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.

Inverse relation between low-density lipoprotein-cholesterol and dehydroisoandrosterone sulfate in human fetal plasma.

A striking inverse correlation was found in umbilical cord plasma between the concentrations of dehydroisoandrosterone sulfate and low-density lipoprotein (LDL)-cholesterol but not high-density lipoprotein-cholesterol or very low density lipoprotein-cholesterol. Dehydroisoandrosterone sulfate is a major secretory product of the human fetal adrenal and the principal precursor of placental estrogen production. The data suggest that the concentrations for LDL-cholesterol in fetal plasma are influenced by the rate of utilization of LDL-cholesterol by the fetal adrenal for steroidogenesis and are not necessarily related to a genetic predisposition for hypercholesterolemia or other lipoprotein disorders.

Conversion of plasma progesterone to deoxycorticosterone in men, nonpregnant and pregnant women, and adrenalectomized subjects.

During the third trimester of human pregnancy the concentrations of deoxycorticosterone (DOC) in maternal plasma are 4-50 times those in nonpregnant women and men. It has been suggested that the increased amount of DOC in maternal plasma originates in the fetal compartment. We considered an alternate explanation for the high levels of DOC in plasma or near-term pregnant women, viz., that DOC may be derived in part from 21-hydroxylation of maternal plama progesterone. To test this hyposthesis we measured the fractional conversion of plasma progesterone to DOC from the relationship between the 3H:14C ratio of the infused tracers, [3H]progesterone and [14C]-DOC, and the 3H:14C ratio or urinary 3 alpha,21-dihydroxy-5 beta-pregnan-20-one (tetrahydro-DOC). The fractional conversion of plasma progesterone to DOC ([rho](BU)P-DOC), measured in this manner, was 0.007 +/- 0.001 (mean +/- SEM, n = 26) in the subjects of this study. The values for [rho](BU)P-DOC varied widely among subjects (0.002-0.022) but the range of values for [rho](BU)P-DOC was similar among women pregnant with an anencephalic or dead fetus, nonpregnant and adrenalectomized women, and men. The transfer constant of conversion of progesterone to DOC in plasma, [rho](BB)P-DOC, remained constant in a nonpregnant woman during the infusion of nonradiolabeled progesterone at rates of 0-14 mg/h. Based on the results of these studied, we conclude that DOC is formed by extra-adrenal 21-hydroxylation of plasma progesterone and that the rate of formation of DOC by this pathway is proportional to the concentration of progesterone in plasma.

Regulation of extraadrenal steroid 21-hydroxylase activity. Increased conversion of plasma progesterone to deoxycorticosterone during estrogen treatment of women pregnant with a dead fetus.

We measured deoxycorticosterone (DOC) and progesterone (P) in plasma of 47 women pregnant with a dead fetus and sequentially throughout gestation in 35 women pregnant with a live fetus. When P levels in plasma were low, the plasma levels of DOC in women pregnant with a dead fetus varied but usually were similar to those in women pregnant with a live fetus. However, when P levels were high, the levels of DOC in some women pregnant with a dead fetus were considerably lower than those in women pregnant with a live fetus. To test whether this finding was due to loss of transfer of DOC from fetus to mother or else loss of extraadrenal steroid 21-hydroxylase activity in the mother after death of the fetus, we conducted several studies. The levels of P and DOC in plasma of one woman remained constant from 30 min after fetal death until delivery occurred 13 h later. Estrogen treatment of four women pregnant with a dead fetus brought about an increase in plasma levels of DOC in three of the women. In one woman the ratio of plasma DOC to P was 0.015, a value similar to that found before fetal death, but was 0.003 after fetal death but before estrogen treatment. In two women pregnant with a dead fetus the transfer constants of conversion of plasma P to DOC were 0.011 and 0.005 before, and 0.024 and 0.013, respectively, during estrogen treatment. In one woman pregnant with a deformed fetus with adrenal agenesis, the metabolic clearance rates of DOC before and during estrogen treatment were similar, whereas the plasma production rates of DOC were 2.75 before and 4.31 mg/24 h during estrogen treatment. We suggest that (a) the DOC in plasma of near-term pregnant women arises in part by extraadrenal 21-hydroxylation of plasma P and (b) estrogen stimulates steroid 21-hydroxylase activity in extraadrenal tissues.

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.

Stimulation of dopamine release into hypophysial portal blood by administration of progesterone.

To test the hypothesis that progesterone is involved in the regulation of dopamine release into hypophysial portal blood, female rats were adrenalectomized and ovariectomized at 1200 h on the day of proestrus and, immediately after the operation, injected sc with sesame oil or progesterone. Approximately 24 h later, hypophysial portal blood was collected and the plasma from this blood was analyzed for dopamine. The concentration of dopamine in portal plasma from rats given sesame oil or progesterone (50 mg) was 1.8 +/- 0.24 or 6.0 +/- 1.2 ng/ml (mean +/- SE), respectively. At the time of collection of portal blood, the progesterone concentration in arterial plasma was less than 0.8 ng/ml in rats given sesame oil and was 93 +/- 11 ng/ml in animals given progesterone. Conversely, the circulating concentrations of PRL was less (25 +/- 3 ng/ml) in animals given progesterone and higher (42 +/- 5 ng/ml) in animals given sesame oil. Twenty-four hours after the injection of progesterone (50 mg) into intact proestrous rats, the dopamine concentration in portal plasma was 5.8 +4- 1.29 ng/ml, a concentration which was significantly (P less thann 0.01) higher than that in untreated proestrous rats (1.1 +/- 0.24 ng/ml) but only slightly higher than that in untreated estrous rats (3.7 +/- 1.0 ng/ml). It is proposed 1) that progesterone administered sc can lead to an increased secretion of dopamine into hypophysial portal blood and 2) that the increased concentration of dopamine in portal plasma of intact pregnant rats relative to that of proestrous rats is, in part, a consequence of augmented progesterone secretion.

Estrogen inhibition of dopamine release into hypophysial portal blood.

The hypothesis that 17 beta-estradiol suppresses dopamine secretion into hypophysial portal blood was tested. Portal plasma concentrations of dopamine were significantly lower in proestrous rats (1.0 +/- 0.1 ng/ml; mean +/- SE) than in estrous rats (1.9 +/- 0.38 ng/ml). To deplete the animal of endogenous steroid hormones, proestrous rats were adrenalectomized (Adx) and ovariectomized (Ovx). Twenty-four hours later, hypophysial portal blood was collected for 60 min, and the plasma from this blood was analyzed for dopamine. Arterial plasma from these rats was assayed for 17 beta-estradiol and progesterone. The concentrations of dopamine in the portal plasma of sham-operated rats and bilaterally Adx-Ovx rats were similar to those in estrous animals. The concentration of dopamine in portal plasma of Adx-Ovs rats injected 24 h earlier with 50 micrograms 17 beta-estradiol was 1.0 +/- 0.31 ng/ml, which was comparable to that in proestrous animals but less than that in the estrous rats. The concentrations of 17 beta-estradiol in arterial plasma were as follows: 24 +/- 8.3 pg/ml in proestrous rats, 40 +/- 2.9 pg/ml in estrous rats, 10 +/- 1.3 pg/ml in Adx-ovx rats, and 96 +/- 17.3 pg/ml in Adx-Ovx rats injected with 50 micrograms 17 beta-estradiol. Twenty-four hours after injection of 25 micrograms 17beta-extradiol into Adx-Ovx rats, the plasma 17beta-estradiol levels were 51 +/- 7.4 pg/ml, and the dopamine concentrations in portal plasma were 1.9 +/- 0.57 ng/ml. It is concluded that an acute effect of 17 beta-estradiol is suppression of hypothalamic secretion of dopamine into hypophysial portal blood.

Studies on the subsynaptosomal localization of luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in the rat hypothalamus.

In the current investigation, subcellular particles (synaptosomes) of hypothalamic homogenates were isolated by differential centrifugation and discontinuous sucrose density gradient fractionation and found to be rich in LHRH, TRH, and the neuronal marker, norepinephrine (NE). Of the total quantity of LHRH, TRH, or NE in the nuclei-free homogenate, 52-65% was recovered in synaptosomes, whereas the cytosol, myelin/microsomes, and mitochondria contained only 1-12%. To determine the subsynaptosomal localization of LHRH and TRH, purified synaptosomes were lysed and the resulting suspensions were fractionated on discontinuous sucrose density gradients. LHRH (30-40%) was found to be localized primarily in subsynaptosomal particles which banded at sucrose densities between 0.6-1.0 M. Electron micorscopic analysis of these particles revealed the presence of dense-cored granules (70-80 nm diameter) and synaptosomal membrane remnants. Norepinephrine was found in two pools within the isolated nerve endings: 15-25% of synaptosomal NE was associated with the synaptic vesicles (45-55-nm diameter); about 40% was in the cytosol. TRH was present primarily as a soluble component of the nerve ending. No apparent association of TRH with dense-cored granules was demonstrable in this study; however, there may be some TRH in synaptic vesicles.

Actions of pregnant mare serum gonadotropin in the immature female rat: correlative changes in blood steroids, gonadotropins, and cytoplasmic estradiol receptors of the anterior pituitary and hypothalamus.

Several blood steroids, serum gonadotropins and cytosol estradiol receptors of the anterior pituitary and hypothalamus were quantified in immature female rats which were induced to ovulate with pregnant mare's serum gonadotropin (PMSG). Studies revealed that serum levels of progesterone, 17-hydroxyprogesterone, testosterone, androstenedione and estradiol were initially elevated at 6 PM (day 30) after administration of 8 IU of PMSG at 10 AM day 30. Serum levels of estradiol and testosterone rose progressively from day 30 through the AM of day 32. A further increase in serum concentrations of progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, and dehydroepiandrosterone occurred on the PM of day 32 whereas serum estradiol levels declined. Serum levels of all steroids declined on the day of estrus (33) and only progesterone levels were further elevated on day 34 (diestrus). Dihydrotestosterone concentrations were minimally altered by PMSG treatment. Saline administration resulted in no significant alterations in levels of any steroid quantified from day 29 to 34 in control animals. A progressive decline in cytosol estradiol receptor content of the anterior pituitary and hypothalamus was documented following PMSG treatment of intact female rats; there was no depletion of receptors following PMSG administration to ovariectomized immature rats. Maximal depletion of cytosol estradiol receptors occurred on day 32 with replenishment of cytosol estradiol receptor levels on estrus (day 33). The preovulatory gonadotropin surge was found to occur on the PM of day 32 after maximal receptor depletion. The cycle of depletion and replenishment of receptors was repeated during a second spontaneous estrous cycle four days later which coincided with a rise and fall in serum estradiol levels. It is suggested that the depletion of cytosol estradiol receptors of the anterior pituitary/hypothalamic unit may be causally related to the preovulatory gonadotropin surge resulting from PMSG administration to immature female rats. In addition, changes in blood steroids and gonadotropins after PMSG treatment are similar to those reported for proestrus-estrus-diestrus I of the normal adult estrous cycle. These findings further demonstrate the validity of the PMSG-primed immature female rat preparation as a model for the estrous cycle of the adult rat.

Metabolism of high density lipoprotein by human fetal adrenal tissue.

The role of lipoproteins as a source of the cholesterol utilized for steroidogenesis by human fetal adrenal (HFA) tissue was investigated previously. It was found that low density lipoprotein (LDL) was the lipoprotein preferred as a source of cholesterol for steroidogenesis by the HFA. [125I]Iodo-LDL was taken up and degraded by HFA tissue in organ culture, and the degradation of [125I]iodo-LDL was stimulated when ACTH (1 microgram X ml-1) was present in the culture medium. Others have shown that high density lipoprotein (HDL) is utilized as a source of cholesterol for steroidogenesis by rat adrenocortical cells in vitro and by the adrenals of the adult rat in vivo. In the present investigation we evaluated the metabolism of [125I]iodo-HDL by HFA tissue. [125I]iodo-HDL uptake by the HFA tissue increased in a linear manner with time and as the concentration of [125I]iodo-HDL in the culture medium was increased. However, there was little degradation of [125I]iodo-HDL by HFA. Moreover, preincubation of HFA tissue in medium containing ACTH (1 microgram X ml-1) or HDL, in various concentrations, did not affect the rate of uptake and degradation of [125I]iodo-HDL. The rate of degradation of [125I]iodo-LDL was found to decrease to low levels as the concentration of nonradiolabeled LDL in the culture medium was increased, whereas nonradiolabeled HDL had little effect on the degradation of [125I]iodo-LDL. HFA tissue fragments were incubated in medium containing ACTH plus lipoprotein-poor serum (LPPS) alone or LPPS plus HDL in various concentrations (50-1000 microgram X ml-1). The medium was changed daily and assayed for dehydroisoandrosterone sulfate and cortisol. In the presence of HDL, steroid secretion rates were no greater than those attained by HFA maintained in medium containing LPPS. It is concluded that the HFA utilizes cholesterol derived from LDL for steroidogenesis and that HDL is not metabolized efficiently by the human fetal adrenal.

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.

Luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in the hypothalamus of women: effects of age and reproductive status.

In the study of the effects of age and reproductive status on LHRH and TRH content in the hypothalamus of women, we found that the amount of LHRH (58 +/- 5.5 ng; mean +/- SE) in the hypothalamus of young women (16-29 yr) was significantly greater (P less than 0.001) than that (28 +/- 3.0 ng) in postmenopausal women (50-78 yr). The hypothalamic content of LHRH (18 +/- 2.4 ng) of bilaterally ovariectomized women (39-47 yr) was significantly less (P less than 0.001) than that (60 +/- 12.6 ng) in younger ovulatory women (30-39 yr) or that (56 +/- 13.5 ng) in ovulatory women of comparable age (40-49 yr). In contrast, the hypothalamic content of TRH (121.4 +/- 32.8 ng) in postmenopausal women were similar to that (122.3 +/- 12.5 ng) in young women. Although aging in women is associated with a significant reduction in the amount of LHRH in the hypothalamus, such a reduction appears to be a consequence of ovarian failure and not of aging per se.

Adrenocorticotropin interferes with transforming growth factor-beta-induced growth inhibition of neocortical cells from the human fetal adrenal gland.

The effects of transforming growth factor-beta (TGF-beta) and ACTH on growth, as indicated by [3H]thymidine incorporation into DNA, of primary cultures of neocortical cells from the human fetal adrenal gland were studied. TGF-beta inhibited, in a dose- and time-dependent manner, the growth of fetal neocortical cells, and ACTH significantly blunted the inhibitory effects of TGF-beta on growth of these cells. ACTH did not block the inhibitory effects of TGF-beta on growth of fetal adrenal fibroblasts or liver cells; neither ACTH nor TGF-beta had any effect on growth of fetal kidney cells. Thus, it appears that growth regulation of the neocortex may differ strikingly from that of the fetal zone of the human fetal adrenal, in which ACTH and TGF-beta have been reported recently to have additive inhibitory effects on cell proliferation.

Effect of burn trauma on adrenal and testicular steroid hormone production.

The effects of burn trauma in men on the production of adrenal and testicular steroids was investigated. Whereas there were significant increases in serum cortisol levels and urinary 17-hydroxycorticosteroid excretion soon after thermal injury, there were significant decreases in serum dehydroepiandrosterone sulfate, dehydroepiandrosterone, androstenedione, and testosterone concentrations during the first 4 weeks following burn trauma. Serum androstenediol and androstenediol sulfate levels also were reduced, though insignificantly, 10-23 days postburn. Serum LH levels were unchanged during the postburn interval. Since urinary 17-ketosteroid excretion was normal or below normal rather than increased, the decline in serum C19-steroid levels probably resulted from decreased glandular secretion rather than increased rates of metabolism and excretion. Low dehydroepiandrosterone sulfate and/or testosterone levels were found in some men several months after recovery from their burns. These data suggest that thermal injury leads to acute inhibition of adrenal and testicular C19-steroid secretion, but stimulation of adrenal glucocorticosteroid production, and that endocrine function in many instances is not normalized after complete healing of the burned surfaces. The mechanisms and physiological consequences of such changes in the steroid milieu of men after burn trauma are unknown.

Puberty in the chimpanzee: somatomedin-C and its relationship to somatic growth and steroid hormone concentrations.

A relationship between sex steroids and the somatomedins (Sms) is well known, but poorly defined. In some primates, including man, there are pubertal increases in Sms, concurrent with increased growth and sex steroid production. In the current studies, indices of somatic growth [body weight, crown-rump length (CRL), and testis size (testicular volume index)] and circulating concentrations of testosterone (T), estradiol (E2), dehydroepiandrosterone sulfate (DHEA-S), cortisol, and Sm-C were determined (n = 208) in 86 male and female chimpanzees during a 1-yr period. In addition, we have attempted to determine whether plasma Sm-C concentrations correlate with serum levels of estrogen and androgens. In male animals between 6 and 8 yr of age, there was a marked increase in testicular size, concurrent with an increase in serum T and preceding slightly an increase in the rate of body weight gain. There were no detectable increases in serum E2 or the CRL slope. In females between 6 and 8 yr of age, serum T increased, concurrent with an increase in the rate of body weight gain much smaller than that in male animals. Serum E2 increased only after 10 yr of age, and no increased linear growth (CRL) was found. In both sexes, increases in serum DHEA-S were found by 4-6 yr of age, in contrast to cortisol concentrations, which were high and remained unchanged from birth to 12 yr of age, except for lower values in the very youngest and very oldest female animals. An increase in Sm-C occurred in both sexes by 4-6 yr of age, with higher values in female than in male animals 0-2, 4-6, and 6-8 yr of age, and when all ages were considered together. In both sexes, plasma Sm-C concentrations correlated with serum T (r = 0.60 and P less than 0.001; r = 0.68 and P less than 0.001; females and males, respectively), although when both sexes were analyzed together, the correlation was not as good (r = 0.36; P less than 0.001). Sm-C concentrations correlated with serum DHEA-S when the two sexes were analyzed separately (r = 0.44 and P less than 0.001; r = 0.54 and P less than 0.001; females and males, respectively) or together (r = 0.49; P less than 0.001). Sm-C correlated poorly with serum E2 levels in females (r = 0.20; P less than 0.05) and did not correlate with E2 in males.(ABSTRACT TRUNCATED AT 400 WORDS)

Age-dependent extinction of thyrotropin-releasing hormone in the human cerebellum.

Immunoreactive TRH was quantified in eight regions of the cerebellum as well as in the medulla, pons, and hypothalamus of the fetal and adult human brain. High levels of TRH were detected in the fetal cerebellum, ranging from 216 +/- 103 pg/mg protein (mean +/- SD) in the deep cerebellar nuclei to 591 +/- 153 pg/mg protein in the anterior vermis. The concentrations of TRH were significantly greater (P less than 0.001) in each of the eight regions of the cerebellum of the fetal brain than in the corresponding regions of the adult brain. The magnitude of the difference between adult and fetal cerebellar levels ranged from an 18-fold difference in the deep cerebellar nuclei to more than a 100-fold difference in the anterior hemisphere. However, the TRH levels in pons and medulla were similar among fetuses and adults, and the TRH concentration in the adult hypothalamus was significantly higher (P less than 0.01) than that in the fetal hypothalamus. The TRH levels in adult rat hypothalami were extremely stable for several hours post mortem. We, therefore, conclude that the differences in cerebellar TRH concentrations of the fetal compared to those of the adult human are not related to a difference in the extent of postmortem degradation of TRH. Rather, we postulate that the decline in cerebellar TRH during maturation is a normal developmental process, and speculate that TRH, which has been found to have diverse effects on the central nervous system, may also influence the development of the human cerebellum.

The role of cyclic adenosine 3',5'-monophosphate in cholesterol metabolism and steroidogenesis by the human fetal adrenal gland.

In the present investigation we studied the role of cAMP as a mediator of ACTH action in human fetal adrenal (HFA) tissue. We have characterized the response to ACTH, dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP), and cholera toxin (CT) with respect to steroidogenesis, low density lipoprotein (LDL) binding, degradation of LDL, and the rate of de novo synthesis of cholesterol. The rate of dehydroisoandrosterone sulfate secretion was similar in HFA tissue maintained in the presence of ACTH, dbcAMP, or CT. In contrast, cortisol secretion by HFA tissue was more sensitive to dbcAMP and CT than to ACTH. In membrane preparations obtained from HFA tissue maintained in the presence of ACTH, dbcAMP, or CT, there was a 2 to 3-fold increase of specific binding of [125I]iodo-LDL. In HFA tissue maintained in the presence of ACTH or CT, the rate of degradation of LDL was significantly increased compared to tissue maintained in the lipoprotein-poor serum alone. Finally, in HFA tissue maintained in the presence of ACTH, dbcAMP, or CT there was a 6- to 10-fold stimulation of the rate of incorporation of [14C]acetate into cholesterol. We conclude that steroidogenesis, LDL binding, and degradation, as well as de novo synthesis of cholesterol, are probably stimulated in HFA tissue via a cAMP-mediated pathway.

The major immunoreactive alpha-melanocyte-stimulating hormone (alpha MSH)-like substance found in human fetal pituitary tissue is not alpha MSH but may be desacetyl alpha MSH (adrenocorticotropin1-13NH2).

Pituitary glands were obtained from human abortuses during the second half of gestation. Acid extracts were made from the anterior and neurointermediate lobes, and alpha MSH immunoreactivity (alpha MSHi) was quantified by RIA. alpha MSHi was found in both lobes of the pituitary gland, with 20-80% of the total pituitary alpha MSHi being present in extracts of the anterior lobe. Anterior and neurointermediate lobe extracts subjected to gel filtration on Sephadex G-50 revealed one peak of alpha MSHi having an elution profile identical to those of alpha MSH and desacetyl alpha MSH (ACTH1-13NH2). To characterize further the alpha MSHi extracts were subjected to high pressure liquid chromatography. No alpha MSH could be identified in extracts of the anterior lobe, and most of the alpha MSHi had an elution profile identical to that of desacetyl alpha MSH. Although small amounts of alpha MSH might be present in the neurointermediate lobe, most of the alpha MSHi in this lobe coeluted with desacetyl alpha MSH. Since alpha MSH was not converted to desacetyl alpha MSH during the extraction and chromatographic procedures, we hypothesize that the predominant form of alpha MSH-like material in the human fetal pituitary gland may be desacetyl alpha MSH.

Regulation of steroid secretion by adrenal tissue of a human anencephalic fetus.

ACTH-stimulated adrenal tissue of a human anencephalic fetus, when maintained in organ culture, secreted appreciable quantities of cortisol but little dehydroisoandrosterone sulfate or pregnenolone sulfate. In the absence of ACTH, cortisol secretion was severely attenuated. Arginine vasopressin or alpha MSH, when added to the culture medium, did not stimulate steroid secretion. When whole human serum was present in the culture medium bathing the adrenal tissue of the anencephalic fetus, the rate of cortisol secretion was similar to that attained when lipoprotein-poor serum was in the medium. Based on these findings, it is concluded that in the presence of ACTH, the adrenals of the anencephalic fetus secrete principally cortisol, and the failure of dehydroisoandrosterone sulfate and pregnenolone sulfate secretion is due to the absence of the fetal zone. The lack of stimulation of cortisol secretion by lipoprotein is probably due to a reduced number of low density lipoprotein receptors resulting from diminished ACTH stimulation before birth.

Acute adrenocorticotropin-(1-24) (ACTH) adrenal stimulation in eumenorrheic women: reproducibility and effect of ACTH dose, subject weight, and sampling time.

Assessment of adrenal reserve and the diagnosis of adrenal insufficiency by acute adrenocortical stimulation with ACTH-(1-24) has been well established. Alternatively, estimation of adrenocortical enzymatic activities by this method for the detection of inherited or acquired biosynthetic abnormalities has been less well characterized. Some of the discrepancies between studies estimating adrenocortical enzymatic activities in different pathological conditions (e.g. hyperandrogenism) may result from the different stimulation protocols used. The objective of this prospective study was to establish the inherent variability of the adrenal response to acute ACTH-(1-24) stimulation and to determine the effect of sampling time, stimulation dose, and subject weight on the same. Forty-one normal female volunteers were recruited (mean age, 29.1 yr), 30 within 90-110% ideal body weight and 11 weighing more than 120% ideal body weight. Three protocols were designed to study 1) the effects of sampling time, ACTH-(1-24) dose, and subject weight on adrenal response; 2) the effect of time of the day on the variability of basal steroid levels and the adrenal response to stimulation; and 3) the long term reproducibility of the adrenal response to ACTH-(1-24). Androstenedione, 17-hydroxyprogesterone, 11-deoxycortisol, dehydroepiandrosterone, and cortisol were measured in serum under basal and stimulated conditions. All subjects had normal basal levels of testosterone, androstenedione, dehydroepiandrosterone sulfate, and PRL. The acute iv administration of 0.10, 0.25, and 1.0 mg ACTH-(1-24) elicited similar and maximal steroid responses, with all steroid levels reaching a plateau 60-90 min poststimulation regardless of subject weight. Sampling of basal steroid levels every 5 min in the morning (AM; beginning 0700-0900 h) or evening (PM; 1500-1700 h) did not reveal any difference in steroid variability. Only the mean basal cortisol level was higher in AM than PM testing (P less than 0.03). Although the mean levels of dehydroepiandrosterone and 17-hydroxyprogesterone 60 min after stimulation were significantly higher in AM than PM studies, these differences were minimal. Ten volunteers underwent an average of four (range, 2-6) adrenal stimulation studies using 1.0 mg ACTH-(1-24) over a 1-yr period. The long term coefficient of variation (CV) for basal steroid levels ranged from 15-28%. Calculations of net adrenal response (delta steroid O-T and area delta steroid O-T) were less reproducible (CV, 0-82%) than measures of absolute response (steroid T, area steroid T, and %steroid T; CV, 7-32%). This difference in CV between the measures of net and absolute adrenal responses was significant for all steroids except androstenedione.(ABSTRACT TRUNCATED AT 400 WORDS)