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J I Mason

Publications and source records attributed to J I Mason.

At least 127 records · Page 7Linked to original sources
PubMed1990

Placental steroid hormones.

The intent of this review was a selective consideration of recent advances in understanding placental steroidogenesis in humans. While we have omitted material, both intentionally and unintentionally, we hope this discourse presents a flavour of the current molecular endocrinology of placental steroidogenesis. In particular, advances in knowledge as it relates to the enzymes involved in progesterone and oestrogen metabolism have been addressed and correlated with placental development. Finally, because relatively less is known about regulation of steroidogenesis in the human placenta beyond the aspects of growth and differentiation, we have discussed regulation in general terms using recent data obtained in animal species.

Androstenedione↗
PubMed1990

Characterization of the steroid-metabolizing capacity of the hepatic cytochrome P450IIC5 expressed in COS-1 cells: 3 beta-hydroxysteroid dehydrogenase/delta 5----4 isomerase type activity.

Cytochrome P450IIC5 (rabbit liver 21-hydroxylase) is unusual among hepatic forms of cytochromes P450 because it catalyzes the conversion of one active steroid hormone (progesterone) to another active hormone (deoxycorticosterone). Another interesting aspect of this steroid-hydroxylating enzyme is the ability to convert delta 5-3 beta-hydroxysteroids to the delta 4-3-ketosteroid configuration. The delta 5-3-beta-hydroxysteroid, pregnenolone, was readily 21-hydroxylated, and this product was further metabolized to the delta 4-3-ketosteroid, deoxycorticosterone. It is suggested that the mechanism of this cytochrome P450-mediated, 3 beta-hydroxysteroid dehydrogenase/delta 5----4 isomerase-like reaction is through a gem-diol formation. In this study, COS-1 cells were transfected with the plasmid encoding cytochrome P450IIC5 to express a functional enzyme within the cell milieu. Transfected COS cells preferentially metabolize pregnenolone compared with all other steroids tested. Progesterone and 17 alpha-hydroxypregnenolone are also 21-hydroxylated, whereas 17 alpha-hydroxyprogesterone is a poor substrate. Substrate preference of this 21-hydroxylase differs from that seen with bovine adrenal P450XXIA1 (formerly P450C21) hydroxylase. Additionally, this study demonstrated that C19 steroids, like dehydroepiandrosterone and androstenedione, are hydroxylated at the 16 alpha position. Contrary to previous reports, no metabolite of estradiol-17 beta was detected, presumably due to the unstable nature of catechol estrogens (2-hydroxyestradiol).

Animals↗
PubMed1990

Immunohistochemical study of 3 beta-hydroxysteroid dehydrogenase in sex cord-stromal tumors of the ovary.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD), which converts pregnenolone to progesterone, was localized immunohistochemically in 18 thecomas, 23 fibromas, 5 granulosa-cell tumors, 5 sclerosing stromal tumors, and 2 steroid-cell tumors. Immunohistochemical study of estrogen, progesterone, and testosterone was also performed in serial sections of thecomas and fibromas. In thecomas, immunoreactivity of 3 beta-HSD was observed only in luteinized theca cells and thecomatous tumor cells with abundant pale to vacuolated cytoplasm but not in spindled tumor cells and thecomatous tumor cells with small to moderate amounts of pale to vacuolated cytoplasm. Immunoreactivity of steroids was not observed in thecomas except for testosterone immunoreactivity in one case. No immunoreactivity of steroids or the enzyme was present in fibromas. No tumor cells were positive for 3 beta-HSD in any of the cases of granulosa-cell tumor examined. Immunoreactivity of 3 beta-HSD was present in cells in steroid-cell tumors and polygonal tumor cells with prominent cytoplasmic vacuoles in two cases of sclerosing stromal tumor. Thus, 3 beta-HSD can be a good immunohistochemical marker of steroidogenesis in functioning ovarian neoplasms.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland.

The fetal zone of the human fetal adrenal (HFA) gland is established to have decreased 3 beta-hydroxysteroid dehydrogenase/delta 4-5 isomerase (3 beta HSD) activity compared to the neocortex or definitive zone. 3 beta HSD activity, however, can be induced in primary cell culture through treatment with ACTH. Therefore, the HFA with two distinct steroidogenic zones with differences in 3 beta HSD activity as well as the capacity to increase 3 beta HSD activity in response to ACTH provides an excellent model to study the regulation of this enzyme. The presence of 3 beta HSD in the fetal and neocortex zones of the HFA was examined using a polyclonal antibody raised against purified human placental microsomal 3 beta HSD. After homogenates of the fetal and neocortical zones of the HFA were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and immunoblotted, the presence of the 3 beta HSD protein with a molecular size of 45 kDa could be demonstrated only in the neocortical zone. ACTH treatment (greater than 2 days) of fetal and neocortical zone explant cultures produced increases in cortisol secretion associated with the respective levels of immunodetectable 3 beta HSD protein. Cortisol and dehydroepiandrosterone sulfate were the respective principal steroid products of neocortical and fetal zone explants. After ACTH treatment, immunodetectable 3 beta HSD was induced to a greater magnitude in the neocortex. These findings provide evidence that the lack of 3 beta HSD activity in the fetal zone, previously considered to be the result of the presence of an endogenous inhibitor, is due to an absence of the protein in this portion of the gland. The lack or minimal expression of 3 beta HSD in the fetal zone of HFA may be due to the action (or lack thereof) of a tissue-specific factor regulating the synthesis of 3 beta HSD.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase from placenta: expression in nonsteroidogenic cells of a protein that catalyzes the dehydrogenation/isomerization of C21 and C19 steroids.

The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase is reported. The insert contains an open reading frame encoding a protein of 372 amino acids, the initial 29 amino acids corresponding to the N-terminal sequence identified from the purified human placental microsomal enzyme. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental microsomal 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, very efficiently oxidized 5 alpha-androstan-3 beta,17 beta-diol to 5 alpha-dihydrotestosterone and, upon addition of NADH, reduced 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol. Thus, the dehydrogenation/isomerization steps of steroid biosynthesis can be catalyzed by a single polypeptide chain, which can metabolize all of the major physiological substrates.

17-alpha-Hydroxypregnenolone↗
PubMed1990

Expression of steroid metabolizing enzymes by aggregating fetal brain cells in culture: a model for developmental regulation of the progesterone 5 alpha-reductase pathway.

In this study, we established an in vitro model system for the study of developmental regulation of steroid enzyme expression in the perinatal brain. Single cell suspensions were prepared from the hypothalamic-olfactory tubercle region of 18-day-old rat fetuses, and aggregates were formed by incubation under constant rotation. On day 0, 3, 6, or 12 of culture, aggregates were incubated for 4 or 20 h with 3H-progesterone (P4) and the profile of 3H-steroids in the medium analyzed. Five major metabolites were formed from 3H-P4: 5 alpha-pregnan-3, 20-dione (DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP), 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-OH-DHP), and two unidentified polar substances designated A and B. Progressively with aggregate-age in culture, there was a decrease in the relative amounts of 3H-P4 recovered in the medium and a sequential increase in DHP, 3 beta-OH-DHP, 3 alpha-OH-DHP, A and B. Aggregates maintained in a chemically defined, serum-free medium metabolized P4 at an accelerated rate compared to those maintained in serum. An inhibitor of the enzyme 5 alpha-reductase completely inhibited P4 metabolism, indicating that 5 alpha-reduction is the primary step in this pathway. Thus, the aggregates express three key enzymes in P4 metabolism: 5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductases and the operation of this pathway is proposed: P4----DHP----3 alpha-OH-DHP + 3 beta-OH-DHP; A and B are derived from one or more of the latter three. Hence, this culture system can serve as a model to study regulatory processes in the developing steroidogenic brain.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Transforming growth factor-beta inhibits steroid 17 alpha-hydroxylase cytochrome P-450 expression in ovine adrenocortical cells.

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of ACTH to promote the synthesis of the various steroid-metabolizing cytochrome P-450 enzymes. In the steroidogenic pathway, 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) is a key enzyme controlling the formation of cortisol and androgens. Recently, we demonstrated that transforming growth factor-beta (TGF beta) is a potent inhibitor of steroid production in ovine adrenocortical cells. In the present study we used a polyclonal antibody to P450(17) alpha to determine adrenal cell P-450(17) alpha enzyme content by Western analysis. In addition, we used a cDNA probe encoding for bovine P-450(17) alpha mRNA to determine levels of P-450(17) alpha mRNA in sheep ovarian adrenocortical cells in primary culture. When cells were cultured in a serum-free medium in the presence of ACTH for 48 h, P-450(17) alpha activity, enzyme content, and mRNA levels for P-450(17) alpha increased by 3- to more than 10-fold. TGF beta decreased the basal level and completely blocked the stimulatory action of ACTH on P-450(17) alpha enzyme activity. The effects of TGF beta on P-450(17) alpha enzyme content and mRNA levels were manifested in a dose-dependent manner, with maximal inhibition observed using 1 ng/ml TGF beta. Importantly, the inhibitory effects of TGF beta on P-450(17) alpha were not overcome by (Bu)2cAMP. These findings indicate that TGF beta is a potent negative regulator of P-450, and the inhibitory action appears to be at the level of P-450(17) alpha gene expression. The ability of TGF beta to suppress the positive stimulatory action of ACTH suggests that TGF beta could play a role in determining the pathway of steroidogenesis and, thereby, the specific steroids secreted by adrenocortical cells.

17-alpha-Hydroxyprogesterone↗
PubMed1990

Expression of messenger ribonucleic acid species encoding steroidogenic enzymes in human follicles and corpora lutea throughout the menstrual cycle.

The levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450(17 alpha], aromatase cytochrome P-450 (P-450AROM), and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were examined in human follicles and corpora lutea (CL) throughout the menstrual cycle. Tissues were obtained from women undergoing hysterectomy and oophorectomy. The largest follicle or the CL was dissected from the ovary depending on whether the surgery was performed in the follicular or luteal phase. The day of the cycle was determined by onset of last menstrual period and was confirmed by endometrial histology. Total RNA was examined by Northern blot analysis, using as probes specific 32P-labeled cDNA inserts encoding each human enzyme. Early follicles demonstrated detectable mRNA for both P450scc and P450(17 alpha), but not for P450AROM or 3 beta HSD. P450AROM was detectable late in the follicular phase and appeared markedly induced in the CL. 3 beta HSD was detectable only in the CL. Levels of P450(17 alpha) mRNA remained relatively unchanged throughout the cycle, whereas P450scc mRNA levels were greatly increased in the CL. The presence of P450(17 alpha) mRNA in the human CL is of interest, since it is absent from the bovine CL, and this is consistent with the ability of the human, but not the bovine, CL to synthesize 17 alpha-hydroxyprogesterone and estrogens. The fact that P450AROM expression is highest in CL is surprising, since plasma estrogen levels are highest during the late follicular phase of the cycle, and may suggest that CL estrogen biosynthesis is limited by 17 alpha-hydroxylase or 17,20-lyase activities.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Proliferating human granulosa-lutein cells in long term monolayer culture: expression of aromatase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase.

The development of long term culture conditions with which to study the regulation of expression of aromatase, cholesterol side-chain cleavage enzyme, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human granulosa-lutein cells is described in this report. Conditions have been established for the dispersal, growth, freezing, and storage of functional human granulosa cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Optimal growth conditions for human granulosa-lutein cells were determined by plating cells at a low density and testing the capacity of a variety of culture conditions to support growth. A combination of fetal bovine serum (FBS), horse serum, and the serum substitute UltroSer G was found to increase cell number to maximal levels, 8- to 10-fold higher than with sera alone. Human granulosa-lutein cells grown under these conditions had a doubling rate of 36-40 h and were morphologically distinct from human theca interna cells grown under similar conditions. Human granulosa-lutein cells treated with forskolin retracted and rounded up, whereas cultures of human ovarian theca interna cells or human fibroblasts treated similarly did not retract. Human granulosa-lutein cells were grown for successive passages and transferred to serum-free medium containing forskolin, LH, hCG, or cholera toxin. Addition of these agents resulted in a time- and dose-dependent increase in aromatase activity and progesterone secretion. In these studies FSH treatment was found not to increase aromatase activity. In a study of the time course of 3 beta HSD activity in the absence of forskolin under serum-free conditions, it was found that 3 beta HSD activity increased 3-fold during the 72-h treatment period. Forskolin-stimulated 3 beta HSD activity also increased in a time-dependent manner, with levels in treated cells 3-fold higher than those in control cells. Northern analysis performed on total RNA obtained from forskolin- or hCG-stimulated granulosa-lutein cells confirmed that the increase in aromatase activity was associated with a corresponding increase in levels of mRNA specific for aromatase cytochrome P-450. Levels of mRNA encoding cholesterol side-chain cleavage cytochrome P-450 were similarly increased in cells treated with forskolin compared with unstimulated values at each of the time points investigated. Under serum-free conditions in the absence of stimulation, the 3.4-kilobase band of aromatase cytochrome P-450 mRNA was detectable.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Structural analysis of the gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase.

The structural gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta HSD) was isolated from a human EMBL3 genomic library. The gene encompasses approximately 8 kilobases of DNA and is comprised of two large introns and three exons encoding amino acid residues 1-48, 49-103, and 104-373, respectively. The exonic sequence is identical to that of the cDNA that we previously isolated and expressed in COS 1 cells. DNA sequence analysis reveals a putative TATA (TATATAA) motif 26 basepairs up-stream of the beginning of exon I, as determined by S1 nuclease protection analysis. However, primer extension analysis using poly(A)+ RNA isolated from both placenta and corpora lutea indicates that the RNA initiates up-stream of the putative TATA motif, and that an additional 53-basepair exon, which is untranslated, is present 5' to the first coding exon. Southern hybridization analysis of genomic DNA using a single exon probe suggests that there may be more than one copy of the gene in the human genome. In addition, we confirm from Southern analysis of genomic DNA isolated from human x hamster somatic cell hybrids that the gene is located on human chromosome 1. These findings will provide a foundation for the characterization of apparent 3 beta HSD clinical deficiencies when these are due to a mutation in the structural gene.

Amino Acid Sequence↗
PubMed1990

Immunolocalization of 3 beta-hydroxysteroid dehydrogenase in human ovary.

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was performed in 55 cases of morphologically normal human ovaries by using a specific polyclonal antibody against purified human placental 3 beta-HSD. In small developing follicles, immunoreactivity was observed only in the theca interna but also became recognizable in the membrana granulosa with development of the follicle. At a late stage of folliculogenesis, the intensity of the 3 beta-HSD activity in the membrana granulosa was nearly equal to that of theca interna in 2 or 3 large follicles examined. One to several layers of theca interna cells just beneath membrana granulosa did not demonstrate any immunoreactivity of 3 beta-HSD or that of cytochrome P-450 17 alpha-hydroxylase. These unstained theca interna cells did not appear to be directly involved in ovarian steroidogenesis and might be designated as 'enzymically inactive theca interna cells.' Marked immunoreactivity was observed in luteinized theca and granulosa cells of the corpus luteum.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

The use of rat Leydig tumor (R2C) and human hepatoma (HEPG2) cells to evaluate potential inhibitors of rat and human steroid aromatase.

The efficacies of 10-propargylestr-4-ene-3,17-dione (PED), 4-hydroxyandrostenedione (4-OHA) and the imidazole broad spectrum antimycotic drugs, econazole, imazalil, miconazole and ketoconazole, to inhibit the steroid aromatase activities of rat Leydig tumor (R2C) cells and human hepatoma (HEPG2) cells have been determined. The analysis of inhibition of steroid aromatase activity of intact cells provided further insight into the potential use of such drugs to block cellular estrogen synthesis. The IC50 values for the inhibition of aromatase activity of R2C cells by econazole, imazalil, miconazole, ketoconazole, 4-OHA and PED were 4, 9, 40, 1100, 11 and 10 nM, respectively. These drugs also inhibited the steroid aromatase activity of HEPG2 cells with corresponding IC50 values of 13, 27, 20, 15000, 2 and 2 nM, respectively; these findings were suggestive that the steroid aromatase of rat has many similarities to the human enzyme in its interaction with putative inhibitory compounds. Importantly, however, ketoconazole inhibited the rat aromatase more effectively than it did the human enzyme, while PED and 4-OHA were less effective inhibitors of the rat enzyme compared to that of human. These findings indicate differences in the potencies of various drugs to inhibit estrogen biosynthesis in human and rat cells. These may relate to differences in the two aromatase systems and/or differences in the stability of the drugs in the human hepatoma and rat Leydig tumor cells.

Androstenedione↗
PubMed1989

Expression of a full-length cDNA encoding bovine adrenal cytochrome P450C21.

Two full-length cDNA clones encoding bovine adrenocortical P450C21 have been constructed in a eukaryotic expression vector using partial-length cDNAs whose structures have been previously reported. Following expression of these cDNAs in COS 1 cells, the substrate specificity of P450C21 was determined. Of the 18 steroids tested, progesterone, 17 alpha-hydroxyprogesterone, and 11 beta,17 alpha-dihydroxyprogesterone were found to be the only steroids to serve as substrates for this adrenal enzyme, a much higher degree of substrate specificity than has been reported for a hepatic 21-hydroxylase. The Vmax for 17 alpha-hydroxyprogesterone was 2.5 times greater than that for progesterone, whereas delta 5-steroids were unable to serve as substrate for this enzyme. A difference between the two cDNAs is located at amino acid 401 where one resultant enzyme contains tyrosine while the other contains histidine. This amino acid difference appears to have no effect on the kinetic properties of adrenal P450C21.

17-alpha-Hydroxyprogesterone↗
PubMed1989

Immunohistochemical studies of steroidogenic enzymes (aromatase, 17 alpha-hydroxylase and cholesterol side-chain cleavage cytochromes P-450) in sex cord-stromal tumors of the ovary.

Aromatase, 17 alpha-hydroxylase, and cholesterol side-chain cleavage P-450 cytochromes (P-450AROM, P-450(17 alpha,) and P-450SCC, respectively) were immunohistochemically localized in nine granulosa cell tumors, 15 thecomas, ten Sertoli-Leydig cell tumors, two steroid cell tumors, five fibromas, and five sclerosing stromal tumors. In the thecomas, P-450SCC and P-450(17 alpha) were positive in luteinized theca cells and in cells with vacuolated cytoplasm, while P-450AROM was not observed. In the steroid cell tumors, all the P-450 cytochromes were intensely stained. P-450SCC and P-450(17 alpha) were present in cells with vacuolated cytoplasm in two cases of sclerosing stromal tumor. P-450AROM was weakly demonstrated in one of the granulosa cell tumors. P-450(17 alpha,) P-450SCC, and P-450AROM were all faintly stained in the Sertoli-Leydig cell tumors. No P-450 cytochrome immunoreactivity was observed in any fibroma.

Aromatase↗
PubMed1989

Immunohistochemical study of cytochrome P-45017 alpha in human adrenocortical disorders.

Cytochrome P-450 specific for steroid 17 alpha-hydroxylation (P-45017 alpha) was immunolocalized in normal and hyperfunctioning adrenal glands of pigs, bovines, and humans, using a specific IgG fraction raised against the enzyme. P-45017 alpha was present in the zona fasciculata (ZF) and zona reticularis (ZR), but not in the zona glomerulosa (ZG), in pig, bovine, and human adrenal glands. In the adrenal glands of patients with Cushing's disease, the positive immunoreactivity to P-45017 alpha was intense in ZF and ZR, particularly in cortical micronodules, corresponding to the sites of active steroidogenesis. Cells of hyperplastic ZG and outer ZF in the adrenal glands of idiopathic hyperaldosteronism were negative for P-45017 alpha. In aldosteronoma, positive immunoreactivity was observed in some tumor cells, which is consistent with cortisol production and its responsiveness to ACTH in aldosteronoma. In the attached adrenal glands of aldosteronoma, the immunoreactive P-45017 alpha was clearly present in the inner ZF and ZR, suggesting persistent androgen production. In Cushing's adenoma, the positive immunoreactivity was intense in tumor cells, and the ZR of the attached adrenal glands was weakly immunoreactive.

Adenoma↗
PubMed1989

Brenner tumor of the ovary: immunoanalysis of steroidogenic enzymes in 23 cases.

Immunohistochemical analysis of cytochromes P-450 aromatase and P-450 17 alpha-hydroxylase, which catalyze the production of estrogens and androgens, respectively, was performed for 23 cases of ovarian Brenner tumor. Immunoreactivity for P-450 aromatase was observed in the epithelial cells of the tumor distinctly in two cases and faintly in four cases, while immunoreactivity for P-450 17 alpha-hydroxylase was seen in two cases. No immunoreactivity was observed in the stromal cells of the tumors, nor were luteinized cells observed in the cases examined. No correlation was observed between the immunoreactivity of cytochromes P-450 in the epithelial cells of the tumor and endometrial abnormalities. These findings, together with a review of the literature, suggest that the usual Brenner tumor is not associated with specific steroidogenesis. Rare massive Brenner tumors may, however, be capable of androgenic or estrogenic activity.

Aromatase↗
PubMed1989

Immunohistochemical analysis of cytochrome P-450 17 alpha-hydroxylase in pig adrenal cortex, testis and ovary.

Cytochrome P-450 specific for steroid 17 alpha-hydroxylation (P-450(17 alpha] was immunohistochemically observed in pig adrenal cortex, testis and ovary by the biotin-streptavidin method using a specific antibody against P-450(17 alpha) purified from neonatal pig testis. In the adrenal cortex, P-450(17 alpha) was present in the zona fasciculata and reticularis while no immunoreactivity was observed in the zona glomerulosa, confirming the absence of 17-hydroxylated steroid synthesis in the zona glomerulosa. In the testis, P-450(17 alpha) was present exclusively in Leydig cells and immunoreactivity was absent in seminiferous tubules. In the ovary, immunoreactivity was observed only in the theca interna but not in the membrana granulosa of follicles. Among the tissues examined, the relative intensity of immunoreactivity was greatest in the Leydig cells, and progressively less in theca interna cells, outer fasciculata cells and inner fasciculata and reticularis cells.

Adrenal Cortex↗