11β-HSD1 Research Articles

Expression of 11 beta-hydroxysteroid dehydrogenase isoenzymes in the human pituitary: induction of the type 2 enzyme in corticotropinomas and other pituitary tumors.

One of the defining biochemical features of Cushing's disease is a relative insensitivity to glucocorticoid (GC) feedback, but an analysis of the GC receptor has failed to detect any major abnormalities. However, two isoenzymes of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD), either by converting cortisone (E) to cortisol (F) (type 1) or conversely by converting F to E (type 2), play an important prereceptor role in regulating corticosteroid hormone action at several sites. 11 beta HSD1 and -2 expression within the anterior pituitary gland itself may modulate GC feedback at an autocrine level, and we have speculated that this may be deranged in Cushing's disease. Detection of 11 beta HSD type 1 and 2 immunoreactive protein was performed using fluorescence immunohistochemistry. Double immunofluorescent studies were undertaken on normal pituitary to define the cellular localization of 11 beta HSD isoenzymes using antisera against GH, ACTH, LH, FSH, PRL, and S100, a nonhormonal marker of folliculo-stellate cells. In normal pituitary, positive staining for 11 beta HSD1-immunoreactive protein was observed in GH- and PRL-secreting cells and in folliculo-stellate cells; gonadotrophs, thyrotrophs, and ACTH-positive cells were negative. 11 beta HSD2 immunoreactivity was absent in all cell types. RT-PCR detected 11 beta HSD1 messenger ribonucleic acid (mRNA) expression in the normal pituitary; 11 beta HSD2 mRNA expression was also seen in most normal tissue. By contrast, in ACTH-secreting adenomas 11 beta HSD2 immunostaining was strongly positive in every case of corticotroph adenoma. 11 beta HSD1 immunoreactivity was also observed occasionally, but to a much lesser extent. In other pituitary tumors, both functional and nonfunctional, 11 beta HSD expression was variable in terms of isoenzyme mRNA and intensity of protein staining. The expression of 11 beta HSD1 (which generates F from E) in somatotrophs and lactotrophs suggests an autocrine role for this isoenzyme in the glucocorticoid regulation of pituitary GH and PRL secretion. 11 beta HSD2 expression is markedly induced in ACTH-secreting pituitary tumors and, by converting F to E, may explain the resetting of glucocorticoid feedback control in Cushing's disease.

Immunohistochemical localization of steroidogenic enzymes in the corpus luteum and the placenta of the ribbon seal (Phoca fasciata) and steller sea lion (Eumetopias jubatus).

To study the luteal and placental function of pinnipeds, we analyzed the localization of steroidogenic enzymes (P450scc, 3 beta HSD and P450arom) in the corpus luteum and the placenta of ribbon seals (Phoca fasciata) and Steller sea lions (Eumetopias jubatus) immunohistochemically. P450scc and 3 beta HSD were present in all luteal cells of both species. Almost all of the luteal cells were immunostained for P450arom, while P450scc and 3 beta HSD were negatively immunostained in placentae and P450arom was present in the syncytiotrophoblast of placentae. These findings suggest that 1) corpora lutea of both species synthesize pregnenolone, progesterone and estrogen during the entire pregnancy period, and 2) like other terrestrial carnivores in the suborder Caniformia, placentae of both species do not have the capability for synthesizing progesterone in the latter half of active pregnancy period.

Male reproductive effect of arsenic in mice.

Arsenic, a known human carcinogen, was given to mice via drinking water as sodium arsenite at a dose 53.39, 133.47, 266.95 and 533.90 micromol 1 for 35 days. A decrease in the activity of 17 beta HSD along with increase in LDH, gammaGT activity were observed at 533.90 micromol l. The observed sperm count, motility and morphological abnormalities in sperm were similar to control at lower dose levels. However at 533.90 micromol l a significant decrease in sperm count and motility along with increase in abnormal sperm were noticed. Significant accumulation of arsenic in testes and accessory sex organs may be attributed to the arsenic binding to the tissues or greater cellular uptake. No effects were observed on indices studied for reproductive effects at 53.39 micromol l arsenic close to which human being are exposed through drinking water under the present set of experimental conditions.

Antisense to Both 11β-Hsd1 and 2 Increase Glucocorticoid (GC) Activity in Vascular Tissue

P69 GC’s amplify the pressor effects of catecholamines and Ang II in VSM. Thus, the level of vasoconstriction in vascular tissue can be influenced by local GC concentrations. GC’s are metabolized to their inactive 11-dehydro derivatives by the enzyme 11β-HSD which exists in at least two isoforms, 11β-HSD1 and 11β-HSD2. 11β-HSD2 is unidirectional, with only dehydrogenase activity (inactivating GC’s) while 11β-HSD1 is bi-directional, also possessing reductase activity and thus the ability to reactivate 11-dehydroGC’s back to the active parent steroid. The aim of the present study was to investigate the role of each isoform in the regulation of GC metabolism in vascular tissue. Aortic rings (2-3mm), from male SD rats, were incubated for 24 hrs in DMEM media with either corticosterone (B) or 11-dehydrocorticosterone (11-dehydroB)(10 -8 -10 -7 M) ± specific Antisense oligos (20 μg/ring) targeted against rat 11β-HSD1 or 11β-HSD2. The contractile responses to graded doses of phenylephrine (PE; 10 -9 - 10 -6 M)in individual rings were then measured. In aortic rings co-incubated with B and 11β-HSD2 Antisense, the contractile responses to PE were amplified 30-50% versus rings incubated with B + nonsense oligonicleotide (n = 21, p

Initial predominance of the oxidative activity of type I 11beta-hydroxysteroid dehydrogenase in primary rat Leydig cells and transfected cell lines.

Glucocorticoids suppress testosterone production in Leydig cells. The level of glucocorticoid action is set within the Leydig cell by the number of glucocorticoid receptors and by the activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD). This enzyme acts either as an oxidase inactivating glucocorticoid or as a reductase amplifying its action. It is currently unknown whether extracellular conditions might cause 11betaHSD oxidative and reductive activities in Leydig cells to change inversely or independently. The aim of the present study was to determine whether extracellular conditions set in vitro by various culture time and media components, such as glucose and pyruvate, affect the relative rates of 11betaHSD oxidation and reduction. Primary rat Leydig cells and cell lines (COS1 and CHOP cells) transfected with 11betaHSD-I complementary DNA (cDNA), were incubated with 25 nmol/L (physiologic range) or 500 nmol/L (stress range) concentrations of radiolabeled substrates, corticosterone or 11-hydrocorticosterone, for 0 to 24 hours. Oxidative activity predominated over reductive activity under initial conditions when product formation increased linearly with time. For example, in Dulbecco's modified Eagle medium/F12 medium (containing 5.5 mmol/L glucose), the peak ratio of oxidation to reduction (with 1 denoting equivalence of oxidative and reductive activities) was 5.5 in rat Leydig cells, 19.7 in COS1 cells, and 20.8 in CHOP cells. Glucose stimulated reductive activity but did not change the predominant direction of 11betaHSD catalysis at earlier times. In COS1 cells transfected with 11betaHSD-I cDNA, oxidative activity rapidly increased during the first 2 hours of the incubation, then gradually decreased while reductive activity increased steadily. The relative ratio of oxidation to reduction rapidly declined to less than 0.5 at 6 hours, and thus the favored direction of catalysis changed from oxidation to reduction. However, in transfected CHOP cells, 11betaHSD oxidative activity rapidly increased during the first 2 hours and continued to increase for 24 hours. The ratio of oxidative to reductive activity rapidly declined but kept above 1 in CHOP cells for 24 hours, and the favored direction of catalysis remained predominantly oxidative. These results revealed that 11betaHSD-I is a predominant oxidase initially in Leydig cells and 2 cell lines, and that the oxidative activity is gradually lost over time. The data suggest that type I 11betaHSD is a predominant oxidase in Leydig cells in vivo.

Congenital adrenal hyperplasia in pregnancy

The congenital adrenal hyperplasias (CAH) are a group of inherited enzymatic defects of adrenal steroid biosynthesis. Deficiencies of each enzyme required in the steroid biosynthesis pathway are known, and these deficiencies are all inherited as autosomal recessive disorders. During pregnancy, maternal and fetal problems are confined to women who have 21-hydroxylase deficiency (P450c21 deficiency), 11-hydroxylase deficiency (P450c11 deficiency), and 3 beta-hydroxysteroid dehydrogenase deficiency (3 beta HSD deficiency), because other adrenal enzyme deficiencies are not compatible with fertility. The interposition of the placenta on the hypothalamic-pituitary-adrenal axis and other endocrine changes during pregnancy impact considerably on the clinical evaluation of the congenital adrenal hyperplasias. Successful management of CAH in pregnancy requires a firm knowledge of normal adrenal anatomic and endocrine changes that occur during gestation. Women with severe forms of CAH have decreased fertility rates because of oligo-ovulation, and successful conception requires a combination of good therapeutic compliance, careful endocrine monitoring, and often ovulation induction. From a fetal and neonatal standpoint, accurate prenatal diagnosis of 21-hydroxylase deficiency and 11-hydroxylase deficiency is now possible, which allows for prenatal treatment in an attempt to minimize clinical problems in the neonates. Prevention of masculinization of affected female fetuses by corticosteroid suppression has been attempted in both 21-hydroxylase deficiency and 11beta-hydroxylase deficiency CAH, with variable degrees of success. This review provides an overview of the congenital adrenal hyperplasias and their management during pregnancy.

The effects of aging on the expression of glutathione S-transferases in the testis and epididymis of the Brown Norway rat.

Glutathione S-transferases (GSTs), a family of isoenzymes, catalyze the conjugation of glutathione to a variety of electrophiles, and protect cellular constituents from electrophilic and oxidative attack. Aging is associated with an overall increase in oxidative stress and thus free radical production. The present study examines the immunocytochemical localization of Ya, Yc, Yb1, Yb2, Yo, and Yf GST subunits in the testis and epididymis of Brown Norway rats aged 3, 12, 18, and 24 months. In the testis, neither Sertoli nor germ cells showed changes in the GST staining pattern during aging. At 24 months, two types of Leydig cells were noted. Some (peritubular) formed a distinct band at the periphery of the tubule while others were seen in the interstitial space. The peritubular cells were identified as Leydig cells by specific staining for 3beta-hydroxysteroid dehydrogenase (3beta-HSD), a Leydig cell-specific marker. Both types of Leydig cells were intensely reactive for all GST subunits at all ages. In the epididymis, principal cells of all epididymal regions, except the proximal cauda region, showed no changes in GST expression at all ages examined. At 24 months, some principal cells of this region became greatly enlarged and vacuolated. These cells were unreactive for Yo, Yb1, Yb2, and Yc, while adjacent normal-appearing principal cells maintained the same intensity of expression as seen in 3-month controls. In contrast, vacuolated principal cells were reactive for the Ya subunit, while adjacent normal principal cells were unreactive. These data indicate that selective changes occur in the expression of GSTs at 24 months in principal cells having both a normal and a vacuolated appearance. The underlying mechanism responsible for these changes with age is unresolved, but we speculate that they lose the ability to handle oxidative stress. Taken together, these data show that aging affects region-specific changes in GST expression in the epididymis and Leydig cell distribution in the testis.

Histopathologic and immunohistochemical study of steroidogenic cells in the stroma of ovarian tumors.

The presence of steroidogenic or enzymatically active stromal cells in ovarian tumors can lead to various endocrine manifestations by the overproduction of sex steroids. We studied the clinicopathologic and immunohistochemical features of histologically identified steroid-type cells or enzymatically active cells in the stroma of ovarian lesions. Among 1,249 ovarian tumors or tumorlike lesions, steroid cells were histologically identified in the stroma in 32 ovarian neoplasms, of which 29 were mucinous cystadenomas. The cells were Leydig- or hilus-type cells in 4 of the tumors and were lutein-type cells in the remaining 28. None of the 32 tumors were immuno-reactive for human chorionic gonadotropin or luteinizing hormone. Lutein-like steroid cells were intermingled with spindle-shaped stromal cells and capillaries, while Leydig-type cells were more often associated with dilated vessels. The transcription factor of steroidogenesis, adrenal 4-binding protein (Ad4BP), was present in nearly all steroid-type stromal cells. However, steroidogenic enzymes, 17 alpha-hydroxylase (C17) and 3 beta-hydroxysteroid dehydrogenase (3 beta SD), were expressed only in 10 of 15 cases of histologically identified steroid cells. Steroid cells in which Ad4BP but not C17 or 3 beta HSD were expressed were all lutein-type. Aromatase was rarely detected in lutein-and Leydig-type cells. The presence of morphologically identified steroid cells does not necessarily indicate expression within these cells of steroidogenic enzymes, a finding that merits further attention.

Effect of Estradiol on DHEAS Production in the Human Adrenocortical Cell Line, H295R

To determine if estradiol regulates DHEA and DHEAS production in a human adrenocortical (H295R) cell line and to determine if this effect is receptor mediated. NCI-H295 (H295R) cells were rinsed and placed in phenol red free Dulbecco's Modified Eagle's-F12 medium supplemented with 0.1% charcoal-stripped serum. After 24 hours, cells were rinsed and treated based on experimental design. The effects of estradiol were investigated by: 1) treatment of cells with increasing concentrations of estradiol (300-3000 nmol/L) with or without forskolin (10 mumol/L), 2) treatment of cells with the nonsteroidal synthetic estrogen diethylstilbestrol (DES) (300-3000 nmol/L) with or without forskolin (10 mumol/L), and 3) treatment of cells with an estradiol antagonist (ICI 182, 780) in the presence of estradiol. Estradiol alone increased the basal production of DHEAS in H295R cells in a concentration-dependent manner with a maximal effect at 1000 nmol/L. Forskolin treatment increased the basal production of DHEAS ten-fold. Estradiol also increased the forskolin stimulation of DHEAS production two-fold. In contrast, DES alone or DES in addition to forskolin did not stimulate DHEAS production. Estradiol, in contrast, inhibited H295R adrenal cell production of cortisol whereas DES exhibited a similar inhibition. The estrogen receptor antagonist ICI 182,780 was unable to inhibit the stimulatory effect of estradiol. Finally, estradiol in a concentration-dependent manner suppressed 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity in H295R adrenal cells. These experiments support the role of estradiol in regulation DHEAS production by inhibiting 3 beta HSD activity; however, the mechanism appears to require high concentrations of estradiol and appears to be independent of the estrogen receptor.

Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells.

The physiological importance of adrenal 21-hydroxylase cytochrome P450 (CYP21) expression is clearly demonstrated by 21-hydroxylase deficiency, which results in adrenal hyperplasia and over-production of C19 steroids, leading to virilization. The mechanisms regulating normal expression of this key enzyme in human adrenocortical cells are ill defined. Herein we examine the role of the calcium, protein kinase C, and protein kinase A signaling pathways in the expression of CYP21 messenger ribonucleic acid (mRNA) using the H295R human adrenocortical cell model. Forskolin (10 mumol/L) treatment caused a progressive increase in CYP21 mRNA levels (maximum, 4-fold; P < 0.05) over 36 h of treatment, whereas angiotensin II (AII; 10 nmol/L) produced a smaller, biphasic rise (maximum, 1.8-fold at 12 h; P < 0.05). K+ (14 mmol/L) also induced a time-dependent (maximal, 1.5-fold at 12 h; P < 0.05) and dose-dependent (P < 0.05 12 mmol/L or above at 20 h) rise in CYP21 mRNA levels. The action of forskolin was reproduced by dibutyryl cAMP, confirming the involvement of cAMP in this response. The action of AII was greater than that of K+ or the calcium channel agonist BAYK8644, suggesting that AII action was not solely through the Ca2+ signaling pathway. The action of AII was reproduced and indeed exceeded by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA; 10 nmol/L; 5.5-fold increase; P < 0.05). The actions of forskolin alone were not significantly increased by combined treatment with AII, suggesting neither synergy nor attenuation of the effects of protein kinase A activation. This was further demonstrated at the level of mRNA and 21-hydroxylase activity by the observation that the effect of forskolin and TPA in combination did not exceed that of TPA alone. Inhibition of protein synthesis with cycloheximide blocked induction of CYP21 as well as type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSDII) mRNA expression in response to AII, forskolin, and dibutyryl cAMP, but had no effect on 17 alpha-hydroxylase cytochrome P450 (CYP17) or cholesterol side-chain cleavage cytochrome P450 (CYP11A) mRNA. Together, these findings were remarkably similar to those of our previous studies regarding mechanisms regulating 3 beta HSDII expression and underline the existence of a subset of steroidogenic enzymes regulated positively (CYP21 and 3 beta HSDII) as opposed to negatively (CYP17 and CYP11A) by the protein kinase C signaling pathway. The additional finding of a small induction of CYP21 expression in response to increased Ca2+, as previously reported for CYP17, but not 3 beta HSDII, expression, also demonstrates that the mechanisms of control of CYP21 and 3 beta HSDII are not identical. This latter finding may also relate to how CYP21 as well as CYP17 expression continues in the zona reticularis after adrenarche, whereas 3 beta HSD expression declines.

Corticosteroid metabolism in human granulosa-lutein cells.

The aims of this study were to determine the type and level of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) in human granulosa-leutein cells (GLE) shortly before ovulation and to correlate activity with the outcome of treatment in patients undergoing in vitro fertilization and embryo transfer (IVF/ET). GLC from 32 patients undergoing IVF/ET were tested for type and level of 11 beta HSD activity in relation to treatment outcome. Periovulatory follicles were aspirated by ultrasound guided transvaginal puncture following a standard controlled ovarian stimulation protocol, approximately 36 h after administering an ovulation-inducing dose of human chorionic gonadotrophin (HCG). GLC were separated from follicular fluid by density-gradient centrifugation and taken for measurement of 11 beta HSD activity in vitro; oocytes were used for IVF/ET. Interconversion of cortisol (F) and cortisone (E), and dexamethasone (D) and 11-dehydrodexamethasone (DHD) was measured in standardized assays comprising incubation of GLC with 3H-labelled substrate, with separation of substrate and product by thin-layer radiochromatography. Conversion of F to E varied from 10.5 to 30.9% while that of E to F was between 2.4 and 44.6%. In the GLC of 25 patients in whom both activities were measured, dehydrogenase (F to E) activity predominated in 13 and reductase (E to F) in 12. By contrast, D (substrate for 11 beta HSD2 but not 11 beta HSD1) showed less than 1% metabolism in this system while DHD (substrate for 11 beta HSD1 and 11 beta HSD2) was converted significantly (65.6-90.5%) to D in the four patients tested. There was no significant difference in the interconversion of F and E between patients who became pregnant and those who did not. The dehydrogenase and oxoreductase reactions catalysed by 11 beta HSD both occur in granulosa-lutein cells at the time of follicular rupture, probably due to 11 beta HSD1. A lack of measurable conversion of dexamethasone to 11-dehydrodexamethasone suggests that dehydrogenation due to 11 beta HSD2 is low or absent. Neither type nor level of 11 beta HSD activity measured under the present assay conditions correlates with IVF outcome.

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.

11 beta-Hydroxysteroid dehydrogenase type II in the human endometrium: localization and activity during the menstrual cycle.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) is a potent inactivator of glucocorticoids and is present in high amounts in the placental syncytiotrophoblast and sodium-transporting epithelia. Placental 11 beta HSD2 is thought to protect the fetus from high circulating levels of maternal glucocorticoids, whereas the renal enzyme is important in conferring aldosterone specificity on the mineralocorticoid receptor. An isoform of 11 beta HSD (11 beta HSD1) is also present in a wide range of tissues, but usually acts as an oxoreductase, converting the biologically inactive cortisone to cortisol. In the present study we have used an immunopurified antibody to the carboxy-terminus of human 11 beta HSD2 (HUH23) to demonstrate localization of the enzyme in luminal and glandular epithelia of human endometrium. In some specimens staining was uniformly distributed, but in others there was clear evidence of heterogeneity both between and within epithelia. Although 11 beta HSD2 was found mainly in the cytoplasm, some cells showed evidence of nuclear staining only. Western blot analysis showed a band at 41 kDa in endometrium and myometrium, confirming the presence of 11 beta HSD2. Measurement of activity throughout the menstrual cycle showed that mean levels (+/- SEM) of activity were 156 +/- 17 and 6.1 +/- 1.1 pmol product/min.g homogenate protein for 11 beta HSD2 and 11 beta HSD1, respectively. Patients taking combined estrogen/progesterone contraceptives had significantly lower activities of both enzymes (76 +/- 19 and 1.9 +/- 0.4; both P < 0.01) compared with the control group. 11 beta HSD2 activity was significantly higher in the secretory than in the proliferative phase of the cycle in controls (193 +/- 22 vs. 120 +/- 23; P < 0.05). All groups contained outliers with elevated enzyme activities, with some patients displaying 11 beta HSD2 levels comparable to those observed in human kidney (> 1000 pmol/min.g). Further analysis showed that there was a statistically significant correlation (r = 0.43; P < 0.001) between the levels of 11 beta HSD1 and 11 beta HSD2. There was no detectable mineralocorticoid receptor binding in endometrial cytosols prepared from patients with a range of 11 beta HSD2 activities. It remains to be determined whether elevated or suppressed levels of either isoform are associated with fertility or endometrial pathology.

Regulation of the 11 beta-hydroxysteroid dehydrogenase in the rat adrenal. Decrease enzymatic activity induced by ACTH.

Patients with ectopic ACTH syndrome often develop hypertension and hypokalemic alkalosis with an abnormal increase in the ratio of plasma cortisol to cortisone, indicating that 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) activity is inhibited. Inhibition of 11 beta HSD allows access of cortisol or corticosterone to the mineralocorticoid receptor where it act as a mineralocorticoid. Two isozymes, 11 beta HSD-1 and 11 beta HSD-2, have been cloned and characterized. The rat adrenal expresses the mRNAs for 11 beta HSD-2 and, in lesser amounts, 11 beta HSD-1. We investigated the effect of ACTH on the 11 11 beta HSD-2 activity in the rat adrenal. Rat adrenal cells zone fasciculata (ZF) were dispersed and incubated separately with increasing concentrations of ACTH for 90 min, and secretion of corticosterone (B) and 11-dehydrocorticosterone (A) in the media was measured by enzyme-linked immunoabsorbent assays (ELISA). The conversion of [3H]B to [3H]A in the presence of 0.5 mM NAD+ was evaluated in microsomes prepared from dispersed cells preincubated for 30 min with cyanoketone and metyrapone followed by incubation for 30 min with the same inhibitors, with and without 10 nM ACTH. The dispersed cells of the ZF produced significant amounts of A which increased with ACTH. The basal B/A ratio was 0.97 +/- 0.05. ACTH caused a concentration-dependent increase in the ratio of B/A with a maximum ratio of 9.58 +/- 0.20. ACTH also inhibited the conversion of [3H]B to [3H]A in microsomes in which endogenous B production was inhibited by cyanoketone and metyrapone. ACTH did not change the K(m) for B conversion, but the Vmax was reduced significantly (1.73 +/- 0.43 pmol/min. mg protein), indicating that ACTH suppressed the 11 beta HSD-2 in a noncompetitive fashion. Dibutyryl cyclic AMP (dcAMP) also produced a concentration-dependent increase in the B/A ratio, but various concentrations of calcium did not affect the enzyme activity. In summary, adrenal cells treated with ACTH results in a significant increase in the ratio of B/A in the ZF owing a noncompetitive inhibition of the 11 beta HSD-2 via the ACTH receptor.

Testosterone decreases 3beta-hydroxysteroid dehydrogenase-isomerase messenger ribonucleic acid in cultured mouse Leydig cells by a strain-specific mechanism.

We previously reported a strain-related difference in basal 3beta-hydroxysteroid dehydrogenase-isomerase (3betaHSD) activity in response to testosterone in cultured Leydig cells. The data suggested that the response to testosterone was androgen receptor mediated and that testosterone was acting via a trans-acting factor distal to the androgen receptor to regulate Leydig cell basal 3betaHSD activity. This study was designed to determine whether the previous reported strain-related difference in basal 3betaHSD activity in response to testosterone was due to a difference at the 3betaHSD protein and/or at the mRNA level. In C57BL/6J Leydig cells, 2.0 microM testosterone significantly decreased basal 3betaHSD immunoreactive mass by day 6 in culture. Treatment with 2.0 microM testosterone and 2.0 microM hydroxyflutamide, an androgen receptor antagonist, negated the inhibitory effect of testosterone on C57BL/6J 3betaHSD immunoreactive mass. Treatment with 2.0 microM testosterone also significantly decreased 3betaHSD mRNA content in C57BL/6J Leydig cells, which was detectable on day 3 in culture. In contrast to Leydig cells from C57BL/6J mice, Leydig cells from C3H/HeJ mice were not susceptible to the inhibitory effect of testosterone on 3betaHSD. Treatment with 2.0 microM testosterone had no detectable effect on C3H/HeJ 3betaHSD immunoreactive mass or mRNA content at any time point in culture. These data indicate that the testosterone-induced loss of basal 3betaHSD activity in C57BL/6J Leydig cells can be accounted for by the loss of 3betaHSD immunoreactive mass, which is preceded by the loss of 3betaHSD mRNA, and that the strain-related difference in the regulation of 3betaHSD is present at all three levels. Thus, the putative trans-acting factor involved in the mechanism whereby testosterone decreases basal 3betaHSD is likely to regulate the amount of 3betaHSD mRNA.

Cell type-specific expression of 17 beta-hydroxysteroid dehydrogenase type 2 in human placenta and fetal liver.

The enzymatic actions of the 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isozymes are crucial in steroid hormone metabolism/physiology. The type 1 isozyme catalyzes the conversion of the biologically inactive C18 steroid, estrone, to the active estrogen, 17 beta-estradiol, and the enzyme is predominantly expressed in the syncytiotrophoblast of the placenta and the granulosa cells of the ovary. 17 beta HSD type 2 is highly expressed in placenta, liver, and secretory endometrium and catalyzes the conversion of bioactive estrogens and androgens to biologically inactive 17-ketosteroid counterparts. The expression pattern of 17 beta HSD type 2 protein was determined in human term placenta and fetal liver by immunohistochemical analysis using monoclonal antibodies directed against distinct epitopes of the 17 beta HSD type 2 protein. In placenta, the protein was detected in the endothelial cells of fetal capillaries, but not in cytotrophoblasts or syncytiotrophoblast. There was dichotomous immunostaining seen among pairs of cotyledonary vessels and chorionic vessels. In the liver, on the other hand, staining was detected in the hepatocytes, but not in the cells lining blood vessels. We conclude that the cell type-specific localization of 17 beta HSD type 2 is in accord with the proposed physiological role of the enzyme, namely to protect tissues, in this case the fetus, from bioactive estrogen and androgen.

Ontogeny of inhibin secretion in the rat testis: secretion of inhibin-related proteins from fetal Leydig cells and of bioactive inhibin from Sertoli cells.

The ontogeny of inhibin secretion in the testis of rats was investigated. Testicular localization, content of immunoactive and bioactive inhibin and its molecular size in fetal and neonatal rats (from 16 days of gestation to 5 days of age) were determined. Strong immunostaining with an antiserum against a polypeptide of porcine inhibin alpha-subunit was noted in testicular interstitial cells from 16 days of gestation. Co-localization of inhibin alpha-subunit and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was observed in the interstitial cells until 2 days of age. Immunoreactive inhibin alpha-subunit in the interstitial tissue had disappeared by 5 days of age, although 3 beta HSD-positive cells were still detected. Weak immunostaining for the inhibin alpha-subunit was detected in the seminiferous tubules, probably in the cytoplasm of Sertoli cells, from 20 days of gestation onward. No inhibin alpha-subunit immunostaining was observed in germ cells throughout the experimental period. Testicular inhibin was detected at 16 days of gestation (49.5 +/- 6.7 pg per testis) by RIA. Testicular immunoreactive inhibin showed a tendency to increase during fetal life and levels were maintained at a similar value after birth (697.0 +/- 46.9 pg per testis at 5 days of age). Inhibin bioactivity and its molecular size in testicular homogenate was examined at 17 days of gestation and 0 and 5 days of age. Although no bioactivity was detected at 17 days of gestation, bioactivity was noted at 0 and 5 days of age (177.7 and 1303.9 pg per testis respectively). Immunoblot analysis with antiserum against inhibin alpha-subunit revealed only approximately 40 kDa molecular masses in the testis at 17 days of gestation, probably inhibin-related proteins, but not inhibin. At 0 and 5 days of age, a protein of 30 kDa molecular mass, possibly inhibin, was detected as well as material of approximately 40 kDa molecular mass. FSH in the plasma was first detected at 19 days of gestation (1197.0 ng/l), increased towards birth, and thereafter decreased (4588.5 +/- 572.3 ng/l at 21 days of gestation and 2400.0 +/- 179.6 ng/l at 5 days of age). These results indicate that Leydig cells in fetal and neonatal rats produce inhibin-related substances with no inhibin bioactivity, whereas Sertoli cells begin to produce inhibin during the perinatal period as a possible regulator of FSH secretion.

Stress, 11beta-HSD, and Leydig cell function.

Stress, 11beta-HSD, and Leydig cell function.

Developmental and Functional Biology of the Primate Fetal Adrenal Cortex*

The unique characteristics of the primate (particularly human) fetal adrenal were first realized in the early 1900s when its morphology was examined in detail and compared with that of other species. The unusual architecture of the human fetal adrenal cortex, with its unique and disproportionately enlarged fetal zone, its compact definitive zone, and its dramatic remodeling soon after birth captured the interest of developmental anatomists. Many detailed anatomical studies describing the morphology of the developing human fetal adrenal were reported between 1920 and 1960, and these morphological descriptions have not changed significantly. More recently, it has become clear that fetal adrenal cortical growth involves cellular hypertrophy, hyperplasia, apoptosis, and migration and is best described by the migration theory, i.e. cells proliferate in the periphery, migrate centripetally, differentiate during their migration to form the functional cortical zones, and then likely undergo apoptosis in the center of the cortex. Consistent with this model, cells of intermediate phenotype, arranged in columnar cords typical of migration, have been identified between the definitive and fetal zones. This cortical area has been referred to as the transitional zone and, based on the expression of steroidogenic enzymes, we consider it to be a functionally distinct cortical zone. Elegant experiments during the 1950s and 1960s demonstrated the central role of the primate fetal adrenal cortex in establishing the estrogenic milieu of pregnancy. Those findings were among the first indications of the function and physiological role of the human fetal adrenal cortex and led Diczfalusy and co-workers to propose the concept of the feto-placental unit, in which DHEA-S produced by the fetal adrenal cortex is used by the placenta for estrogen synthesis. Tissue and cell culture techniques, together with improved steroid assays, revealed that the fetal zone is the primary source of DHEA-S, and that its steroidogenic activity is regulated by ACTH. In recent years, function of the human and rhesus monkey fetal adrenal cortical zones has been reexamined by assessing the localization and ontogeny of steroidogenic enzyme expression. The primate fetal adrenal cortex is composed of three functionally distinct zones: 1) the fetal zone, which throughout gestation does not express 3 beta HSD but does express P450scc and P450c17 required for DHEA-S synthesis; 2) the transitional zone, which early in gestation is functionally identical to the fetal zone but late in gestation (after 25-30 weeks) expresses 3 beta HSD, P450scc, and P450c17, and therefore is the likely site of glucocorticoid synthesis, and 3) the definitive zone, which lacks P450c17 throughout gestation but late in gestation (after 22-24 weeks) expresses 3 beta HSD and P450scc, and therefore is the likely site of mineralocorticoid synthesis. Indirect evidence, based on effects of P450c21 deficiency and maternal estriol concentrations, indicate that the fetal adrenal cortex produces cortisol and DHEA-S early in gestation (6-12 weeks). However, controversy exists as to whether cortisol is produced de novo or derived from the metabolism of progesterone, as data regarding the expression of 3 beta HSD in the fetal adrenal cortex early in gestation are conflicting. During the 1960s, Liggins and colleagues demonstrated that in the sheep, cortisol secreted by the fetal adrenal cortex late in gestation regulates maturation of the fetus and initiates the cascade of events leading to parturition. Those pioneering discoveries provided insight into the mechanism underlying the timing of parturition and therefore were of particular interest to obstetricians and perinatologists confronted with the problems of preterm labor. However, although cortisol emanating from the fetal adrenal cortex promotes fetal maturation in primates as it does in sheep, its role in the regulation of primate parturition, unlike that in sheep

Localization of steroidogenic enzymes in macaque luteal tissue during the menstrual cycle and simulated early pregnancy: immunohistochemical evidence supporting the two-cell model for estrogen production in the primate corpus luteum.

It is hypothesized that the two-cell model for estrogen production by the ovarian follicle is preserved in the primate corpus luteum, but there is little direct evidence to support this theory. To determine the sites of androgen and estrogen synthesis within the primate corpus luteum and to ascertain whether changes in steroid hormone levels are related to steroidogenic enzyme expression, the enzymes converting progesterone to androgen (cytochrome P450 17alpha-hydroxylase/17,20 lyase; P450(c17)) and then to estrogen (aromatase; P450(arom)), as well as P450 side-chain cleavage (P450(scc)) and 3beta-hydroxysteroid dehydrogenase (3beta HSD), were detected by immunohistochemistry in macaque luteal tissue throughout the menstrual cycle and simulated early pregnancy. Corpora lutea were collected from rhesus monkeys in the early (Days 2-4 post-LH surge), mid (Days 6-8), mid-late (Days 10-12), and late (Days 14-15) luteal phase and after 1, 3, 6, or 9 days of hCG treatment that began on Day 9 of the luteal phase. Specific cytoplasmic staining for P450(c17), P450(arom), P450(scc), and 3beta HSD was present in luteal cells, but not in the microvasculature, within all luteal tissues examined. P450(c17)-stained luteal cells were located along the vascular tracts and around the periphery of the corpus luteum. Intensely stained luteal cells were associated with blood vessels entering from the outer surface of the corpus luteum, but not with blood vessels returning from the connective tissue centrum. In contrast, P450(arom)-stained luteal cells were distributed throughout the luteal parenchyma. P450(c17) staining intensity was similar at all stages of the luteal phase; however, the number and intensity of P450(arom)-stained cells decreased by late luteal phase. In simulated early pregnancy, cells stained for P450(c17) were present near blood vessels, with some positive cells scattered throughout the corpus luteum. P450(arom) immunostaining was heterogeneous within the corpus luteum; many intensely stained cells were interspersed among others that were only lightly stained. Overall, cellular staining for P450(c17) and P450(arom) remained intense through 9 days of simulated early pregnancy. In contrast, P450(scc) and 3beta HSD immunoreactivity were not located in distinct luteal compartments. These results are consistent with a two-cell model for steroid hormone production in the primate corpus luteum, whereby paraluteal (theca-luteal) cells produce androgen substrate that is converted to estrogens by true (granulosa-) luteal cells. The divergence in enzyme detection as the luteal phase progresses, with P450(c17) labeling high and P450(arom) staining having decreased, suggests a shift in the function of the corpus luteum as it ages. Enzyme localization during chorionic gonadotropin exposure simulating early pregnancy demonstrates the continued capacity of the primate corpus luteum to produce steroid hormones.