Interactive stimulation by luteinizing hormone and insulin of the steroidogenic acute regulatory (StAR) protein and 17alpha-hydroxylase/17,20-lyase (CYP17) genes in porcine theca cells

LH and insulin are postulated to jointly stimulate theca-cell androgen biosynthesis in patients with hyperthecosis or polycystic ovarian syndrome. To explore the mechanisms of putative LH and insulin steroidogenic synergy in primary culture of normal theca cells, we have implemented an in vitro serum-free monolayer culture system of Percoll-purified, porcine theca cells harvested from immature ovaries. Initial dose and time course analyses revealed that a maximally effective concentration of LH (100 ng/ml) or insulin (100 ng/ml) individually will drive androstenedione production (at 6 to 48 h) by 1.5- to 2.6- and 1.1- to 1.7-fold, respectively, while combined agonists act synergistically over the interval 12 to 48 h yielding a 3- to 4-fold joint effect. Coadministration of LH and insulin can augment theca-cell concentrations of CYP17 and StAR messenger RNA (mRNA) resulting in 3.4- to 3.9- and 3.8- to 4.1-fold increases at 24 to 48 h, respectively (P < 0.01). Combined LH and insulin stimulation also amplified the nuclear content of intron-specific heterogeneous nuclear (hn)RNAs encoding CYP17 and StAR. Insulin significantly enhanced LH-driven but not basal cAMP accumulation (14-18 vs. 3-5.5 pmol/microg DNA/12-48 h) (P < 0.01). A stable exogenous analog of cAMP, 8 Br-cAMP, mimicked LH's effect on steroidogenesis and StAR and CYP17 gene expression and with insulin stimulated StAR mRNA and hnRNA accumulation synergistically. However, unlike LH, 8 Br-cAMP did not synergize with insulin on theca-cell androstenedione biosynthesis or CYP17 mRNA and hnRNA expression. In summary, the present in vitro data identify molecular interactions of LH and insulin on StAR and CYP17 gene expression, thus establishing potent signaling interfaces between these distinct hormonal agonists in regulating theca-cell steroidogenesis.     

Oxytocin inhibits LH-stimulated production of androstenedione by bovine theca cells

Oxytocin secretion by bovine granulosa cells increases dramatically after the LH/FSH surge. We have shown that oxytocin stimulates progesterone secretion and inhibits FSH-stimulated estradiol secretion in vitro by granulosa cells from bovine preovulatory follicles obtained before the LH/FSH surge. To determine if oxytocin regulates LH-stimulated steroid production by bovine theca interna cells, theca cells were isolated from preovulatory follicles obtained before the LH surge and were cultured for 4 days in the presence or absence of LH (2 or 4 ng/ml), without or with graded doses of oxytocin (125-1000 ng/ml). LH increased accumulation of androstenedione and progesterone. Oxytocin inhibited LH-stimulated androstenedione production, but had no effect on LH-stimulated progesterone production by cultured theca interna. The next objective was to determine if oxytocin regulates LH-stimulated steroidogenesis by modulating the levels of mRNA for steroidogenic enzymes and/or Steroidogenic Acute Regulatory protein (StAR). Low doses of LH alone increased the levels of mRNA for P450 17 alpha-hydroxylase (17 alpha-OH), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450 side-chain cleavage, but not for StAR. In contrast, the effects of oxytocin on LH-stimulated androstenedione production were not associated with changes in the levels of mRNA for steroidogenic enzymes or StAR. These results suggest that oxytocin may play a paracrine role in regulating the follicular/luteal phase shift in steroidogenesis by decreasing androstenedione secretion by theca cells of the ovulatory follicle and that this effect is not mediated by changes in the levels of mRNA for steroidogenic enzymes and StAR.     

A novel compound heterozygous mutation in the CYP17 (P450 17alpha-hydroxylase) gene leading to 17alpha-hydroxylase/17,20-lyase deficiency

Mutations in the CYP17 gene impair steroid biosynthesis in the adrenals and gonads and often cause 17alpha-hydroxylase/17,20-lyase deficiency, leading to amenorrhea, sexual infantilism, and hypokalemic low aldosterone hypertension. Several CYP17 mutations resulting in 17alpha-hydroxylase/17,20-lyase deficiency have been reported previously. In the present study, we found a novel CYP17 mutation from the molecular analysis of a Korean patient with primary amenorrhea with a 46,XX karyotype, and hypokalemic hypertension. We sequenced all 8 exons of the CYP17 gene that were amplified from patient's genomic DNA using polymerase chain reaction (PCR) and found a compound heterozygous mutation in the CYP17 structural gene; a 1-base deletion and a 1-base transversion (TAC-->AA) at codon 329, leading to the production of a truncated protein (1-417 amino acids), and a 3-base deletion (TCC, either 350-351 or 351-352 codon) in the other allele. Restriction enzyme digestion analysis of patient's and parental DNA showed that the 1-base deletion and the 3-base deletion are inherited from mother and father, respectively. Here we conclude that these novel compound heterozygous mutations might account for the patient's clinical manifestations of 17alpha-hydroxylase/17,20-lyase deficiency.     

17alpha-hydroxylase/17,20-lyase deficiency caused by a novel homozygous mutation (Y27Stop) in the cytochrome CYP17 gene

CONTEXT: 17alpha-Hydroxylase/17,20-lyase deficiency, a rare autosomal recessive form of congenital adrenal hyperplasia, is caused by mutations in the cytochrome P450c17 (CYP17) gene. We report on a case of complete 17alpha-hydroxylase/17,20-lyase deficiency due to a novel homozygous mutation of CYP17. DESIGN: A 20-yr-old female Turkish patient (46,XX) presented with primary amenorrhea, sexual infantilism, and easy fatigability. RESULTS: The patient's steroid metabolism showed increased levels of mineralocorticoid precursors and low or undetectable plasma concentrations of 17alpha-hydroxycorticoids, androgens, and estrogens before and after ACTH stimulation. The gas chromatography-mass spectrometry urinary steroid profile was dominated by metabolites of corticosterone and its precursors, while cortisol and C(19)-steroid metabolites were lacking. ACTH, FSH, and LH levels were elevated. These hormonal findings were consistent with a combined and total 17alpha-hydroxylase/17,20-lyase deficiency. A therapy with hydrocortisone and a cyclic estrogen/gestagen substitution was initiated. CONCLUSION: The CYP17 gene analysis revealed homozygosity of the mutation Y27Stop (TAC-->TAA) in exon 1, a mutation that has not been previously described. This novel mutation leads to a stop codon causing a total loss of 17alpha-hydroxlyase/17,20-lyase activity, as reflected biochemically by the detected concentrations of the steroid metabolites.     

Kinetic studies on ovarian C-17,20-lyase activity: effect of luteinizing hormone surge

It has been shown that 3 h after the preovulatory gonadotropin surge, an abrupt decrease occurs in follicular C-17,20-lyase (lyase) activity which causes a decrease in C19-steroid production. To determine the reason for the reduced lyase activity, we used rats that were induced to ovulate by means of PMSG administration. In these rats, a 54% decrease in lyase activity occurred at the peak of the LH surge. When the gonadotropin surge and ovulation were blocked by pentobarbitone the decrease was prevented. Administration of LH to the pentobarbitone-blocked rats reduced lyase activity to well below the level reached after the endogenous gonadotropin surge. In cycling proestrous rats as well, human CG (hCG) decreased the lyase activity, as measured in isolated follicles 3 h after hCG administration. Out of three potential inhibitory steroids for lyase activity; progesterone, 3 alpha,17 alpha-dihydroxy-5 alpha-pregnen-20-one, and 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, only the last compound inhibited competitively ovarian lyase activity. The inhibition constant (Ki) value was 29 microM. In order to explore which of the two activities of the lyase complex is regulated by the gonadotropin, a double label double substrate experiment was conducted using [14C]progesterone with [3H]17 alpha-hydroxyprogesterone (17 alpha-OHP). With this assay procedure, we could determine in the same experiment the site of stimulation, the preferred substrate, and the amount of conversion. The conversion of progesterone to 17 alpha-OHP, as well as the conversion to androstenedione were significantly inhibited throughout the reaction by the gonadotropin. Thus, the changes in ovarian lyase after hCG mimic those of 17 alpha-hydroxylase. The labeling pattern of androstenedione showed that the ovarian lyase complex catalyzed the conversion preferentially from progesterone. Whereas the 3H/14C ratio in androstenedione varied between 0.29 to 0.76, the ratios in the 17 alpha-OHP were from 5 to 22. Thus, the exogenous 17 alpha-OHP did not equilibrate with the product formed from progesterone. The effect of the hCG was to decrease the preference of progesterone over 17 alpha-OHP as substrate. It is concluded that: the LH of the surge inhibits both the 17 alpha-hydroxylase and lyase activities. The ovarian lyase complex shows a preference for progesterone as a substrate rather than 17 alpha-OHP. 17 alpha-OHP is not an obligatory intermediate in androstenedione production in ovarian tissue. hCG affects the ovarian lyase complex by shifting the relative preference of substrates towards 17 alpha-OHP.     

The cytochrome P450(17) alpha (17 alpha-hydroxylase/C17,20-lyase) activity of the junctional zone of the rat placenta

The cytochrome P450 responsible for androgen synthesis by the placenta during the second half of pregnancy in the rat was studied in intact and hypophysectomized animals. The two activities of P450(17) alpha, 17 alpha-hydroxylase and C17,20-lyase, were limited to the junctional zone. C17,20-Lyase activity was greater with progesterone than with 17-hydroxyprogesterone as substrate. Although the apparent Michaelis constants were similar, progesterone had a higher maximum velocity than 17-hydroxyprogesterone. Regardless of substrate, C17,20-lyase activity was greater with NADPH than with NADH as an electron donor, and there was no additive effect using both cofactors. Administration of human chorionic gonadotrophin (hCG; 10 IU at 09.00 and 21.00 h on days 13 and 14 and at 09.00 h on day 15) to intact females resulted in more than a 50% reduction of enzyme activity when measured on day 15. The same dose of hCG given to hypophysectomized animals with delayed implantation, i.e. pituitary removal on day 3 and implantation induced by oestrone 5 days later, had no effect on placental enzyme activity, but increased that in the ovary. Administration of ovine LH by osmotic minipump (days 11-15) to intact females resulted in abortion in all animals. The same treatment to animals hypophysectomized on day 11 produced abortion in three of four rats; enzyme activity was greatly reduced in the single animal with placentas. In contrast, infusion of LH into hypophysectomized animals with delayed implantation increased placental enzyme activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combined 17 alpha-hydroxylase/17,20-lyase deficiency due to a stop codon in the N-terminal region of 17 alpha-hydroxylase cytochrome P-450

Steroid 17 alpha-hydroxylase (cytochrome P-450(17)alpha) mediates both 17 alpha-hydroxylase and 17,20-lyase activities. A relatively rare disease, 17 alpha-hydroxylase deficiency is characterized by defects in either or both of these activities. The molecular basis for variability of the defect is not well understood. We have determined the exonic sequence of the mutant P-450(17)alpha gene from one Japanese patient with combined 17 alpha-hydroxylase/17,20-lyase deficiencies. A stop codon (TGA) due to a single point mutation was found at the position of amino acid 17 in exon 1 of the P-450(17)alpha gene. The presence of a stop codon in the N-terminal region of this gene leads to the absence of a functional P-450(17)alpha protein in adrenal cortex and ovary, and consequently hypertension, primary amenorrhea and osteoporosis in this patient.     

A compound heterozygous mutation in the CYP17 (17alpha-hydroxylase/17,20-lyase) gene in a Chinese subject with congenital adrenal hyperplasia

Mutations in the CYP17 gene impair steroid biosynthesis in the adrenals and gonads, resulting in 17alpha-hydroxylase/17,20-lyase (P450c17) deficiency, leading to amenorrhea, sexual infantilism, hypokalemia, and hypertension. To date, more than 50 mutations in the CYP17 gene associated with congenital adrenal hyperplasia have been described. In this study, we analyzed a 36-year-old phenotypic female, genotypic male, with P450c17 deficiency to compare with an additional group of 50 Chinese subjects without P450c17 deficiency in Taiwan. DNA sequence analysis of the CYP17 gene was performed. The result showed that the proband had a compound heterozygous mutations in exon 6 (CGC-->TGC) that resulted in the substitution of arginine by cysteine at codon 362, and in exon 7 (CCG-->CGG) that resulted in the substitution of proline by arginine at codon 409. In conclusion, we have identified a compound heterozygous mutation in the CYP17 gene in one patient with congenital adrenal hyperplasia in Taiwan.     

17alpha-hydroxylase/17,20-lyase deficiency as a model to study enzymatic activity regulation: role of phosphorylation

Cytochrome P450 17alpha-hydroxylase (CYP17) is a single gene-encoded protein with two activities: 17alpha-hydroxylase and 17,20-lyase. The two catalytic activities are differentially regulated in health and disease. We took advantage of naturally occurring human mutations to understand the molecular bases of this differential regulation. We identified eight novel mutations in the CYP17 gene, different in nature and spread throughout the gene. As posttranslational modifications appear to be important for activity control, we investigated the phosphorylation state of wild-type and mutant CYP17 proteins. Although phospholabeled protein was seen when the wild-type and most mutant proteins were expressed, no phosphorylation was detected for the F417C mutant. F417C is the only 17,20-lyase deficiency case confirmed at the molecular level and represents the first phosphorylation CYP17-deficient mutant. In search of the physiological agents involved in this process, the effect of cAMP was tested on activity and phosphorylation state of our mutant CYP17 proteins. cAMP stimulates activity and phosphorylation in all cases, except in the F417C and R35L mutants. The lack of response to the physiological second messenger might explain the different phenotypes. The F417C mutant protein, which is already shown to be associated with the lack of electron transfer, provides for the first time a link between the electron transfer system and the phosphorylation state of the CYP17 enzyme in the control of 17,20-lyase activity.     

The steroid C-17,20-lyase complex in isolated Graafian follicles: effects of human chorionic gonadotropin

Androstenedione synthesis was studied in isolated rat preovulatory follicles and compared with that of rat testicular tissue using [14C]progesterone together with 17 alpha-hydroxy-[3H]progesterone as substrates in the presence of NADH or NADPH as cofactors. The amount of androstenedione formed was measured by addition of carrier, reisolation, and crystallization to constant specific activity. The labeling patterns of androstenedione and 17 alpha-hydroxyprogesterone (17-OHP) confirmed that both tissues preferentially catalyzed the synthesis of androstenedione from progesterone rather than from 17-OHP. It appears, therefore, that free 17-OHP was not an obligatory intermediate in this reaction. When hCG (5 IU) was administered sc and the follicles were isolated 3 h later, androstenedione synthesis was inhibited whether NADH or NADPH was added as cofactors. By contrast, 17-hydroxylase activity was inhibited only with NADH as cofactor. Hence, the gonadotropin, with NADH as cofactor, specifically reduced progesterone incorporation into androstenedione without affecting incorporation of 17-OHP. Thus, hCG appears to affect androstenedione production from progesterone at two different sites of the lyase complex.     

Differential inhibition of 17alpha-hydroxylase and 17,20-lyase activities by three novel missense CYP17 mutations identified in patients with P450c17 deficiency

The microsomal enzyme cytochrome P450c17 is an important regulator of steroidogenesis. The enzyme has two functions: 17alpha-hydroxylase and 17,20-lyase activities. These functions determine the ability of adrenal glands and gonads to synthesize 17alpha-hydroxylated glucocorticoids (17alpha-hydroxylase activity) and/or sex steroids (17,20-lyase activity). Both enzyme functions depend on correct steroid binding, but it was recently shown that isolated lyase deficiency can also be caused by mutations located in the redox partner interaction domain. In this article we present the clinical history and molecular analysis of two patients with combined 17alpha-hydroxylase/17,20-lyase deficiency and four patients with isolated 17,20-lyase deficiency. In these six patients, four missense CYP17 mutations were identified. Two mutations were located in the steroid-binding domain (F114V and D116V), and the other two mutations were found in the redox partner interaction domain (R347C and R347H). We investigated the activity of these mutated proteins by transfection experiments in COS-1 cells using pregnenolone, progesterone, or their hydroxylated products as a substrate and measuring 17alpha-hydroxylase- and 17,20-lyase-dependent metabolites in the medium. The mutations in the steroid-binding domain (F114V and D116V) of P450c17 caused combined, complete (F114V), or partial (D116V) 17alpha-hydroxylase and 17,20-lyase deficiencies, whereas mutations in the redox partner interaction domain (R347C and R347H) displayed less severe 17alpha-hydroxylase deficiency, but complete 17,20-lyase deficiency. These findings are consistent with the clinical data and support the observation that the redox partner interaction domain is essential for normal 17,20-lyase function of P450c17.     

Combined 17 alpha-hydroxylase/17,20-lyase deficiency due to a 7-basepair duplication in the N-terminal region of the cytochrome P45017 alpha (CYP17) gene

17 alpha-Hydroxylase deficiency is characterized by defects in either or both of the 17 alpha-hydroxylase/17,20-lyase activities. We have elucidated the molecular basis of the combined deficiency of these activities in a Japanese female who is genotypically male and the child of a consanguineous marriage. The complete exonic sequence of the patient's CYP17 (P45017 alpha) gene revealed a seven-basepair duplication (GCGCACA) in exon 2 which leads to a frame shift and, subsequently, a premature stop codon. Because this stop codon occurs N-terminal to the heme-binding sequence, the presence of this mutation leads to the absence of a functional P45017 alpha-protein in adrenal cortex and testis. This, in turn, leads to an absence of sex steroids and excessive secretion of steroids with mineralocorticoid activity and, consequently, female external genitalia and hypertension in this 46XY patient.     

Resistin stimulation of 17alpha-hydroxylase activity in ovarian theca cells in vitro: relevance to polycystic ovary syndrome

CONTEXT: A newly discovered hormone resistin has been shown to be increased in women with polycystic ovary syndrome (PCOS). OBJECTIVE: The purpose of this study was to confirm increased resistin concentrations in women with PCOS and to test the direct effect of resistin on human theca cell androgen production. DESIGN: Resistin was measured in fasting serum samples by RIA. To test the direct effects of resistin on ovarian androgen biosynthesis, human theca cells were cultured with resistin for 3 d in the presence and absence of forskolin and insulin. PATIENTS: Fasting serum samples were obtained from 45 women with PCOS and 74 regularly cycling premenopausal control women in the follicular phase of their menstrual cycles, and ovarian theca cell cultures were established from two control women. RESULTS: The mean serum resistin concentration was increased (40%) in women with PCOS. Serum resistin concentrations correlated positively with body mass index and testosterone in PCOS women but not in controls. There were no significant correlations between resistin and fasting insulin or indicators of insulin resistance when corrected for body mass index. In cultured human theca cells, basal 17alpha-hydroxylase activity was unchanged by resistin alone, but resistin enhanced 17alpha-hydroxylase activity in the presence of forskolin or a combination of forskolin plus insulin. Resistin (> or =1 ng/ml) augmented forskolin and forskolin plus insulin stimulation of CYP17 mRNA expression in a concentration-dependent manner. CONCLUSION: These data indicate that abnormal resistin secretion in PCOS may play a role in causing ovarian hyperandrogenism.     

Hormonal regulation of messenger ribonucleic acids for P450scc (cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase) in cultured human fetal adrenal cells

ACTH has acute and long term effects on adrenal steroidogenesis by week 14 of fetal life. We used human fetal adrenal cells to investigate the long term effect of physiological doses of ACTH on mRNAs for P450scc (the cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase). Monolayer cultures of 18- to 24-week gestation fetal zone adrenal cells were maintained in the presence and absence of 10(-9) or 10(-8) M ACTH for up to 12 days. As assessed by RNA dot blots probed with cloned homologous human cDNAs, ACTH increased P450scc and P450c17 mRNAs 4- and 9-fold, respectively, over control values on day 7 of culture. ACTH-mediated stimulation was slightly less on day 12 of culture. The ACTH-mediated accumulation of those mRNAs were time dependent. When cells were exposed to a single 10(-8)-M dose of ACTH, the amount of P450scc and P450c17 mRNA was increased by 24 h, reaching a maximum at 48 h and diminishing by 72 h. When cells were maintained in 10(-8) M ACTH continuously, mRNA for both enzymes accumulated in a similar pattern, reaching a peak at 48 h but remaining at nearly maximal values thereafter, up to 96 h. Dibutyryl cAMP (10(-3) M) mimicked these stimulatory actions of ACTH, although its effect was greater at 24 h and more stable up to 96 h. Angiotensin II (1-100 ng/mL) and hCG (1-100 ng/mL) had no effect on accumulation of P450scc and P450c17 mRNAs. The production of both dehydroepiandrosterone sulfate and cortisol also was stimulated by ACTH, suggesting that the increased mRNAs were translated into active enzymes. These results indicate that ACTH induces human fetal adrenal cells to accumulate mRNAs for both P450scc and P450c17; this effect of ACTH is probably mediated by cAMP. Chronic 96-h stimulation of human fetal adrenal cells did not diminish their responsiveness to ACTH. Together with our earlier studies of the human fetal adrenal, these data indicate that fetal adrenal tissue does not exhibit the desensitization to trophic hormone stimulation characteristic of adult tissue.     

Molecular target of endocrine disruption in human luteinizing granulosa cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin: inhibition of estradiol secretion due to decreased 17alpha-hydroxylase/17,20-lyase cytochrome P450 expression

Estradiol (E2) production by human luteinized granulosa cells (hLGC) is inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The molecular target of TCDD toxicity has not been identified. The decrease in E2 is ameliorated by androgen substrate addition and is not associated with changes in aromatase cytochrome P450 (P450arom) activity or protein expression. An antihuman 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) antisera and a direct radiometric assay of 17,20-lyase activity were used to test the hypothesis that TCDD targets P450c17, thereby decreasing substrate availability for E2 synthesis by hLGC. P450c17 expression and 17,20-lyase activity were detected in hLGC with high levels of E2 secretion. Western immunoblot analysis demonstrated that TCDD treatment of hLGC decreased the expression of P450c17 by as much 50% (P < 0.05). TCDD exposure induced a 65% decrease in 17,20-lyase activity (P < 0.05), but no changes were seen in P450arom or in nicotinamide adenine dinucleotide phosphate (reduced)-cytochrome P450 oxidoreductase (reductase). Furthermore, the decreases in P450c17 and 17,20-lyase were proportional to the inhibition of E2 secretion. We conclude that the molecular target for endocrine disruption of hLGC by TCDD is P450c17, specifically decreasing the supply of androgens for E2 synthesis, and that it does not involve either P450arom or the redox partner protein reductase.     

Cloning and gene expression of P450 17alpha-hydroxylase,17,20-lyase cDNA in the gonads and brain of the fathead minnow Pimephales promelas

P450 17alpha-hydroxylase,17,20-lyase (P450c17) is a key steroidogenic enzyme in the production of androgens and, therefore, is also indispensable for the production of oestrogens (that are produced from the aromatisation of androgens). In this study, P450c17 cDNA was cloned from the ovary of the fathead minnow (FHM) and its gene expression was examined in the gonads and brains of male and female FHM at different stages of gonadal development with a view to developing an understanding of its involvement in the reproductive physiology in this species. The FHM-P450c17 cDNA sequence cloned was 1812 bp in length, with an open reading frame of 1554 nucleotides encoding a protein of 518 amino acids. Amino acid identity of FHM-P450c17 with P450c17s in other animals was up to 81.8% in other teleosts (channel catfish), 62% in elasmobranches (spiny dogfish), 64% in birds (chicken), and up to 48.8% in mammals (human). FHM-P450c17 gene expression occurred in the ovary, testis, and also in the brain (both male and female) at all stages of sexual development studied. Expression in the brain was at least 30-fold lower than in the gonads, but consistent in all fish life stages studied. In the testis, FHM-P450c17 gene expression was negatively correlated with gonadal development, but there was no obvious association between P450c17 gene expression and sexual development in the ovary, or brain (in both males and females). The results from this study demonstrate the expression of P450c17 in the brain for the first time in fish. Enzymatic studies are now needed to investigate the possible role of P450c17 in neurosteroid production in teleosts.