Development of adrenal cortical zonation and expression of key elements of adrenal androgen production in the chimpanzee (Pan troglodytes) from birth to adulthood.

The basis for the pattern of adrenal androgen production in the chimpanzee, which resembles that of humans, is poorly defined. We characterized the developmental zonation and expression of elements of the androgen biosynthetic pathway in the chimpanzee adrenal. The newborn adrenal contained a broad fetal zone (FZ) expressing CYP17, SULT2A1, and Cytochrome B5 (CB5) but not HSD3B; the outer cortex expressed HSD3B but not SULT2A1 or CB5. During infancy, the FZ involuted and the HSD3B-expressing outer cortex broadened. By 3years of age, a thin layer of cells that expressed CB5, SULT2A1, and CYP17 adjoined the medulla and likely represented the zona reticularis; the outer cortex consisted of distinct zonae fasiculata and glomerulosa. Thereafter, the zona reticularis broadened as also occurs in the human. The adult chimpanzee adrenal displayed other human-like characteristics: intramedullary clusters of reticularis-like cells and also a cortical cuff of zona fasiculata-like cells adjoining the central vein.

Effect of ovarian aging on androgen biosynthesis in a cynomolgus macaque model.

OBJECTIVE: The role of androgens in chronic disease pathogenesis, cognitive function and libido during menopause is of increasing interest. The aim of this study was to characterize the distribution and expression of androgenic proteins in the macaque ovary and to investigate the relationship between serum androgen concentrations, follicle number, and the persistence of androgenesis in the aging macaque ovary. METHODS: The subjects were 26 adult female cynomolgus macaques. Ovaries were immunostained for cytochrome P450 17α-hydroxylase/17-20 lyase (P450c17), 3ß-hydroxysteroid dehydrogenase (3ßHSD), and cytochrome b5 (cytb5). Based on primordial follicle counts, animals were divided into tertiles (low (≤200), intermediate (226-1232), and high (2372-4356)) to evaluate differences in androgen staining and changes in serum androgen concentrations following ovariectomy. RESULTS: Positive immunostaining for P450c17 and cytb5 within the theca interna layer of growing follicles persisted in advanced atretic follicles and secondary interstitial cells (residual stromal cells). Ovaries with low follicle numbers had less staining for all androgenic proteins compared to ovaries with higher numbers of growing follicles. Immunostaining for cytb5 was the most reliable marker for persistent androgenesis in ovaries with minimal primordial follicle numbers (<100) and residual stromal cells. Following ovariectomy, a significant decrease in testosterone (-27.7%, -30.8%, -27.5%; p < 0.01) and androstenedione (-33.4%, -35.7%, -46.0%; p < 0.01) was observed in monkeys with low, intermediate, and high primordial follicle counts, respectively. CONCLUSIONS: Despite low follicle numbers, the aging macaque ovary retains the necessary proteins for androgenesis within residual stromal cells and contributes to peripheral androgen concentrations.

Intrapartum stress and lipid status of term infants: relation to fetal adrenal production of dehydroepiandrosterone sulphate.

OBJECTIVE: Evaluate the effect of acute intrapartum stress on umbilical cord plasma levels of cholesterol, triglyceride and dehydroepiandrosterone sulphate (DS) in term infants. METHODS: Umbilical cord plasma levels of cholesterol, triglyceride and DS were measured in 176 infants that delivered >or=37 weeks' gestation. Eighty-eight infants experienced acute intrapartum stress while the remaining infants were non-stressed controls who were case-matched by gestational age and delivery method. RESULTS: The umbilical cord plasma levels of cholesterol in the stressed infants (71.7 +/- 16.1 mg/dL, mean +/- SD) were similar to those of the control infants (70.9 +/- 16.0 mg/dL). Triglyceride levels in stressed infants were significantly higher (p = 0.003) than those of control infants. Both stressed and control infants with increased triglyceride levels had normal DS levels; however, those with increased cholesterol levels had reduced umbilical cord plasma levels of DS. Stressed infants with increased cholesterol levels had significantly lower DS levels than control infants (p = 0.008). CONCLUSIONS: Intrapartum stress leads to increased fetal plasma levels of triglycerides; yet, cholesterol levels are usually unaffected. Infants with increased cholesterol levels also have reduced DS levels, suggesting that the rise in cholesterol was due to a reduction in fetal adrenal utilisation of plasma cholesterol as substrate for steroid formation.

Alendronate in the treatment of primary hyperparathyroid-related osteoporosis: a 2-year study.

We investigated the effect of alendronate on calcium, PTH, and bone mineral density in 27 female and 5 male patients with primary hyperparathyroidism. The treatment group [n = 14; T score < or = -2.5 SD at the femoral neck (FN) or T < or = -1.0 SD plus previous nonvertebral fracture] was given alendronate 10 mg/d for 24 months. The second group (n = 18; T score > -2.5 SD at the FN) was untreated. Biochemistry was repeated at 1.5, 3, 6, 12, 18, and 24 months, and dual-energy x-ray absorptiometry at 12 and 24 months. There were no significant between-group baseline differences in calcium, creatinine, or PTH. Alendronate-treated patients gained bone at all sites [lumbar spine (LS), 1 yr gain, +7.3 +/- 1.7%; P < 0.001; 2 yr, +7.3 +/- 3.1%; P = 0.04). Untreated patients gained bone at the LS over 2 yr (+4.0 +/- 1.8%; P = 0.03) but lost bone elsewhere. Calcium fell nonsignificantly in the alendronate group between baseline (2.84 +/- 0.12 mmol/liter) and 6 wk (2.76 +/- 0.09 mmol/liter), with a nonsignificant rise in PTH (baseline, 103.5 +/- 14.6 ng/liter; 6 wk, 116.7 +/- 15.6 ng/liter). By 3 months, values had reverted to baseline. In primary hyperparathyroidism, alendronate is well tolerated and significantly improves bone mineral density at the LS (with lesser gains at FN and radius), especially within the first year of treatment. Short-term changes in calcium and PTH resolve by 3 months.

Gene profiling of human fetal and adult adrenals.

The mechanisms that lead to the steroidogenic differences in the human fetal adrenal (HFA) and adult adrenal gland are not known. However, gene expression clearly plays a critical role in defining their distinct steroidogenic and structural phenotypes. We used DNA microarrays to compare expression levels of several thousand transcripts between the HFA and adult adrenal gland. Total RNA was isolated from 18 HFA and 12 adult adrenal glands. Samples of total RNA were used to make five pools of poly A+ RNA (mRNA). Gene profiling was done using five independent microarrays that contained between 7075 and 9182 cDNA elements. Sixty-nine transcripts were found to have a greater than 2.5-fold difference in expression between HFA and adult adrenals. The largest differences were observed for transcripts that encode IGF-II (25-fold higher in HFA) and 3beta-hydroxysteroid dehydrogenase (24-fold higher in adult). Among the other genes, transcripts related to sterol biosynthesis or to growth and development were higher in the HFA than adult adrenals. Transcripts concerned with cellular immunity and signal transduction were preferentially expressed in the adult adrenal. The vast majority of the 69 transcripts have not been studied with regard to adrenal function. Thus, these gene profiles provide valuable information that could help define the mechanisms that control adrenal function.

Adrenocortical secretion of dehydroepiandrosterone in healthy women: highly variable response to adrenocorticotropin.

Excess adrenal androgen (AA) levels are observed in 25--50% of women with the polycystic ovary syndrome (PCOS), and AA excess in PCOS may represent selection bias. Thus, it is possible that AA secretion among the general population is highly variable, and that those women who are predisposed to secreting greater amounts of AA have a greater probability of having PCOS. We now hypothesize that the levels of AAs are highly variable among normal nonhyperandrogenic women, and that this heterogeneity is the result of a variable response of AAs to ACTH stimulation. To test this hypothesis we prospectively studied the response of dehydroepiandrosterone (DHA) and cortisol (F) to a 60-min acute stimulation with ACTH-(1--24) in 56 healthy eumenorrheic nonhirsute healthy women with a mean age of 28.9 yr (range, 20--37 yr.) and a mean body mass index (BMI) of 29.2 kg/m(2) (18.2--46.2 kg/m(2)). Baseline samples and poststimulation samples were assayed for DHA and F. The basal and ACTH-stimulated levels of DHA, but not those of F, were negatively correlated with age, although neither the basal nor ACTH-stimulated responses of DHA and F varied with BMI. After controlling for age, the basal F level was negatively correlated to its net increment (i.e. Delta F; r = -0.54; P < 0.001), whereas there was no significant relationship between basal DHA and Delta DHA. We also compared the intersubject variability (coefficient of variation) for basal and stimulated levels of DHA and F. For basal (DHA(0)), 60 min (DHA(60)), and net increment in (Delta DHA) DHA levels, the coefficients of variation were 67.9%, 61.4%, and 76.0%, respectively; for F(0), F(60), and Delta F, they were 40.4%, 16.9%, and 31.3%, respectively. The variance in Delta DHA was significantly higher, and the variance in F(60) was significantly lower than that in all other variables; DHA(0), DHA(60), F(0), and Delta F had similar variances. In conclusion, in our population of healthy reproductive-aged women we observed that both basal and ACTH-stimulated levels of DHA after ACTH-(1--24) stimulation had significantly greater intersubject variance (approximately 60--70%) compared with the basal and poststimulation levels of F (approximately 15--40%). These data support the hypothesis that among normal women, AA (i.e. DHA) levels are highly variable compared to those of F. In addition, the intersubject variability in DHA levels is at least in part due to a variable response of AAs to ACTH stimulation. Whether the AA excess frequently observed in PCOS is due to the greater risk of those women with higher AA levels, basally and after ACTH stimulation, remains to be confirmed.

Umbilical cord serum levels of thromboxane B2 in term infants of women who participated in a placebo-controlled trial of low-dose aspirin.

OBJECTIVE: Our aim was to quantify thromboxane B2 (TXB2) in umbilical cord serum of term infants of nulliparous, low-risk women who were randomly assigned to either placebo or low-dose (60 mg) aspirin (ASA) on a daily basis from 24 weeks' gestation through delivery as part of a randomized clinical trial for prevention of preeclampsia. METHODS: Umbilical cord sera from 230 singleton, term infants whose mothers were involved in our low-dose ASA trial were assayed for TXB2, the stable metabolite of thromboxane A2, without knowledge of treatment or outcome data. The data were related to assigned treatment group, longitudinal pattern of maternal serum TXB2 levels, and other maternal and newborn characteristics. The data also were analyzed according to whether or not maternal serum levels of TXB2 at 29-31, 34-36, and delivery were reduced > or =50% compared to values prior to initiation of the trial. RESULTS: Umbilical cord TXB2 levels (ng/ml, mean +/- SE) were significantly lower at term in the ASA group (36.1 +/- 3.3, n = 111) than in the placebo group (56.6 +/- 5.7, n = 119; P = 0.002). Umbilical cord TXB2 levels were correlated to those in maternal serum at delivery in the ASA group (r = 0.3441; P = 0.0005) but not in the placebo group (r = 0.0626; P = 0.53). Regardless of assigned treatment group, infants whose mothers had a > or =50% longitudinal reduction in serum TXB2 had lower umbilical cord TXB2 levels (39.2 +/- 3.6, n = 114) than infants whose mothers had <50% reductions in TXB2 (54.6 +/- 5.9, n = 116; P = 0.027). Birthweights of these infants correlated inversely (r = 0.1678, P = 0.017) with maternal serum TXB2 at delivery but not to umbilical cord TXB2 levels; the best correlation between birthweight and maternal serum TXB2 was noted in pregnancies assigned to receive placebo (r = -0.2558, P = 0.009). CONCLUSIONS: Umbilical cord serum levels of TXB2 1) are reduced in instances of long-term maternal ingestion of ASA, 2) correlate well with maternal serum levels of TXB2 at delivery when there is evidence for consistent maternal use of ASA, but 3) do not correlate with maternal serum TXB2 levels when there is no evidence for frequent maternal ingestion of cyclooxygenase inhibitors. These data suggest that the capacity for platelet production of TXA2 in fetal and maternal compartments are regulated independently. Finally, there is an inverse relationship between maternal serum TXB2 levels at delivery and birthweight of newborn infants that is most evident among the pregnancies assigned to placebo and also among pregnancies in which there was little evidence to suggest a pattern of cyclooxygenase inhibitor use during pregnancy.

Effects of aging on adrenal function in the human: responsiveness and sensitivity of adrenal androgens and cortisol to adrenocorticotropin in premenopausal and postmenopausal women.

We sought to determine the effects of aging on several aspects of adrenal steroidogenesis in the hopes of characterizing the possible causes of adrenal androgen deficiency in elderly women. To this end, we quantified basal morning concentrations of cortisol (F), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DS), and androstenedione (A4) and then evaluated the effects of overnight dexamethasone (DEX) suppression followed by adrenal responses to graded hourly infusions of ACTH, ranging from 20-1280 ng/1.5 m2 x h. Finally, we performed a standard 0.25-mg ACTH bolus stimulation test, with sampling at 1 h thereafter. Basal serum levels of DHEA, DS, and A4 were significantly reduced (approximately 50% each) in a group of 35 healthy postmenopausal women, 55-68 yr old, compared to those in 30 healthy, regularly menstruating women, 20-25 yr old. Post-DEX levels of these C19 steroids also were significantly lower in the older women than in the younger women; the percent decrease after DEX for A4 was greater in the older women, whereas those in DHEA and DS were not age related. Basal and post-DEX levels of F were similar in both groups. Secretory responses of DS to ACTH were not informative due to its large plasma pool and slow clearance rate. The maximally stimulated levels of DHEA after ACTH bolus were significantly lower in the older women than in younger women; those of A4 were similar in both age groups, and the maximally achieved levels of F were higher in the older women than in the younger women. The sensitivity of adrenal DHEA, A4, and F to ACTH (defined as the minimal dose of ACTH required to significantly increase the steroid levels above basal post-DEX values) was similar in older and younger women. The responsiveness of the steroids of interest to ACTH (defined as the slope of the dose-response curve over the linear portion of the dose-response curve) also was similar among younger and older women. These data demonstrate that the deficiency in adrenal androgen production in women is restricted to the delta5-pathway steroid products (DHEA and DS), whereas there is no reduction in the capacity of the adrenal to produce A4 or cortisol. As DHEA and DS are likely to be produced mainly in the zona reticularis of the adrenal cortex, we propose that these data point to an alteration in that cortical zone as the cause of adrenal androgen deficiency in aging. The reductions in A4 in aging are probably due to reduced ovarian secretion after menopause.

The localization of DHEA sulfotransferase in steroidogenic and steroid metabolizing tissues of the adult rhesus macaque monkey.

Dehydroepiandrosterone sulfate is a major secretory product of the human adrenal cortex during intrauterine development as well as during adulthood. There are few animal experimental models that share this characteristic pattern of adrenal cortical steroidogenesis, which probably accounts for the relative paucity of information about the control of development and function of the adrenal androgen secretory apparatus. Adrenal androgen production in the rhesus macaque shares many similarities with that of the human. We sought to determine the tissue distribution of the enzyme DHEA sulfotransferase (DST) in the rhesus. Tissues were harvested at the time of autopsy from 7 adult monkeys (5 M, 2 F) ranging from 8-15 yrs old, and were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 mu thickness. Tissue sections were immunostained for DST with an anti-human liver DST antibody. DST was not detected in the testis or ovary. In the liver, immunoreactive DST was detected only in hepatocytes and in the kidney, DST was found only in the epithelial cells lining the collecting tubules. In the adrenal, DST was present in the cells of the zona reticularis but was not found in the cells of the medulla, zona fasciculata or in the very prominent zona glomerulosa. DST was most prominent in cells that were closest to the reticularis-medullary border. In most adrenals evaluated, the immunopositive cells were scattered, rather than forming a continuous band of cells around the medulla. The tissue distribution of DST in the adult rhesus macaque is qualitatively similar that observed in the adult human. These data aresuggestive that the rhesus might be an excellent model for the exploration of factors that regulate adrenal androgen production during development, aging, and in response to illness and stress, all of which have been found to be associated with variations in DHEA and DHEA sulfate production in the human.

Effects of maternal infections on fetal adrenal steroid production.

We sought to determine the effect of maternal infections on the fetal hypothalamic-pituitary-adrenal axis. Umbilical cord blood was collected at vaginal delivery after labor (24-44 wk. gestation) from 361 infants of women having normal pregnancy ( apart from preterm delivery in some) and 110 infants of women diagnosed with infections: 86% of these women had amnionitis. Infants exposed to antenatal corticosteroids, being growth retarded, or having developmental abnormalities that would be expected to alter function of the hypothalamic-pituitary unit were excluded. Umbilical cord serum was assayed for dehydroepiandrosterone sulfate (DS) and for cortisol. The data were analyzed by use of SAS. The gestational age of the infants of normal women (35.8+/-0.2 wk., Mean +/- SE) was greater than that of the infants of women having infections (34.3+/-0.4 wk., P = 0.003). Umbilical cord serum levels of DS and cortisol rose as a function of gestational age in both groups of infants (P<0.01). Despite being, on average, 1 wk. younger than the normal infants are, the infants of women having infections during pregnancy had higher serum levels of cortisol and DS than did those infants of the normal women. These data are consistent with activation of the fetal hypothalamic-pituitary-adrenal axis in pregnancies complicated by maternal infections. Such a fetal response could be the consequence of transplacental passage of products of the activated maternal immune system.

Developmental changes in molecular forms of immunoreactive adrenocorticotropin in the anterior pituitary gland of humans.

In extracts of anterior pituitary tissue obtained at autopsy of fetal, infant, and adult humans, five molecular forms of immunoreactive ACTH (ACTHi) were detected that had apparent molecular weights of approximately 4044, 30-34, 24-28, 16-18, and 4.5 kilodaltons (K). The relative proportion of each molecular form of ACTHi was similar in tissues that were extracted at the time of autopsy and in tissues that were stored frozen (-20 degrees C) for up to 2 years prior to extraction. We found that 40-44K ACTHi comprised a significantly greater proportion of total ACTHi in fetuses (12.3+/-3.5%) than in adults (3.8+/-0.8%); intermediate amounts of this form of ACTHi (8.0+/-4.1%) were found in tissues obtained from infants. On the other hand, the proportion of 4.5 K ACTHi in fetal pituitaries (67 %) was less than that in those of adults (84 %). The ratio of 40-44/30-34K ACTHi was significantly greater (P<0.001) in fetuses (1.46+/-0.12) and infants (1.31+/-0.07) than in adults (0.52+/-0.07). The ontogenetic differences in molecular forms of pituitary ACTHi are thought to reflect alterations in the processing of proopiomelanocortin as a function of human development.

Corticotropin-releasing hormone stimulates steroidogenesis in cultured human adrenal cells.

The effects of corticotropin releasing hormone (CRH) on steroid production by cultures of human fetal adrenal cells was investigated. We found that CRH, at concentrations that have been reported to exist in human fetal serum, stimulated dehydroepiandrosterone sulfate (DS) and cortisol production by cultured fetal zone and neocortical zone cells. A dose-dependent increase in secretion of both steroids was noted, with the cortisol pathway being preferentially enhanced by CRH at high concentrations. Pretreatment of adrenal cells for 3 days made them more responsive to ACTH stimulation and such effects were dose-dependent also. Inclusion of the antagonist, alpha-helical CRH (9-41) blocked CRH-induced stimulation of DS and cortisol over a broad dose range and also interfered with the augmentation of cortisol secretion noted after ACTH in CRH treated cells. CRH had no effects on adrenal cell proliferation or total cell protein. These studies are suggestive that CRH, either of systemic origin or else produced within the adrenal itself, has the potential to be a modulator of adrenal steroid production in the human.

Cross-sectional analysis of renal transplantation osteoporosis.

We report a cross-sectional study of 54 adult female renal transplant recipients. We measured bone mineral density (BMD) of the lumbar spine, femoral neck, total hip, and mid- and total radius, and 38 patients underwent transiliac crest bone biopsy. Osteopenia was widespread with 31/54 (57%) of patients osteoporotic at one or more sites. Seventeen out of 54 (32%) of the patients had a prevalent low-trauma fracture. There was a clear trend in BMD reduction across spine, hip and midradius, with the predominantly cortical midradial site showing the greatest loss. We found no relationship between BMD and body mass index, parathyroid hormone (PTH), dose of immunosuppressant, years since transplantation, age at menopause, or years since menopause. Histologically, abnormal biopsies could be classified into three categories: hyperparathyroid (n = 20), adynamic (n = 14), and osteomalacic (n = 2). Mean PTH was lower (p = NS) and mean cumulative prednisolone dose was higher (p = 0.04) in the adynamic group compared with the hyperparathyroid group, but because of overlap between groups neither was an effective discriminator of histology. We suggest that bone biopsy is indicated in these patients to direct appropriate treatment. At the cellular level, there were significant negative correlations between osteoclast function (eroded surface, r = 0.47, p = 0.003) and osteoblast numbers (osteoblast surface, r = -0.40, p = 0.01) and cumulative exposure to prednisolone. We postulate that suppression of osteoblast function by prednisolone with unopposed bone resorption may result in relative hypercalcaemia and low PTH. This progressive reduction in bone turnover may promote or prolong the adynamic state.

Dehydroepiandrosterone and dehydroepiandrosterone sulfate production in the human adrenal during development and aging.

Dehydroepiandrosterone (DHEA) is produced in prodigious quantities by the human adrenal, principally as the 3-sulfoconjugate DHEA sulfate (DS) during intrauterine life. The fetal zone and neocortex cells of the fetal adrenal express large amounts of DHEA sulfotransferase and minimal amounts, at least until very near the end of gestation, of 3beta-hydroxysteroid dehydrogenase. This pattern of enzyme expression favors substantial secretion of DHEA/DS with minimal cortisol produced; the DHEA/DS serves as the major precursor for placental estrogen formation in human pregnancy. Aside from adrenocorticotropin, other physiologic regulators of growth and steroidogenesis in the fetal adrenal have been postulated to exist, but have yet to be identified. Whereas intrauterine stressors may activate adrenal cortisol secretion, the fetal adrenal responds to many pregnancy conditions by suppressing DHEA/DS formation. After birth, the human adrenal undergoes reorganization whereby the large, inner fetal zone regresses, and DHEA/DS production is diminished. Just prior to gonadal maturation, the human adrenal undergoes morphologic and functional changes (adrenarche) that give rise to a prominent zona reticularis that is characterized by the presence of DHEA sulfotransferase, the absence of 3beta-hydroxysteroid dehydrogenase, and an enhancement of DHEA/DS production. The adrenal of the adult responds to stress in many instances like that of the fetus: increased cortisol secretion and diminished DHEA/DS secretion. The mechanisms for this divergence in the adrenocortical pathway is unknown. With aging, there is suppression of DHEA/DS secretion, possibly as the consequence of an involution of the zona reticularis, but corticosteroid production continues unabated.

The relationship between salivary cortisol concentrations in frozen versus mailed samples.

Saliva, popular for the measurement of cortisol concentrations, can be easily and painlessly obtained, so that study participants or medical patients may collect their own samples. This raises the question of whether cortisol concentrations are stable if samples are mailed unfrozen. Seventeen adult subjects (five males, 12 females, mean age = 27.82, SD = 7.55) participated in this study. One saliva sample from each subject was split. Half were frozen within 1 h. The other was exposed to conditions that would mimic a postal trip, including wide variations in temperature and movement over 5 days. A statistically significant positive correlation between cortisol concentration in the frozen and nonfrozen saliva samples was found (R2 = 0.92, p < .001). A paired t-test revealed no significant difference between samples (t(16) = 1.56, n.s.). This indicates that cortisol concentrations are stable during extended periods without freezing when exposed to widely varying temperatures and movement.

The effects of dehydroepiandrosterone (DHEA) on the thymus, spleen, and adrenals of prepubertal and adult female rats.

Administration of dehydroepiandrosterone (DHEA) to female rats produces a condition of reproductive failure and ovarian cysts similar to that seen in women having polycystic ovarian disease. On the other hand, DHEA may have beneficial effects on the immune system. We sought to determine the effect of DHEA, when administered in pharmacological amounts, on the thymus and spleen of prepubertal (25 day old) and young adult (60 day old) female rats. Since the adrenal, by means of its production of corticosteroids, also is known to modulate the immune system, we also evaluated the effects of DHEA administration on this gland. The daily SC administration of DHEA (6 mg/100g BW) to young adult female rats led to progressive and striking reductions in thymic weights (greater than 85% suppression after 20 days compared to vehicle treated animals). There were no effects of DHEA on body weights or the weights of the spleen. DHEA treatment also led to significantly reduced weights of the adrenals , which was sustained at about 15-20% below normal over 5-20 days treatment. Ovariectomy of the rats 5 days before initiation of DHEA or vehicle treatment gave rise to significant increases in thymic and spleenic weights in control animals and strikingly blunted the inhibitory effects of DHEA treatment for 10 days on the thymus; DHEA had no effect on the ovariectomy-induced rise in the weight of the spleen. Ovariectomy also had no effect on the inhibitory effects of DHEA on adrenal weight. Similar, albeit quantitatively less striking, responses were noted to occur after DHEA treatment in immature female rats. These data indicate that DHEA in doses sufficient to interfere with ovarian cyclicity also has potentially adverse effects on the adrenal and thymus. The ovary appears to play an independent role in maintenance of the size of the thymus and spleen and also may mediate some of the effects of DHEA on the thymus but not those on the adrenals.

The effect of 17 beta-estradiol on adrenocortical sensitivity, responsiveness, and steroidogenesis in postmenopausal women.

Aging in women is associated with reduced production of adrenal androgens (AAs); this decrease may in part be the result of menopausal hypoestrogenism. To determine the effects of physiological concentrations of estradiol (E2) on adrenocortical sensitivity and responsiveness in postmenopausal women, we determined steroid responses to a continuous incremental ACTH-(1-24) infusion (0, 20, 40 80, 160, 320, 640, and 1280 ng/1.5 m2/h), followed by an ACTH-(1-24) bolus of 0.25 mg, after overnight dexamethasone suppression before and after 3 months of E2 therapy (transdermal E2, 0.05 mg/day) in 14 postmenopausal women. After E2 treatment, subjects demonstrated an increase in serum E2 concentrations from 29.8 +/- 2.6 to 49.9 +/- 6.0 pg/mL (P < 0.005) and a decline in mean FSH levels from 83.1 +/- 24.4 to 57.5 +/- 17.3 mIU/mL (P < 0.004). E2 administration had no effect on basal, postdexamethasone, or maximally stimulated serum levels of cortisol (F), dehydroepiandrosterone (DHEA), androstenedione (A4), or 17-hydroxyprogesterone (17-OHP). Furthermore, E2 did not affect adrenal sensitivity or responsiveness to ACTH-(1-24) stimulation. Finally, the steroid ratios reflecting 3 beta-hydroxysteroid dehydrogenase (i.e. the A4/DHEA ratio) and delta 417,20-lyase (i.e. the A4/17-OHP ratio) activities also were unaffected by E2 therapy. The responsiveness of F to ACTH was significantly greater than that of DHEA, A4, or 17-OHP regardless of the circulating E2 levels. Furthermore, F and A4 were significantly more sensitive to ACTH stimulation than were 17-OHP and DHEA, and this was not altered by E2 administration. We conclude that transdermal E2 replacement to postmenopausal women does not significantly alter AA sensitivity or responsiveness to ACTH. Hence, it is unlikely that the hypoestrogenism of menopause contributes to the decline in AAs noted with age. Furthermore, menopausal estrogen replacement, at least in physiological amounts administered transdermally, cannot be expected to reverse the suppressed production of these androgens.

Effects of ACTH and cytokines on dehydroepiandrosterone sulfotransferase messenger RNA in human adrenal cells.

Dehydroepiandrosterone sulfate (DS) is the major adrenal androgen produced in the fetal and adult human; its formation is dependent upon the action of dehydroepiandrosterone sulfotransferase (DST). Since the factors that regulate DST are poorly characterized, we investigated the effects of ACTH, which stimulates DS production, and the cytokines transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) , both of which are inhibitory to adrenal steroidogenesis, on cultured human fetal adrenal cells. Cellular levels of DST mRNA were increased in a dose dependent fashion in response to ACTH; DST mRNA was less responsive to ACTH stimulation than was 17 hydroxylase (CYP 17) mRNA. The stimulatory effects of ACTH on DST mRNA levels were blunted by both TGF-beta and TNF-alpha; the inhibitory effects of TNF-alpha on DST mRNA were more striking than were those on CYP 17 mRNA. These data suggest that DS production can be altered by several agents acting on the DST gene.

Expression and subcellular distribution of the beta-isoform of the human glucocorticoid receptor.

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two highly homologous protein isoforms, termed hGR alpha and hGRbeta, that differ at their carboxy-termini. In contrast to the well characterized hGR alpha isoform, which modulates gene expression in a hormone-dependent fashion, the biological significance of hGRbeta has only recently begun to emerge. We and others have shown that the hGRbeta messenger RNA transcript is widely expressed in human tissues and that the hGRbeta protein functions as a dominant negative inhibitor of hGR alpha in transfected cells. Unfortunately, these initial studies did not determine whether the hGRbeta protein was made in vivo. Such analyses are hindered because available anti-hGR antibodies cannot discriminate between the similarly sized hGR alpha and hGRbeta proteins. Therefore, to investigate the expression of the hGRbeta protein, we have produced an antipeptide, hGRbeta-specific antibody termed BShGR. This antibody was made against the unique 15-amino acid peptide at the carboxy-terminus of hGRbeta and recognizes both the native and denatured conformations of hGRbeta, but does not cross-react with hGR alpha. Using BShGR on Western blots and in immunoprecipitation experiments, we detected the hGRbeta protein in a variety of human cell lines and tissues. Immunocytochemistry was then performed with BShGR on HeLa S3 and CEM-C7 cells and on tissue sections prepared from lung, thymus, and liver to assess the cellular and subcellular distribution of hGRbeta. In all immunopositive cells, hGRbeta was found in the nucleus independent of glucocorticoid treatment. Within tissues, the hGRbeta protein was expressed most abundantly in the epithelial cells lining the terminal bronchiole of the lung, forming the outer layer of Hassall's corpuscle in the thymus, and lining the bile duct in the liver. As a potential in vivo inhibitor of hGR alpha activity, expression of hGRbeta may be an important factor regulating target cell responsiveness to glucocorticoids.