ene-3,20-dione (progesterone) to 21-hydroxypregn-4-ene-3,20-dione (deoxycorticosterone). Previous reports have indicated that 21-hydroxylation occurs within the rat brain, however, the precise identity of the cells expressing 21-hydroxylase has not yet been determined. Several metabolites, such as 5alpha-pregnan-3alpha-ol-20-one (tetrahydroprogesterone) and 3alpha,21-dihydroxy-5-pregnan-20-one (tetrahydrodeoxycorticosterone) were of particular interest because of their modulatory role in neuronal function, such as their agonist activity at gamma-aminobutyric acid (GABA(A)) receptors. Evidence was obtained for the expression of peripheral 21-hydroxylase enzyme (P450c21) in cultured rat brain astrocytes by a combination of mass spectroscopy and molecular biology techniques. This is a significant finding as expression of 21-hydroxylase within astrocytes may be indicative of a wider role for these cells in modulating neuronal behaviour.

Molecular cloning and expression of guinea pig cytochrome P450c21 cDNA (steroid 21-hydroxylase) isolated from the adrenals.

In mammals, the P450c21 enzyme mediates 21-hydroxylase activity by transforming progesterone and 17-hydroxyprogesterone into deoxycorticosterone (DOC) and 11-deoxycortisol (11-DOC), respectively. Previous studies have shown that among the adrenal steroid hydroxylase enzymes involved in C19 steroid and glucocorticoid syntheses, P450c21 plays an important role, because it is localized at the key branch between glucocorticoids and C19 steroid production. Its implication in congenital adrenal hyperplasia is also of great clinical interest. In this study, in addition to describing the isolation of the P450c21 cDNA from guinea pig (GP) adrenal and comparing it to those from other species, we report on its tissue-distribution and on the activity of the recombinant protein towards progesterone and 17-hydroxyprogesterone. The guinea pig P450c21 includes the full-length coding region (1464 nucleotide) that is translated to a protein of 488 amino acids. The clone shares highly conserved regions with other species. The guinea pig P450c21 cDNA hybridized with a major transcript of 2.1kb and with two minor related transcripts of 1.8 and 1.5 kb and was found to be adrenal-specific among the various tissues analyzed. Characterization of the enzymatic activity by transient transfection of the guinea pig P450c21 cDNA in human embryonic kidney 293 cells indicated a net preference for the 21-hydroxylation of 17-hydroxyprogesterone in comparison to the progesterone substrate. Assays showed a maximum conversion rate of 12.5% for the conversion of progesterone into deoxycorticosterone (mineralocorticoid pathway), whereas the guinea pig P450c21 demonstrated a higher activity with 17alpha-hydroxyprogesterone, with 55% of 11-deoxycortisol formation (glucocorticoid pathway) after 48 h. Adrenocorticotropin and an analogue of the second messenger cyclic adenosine monophosphate specifically increased the abundance of P450c21 mRNA levels in guinea pig adrenal cells.