Prostaglandin secretion by the neocortex and fetal zones of the human fetal adrenal gland

Previously, we reported that the human fetal adrenal (HFA) gland secretes various prostaglandins (PGs) in vitro and that PG secretion is inhibited by endogenously synthesized glucocorticosteroids. In this investigation, the neocortex (NC) and fetal zone (FZ) of the HFA gland were separated by microdissection and maintained as tissue fragments in organ culture. The rate of PG secretion into the culture medium was determined by measuring various PGs using specific RIAs in media collected at 24-h intervals. During the first 24 h in culture, the secretion rates of PGF2 alpha and PGE2 were 6- and 7-fold greater by NC [14 +/- 5 and 9.9 +/- 3 ng mg protein-1 24 h-1 (mean +/- SE)], respectively, than by FZ tissue (2.5 and 1.4 ng mg protein-1 24 h-1). The secretion rates of PGFM and PGD2 were 2-fold greater in NC tissue than in FZ tissue, but the secretion rates of thromboxane B2 were similar in both zones of HFA tissue. In another study, the patterns of secretion of PGF2 alpha and PGE2 were determined as a function of days in culture. The secretion rates of PGF2 alpha and PGE2 fell rapidly in NC from 19.0 +/- 11 and 38.3 +/- 9.7 ng mg protein-1 24 h-1, respectively, to 1.3 +/- 7.2 and 4.8 +/- 3.3 by day 4. In contrast, the secretion rates of PGF2 alpha and PGE2 rose 8- and 3-fold in FZ tissue (from 0.7 +/- 0.2 and 0.9 +/- 0.6 ng mg protein-1 24 h-1, respectively, to 5.9 +/- 0.5 and 3.1 +/- 1.2 by day 4). The addition of ACTH or dexamethasone inhibited PG secretion in both zones, but to a greater degree in FZ tissue than in NC tissue. In summary, the NC secretes larger quantities of PG than the FZ, and the patterns of secretion are different in the two zones. The secretion of PGs is inhibited more in FZ than in NC tissue by ACTH and glucocorticosteroids.     

Development of the baboon fetal adrenal gland: regulation of the ontogenesis of the definitive and transitional zones by adrenocorticotropin.

Throughout gestation, the primate fetal adrenal gland is comprised of the fetal zone, which expresses the P-450 17alpha-hydroxylase-C17,20 lyase (P-450c17) enzyme that catalyzes the synthesis of C19 steroids used for placental estrogen production. The development of the transitional zone comprised of cortical cells that express the P-450c17 and the 3beta-hydroxysteroid dehydrogenase-isomerase (3betaHSD) enzymes for cortisol production, and the definitive zone, which expresses 3betaHSD, but not P-450c17, for mineralocorticoid synthesis, does not occur until relatively late in gestation. Although ACTH is considered essential to fetal adrenal growth and function, the role that ACTH has in the development of the transitional and definitive zones, is less clear. To answer this question, the width of these zones was determined by immunocytochemical expression of P-450c17 and/or 3betaHSD in fetal adrenal glands obtained on day 100 (mid) of gestation (term = day 184) from baboons in which ACTH was administered to the fetus on days 95-99 of gestation or on day 165 (late) of gestation from baboons in which fetal ACTH was suppressed by treatment of the mother and fetus with betamethasone on days 150-164 of gestation. At midgestation, the fetal adrenal was comprised almost exclusively of fetal zone cells and a small definitive zone (38 +/- 2 microm in width), but was essentially devoid of a transitional zone (7 +/- 2 microm). Treatment with ACTH enhanced (P < 0.05) the width of the transitional zone (67 +/- 4 microm), but not the size of the definitive zone (10 +/- 4 microm). In late gestation, the width of the definitive zone, although 2-fold greater than that on day 100, was smaller (P < 0.05) than that of the transitional zone (120 +/- 15 microm), which greatly exceeded that at midgestation. Treatment with betamethasone in late gestation eliminated the transitional zone, but had no effect on the size of the definitive zone (120 +/- 8 microm). These findings indicate that the development of the baboon fetal adrenal transitional zone late in gestation is dependent on fetal pituitary ACTH. In contrast, the ontogenesis of the definitive zone at midgestation and its growth in late gestation occur in the relative absence of ACTH.     

The control of steroidogenesis by human fetal adrenal cells in tissue culture. II. Comparison of morphology and steroid production in cells of the fetal and definitive zones

Preparations of dispersed human fetal adrenal cells from the inner third of the gland and from the subcapsular area were maintained in culture, and their ultrastructure and steroid production were studied. The former type of preparation contained only fetal zone cells, while the latter contained definitive zone cells together with varying numbers of fetal zone cells. Both types could be cultured with equal ease, but during short term culture, fetal and definitive zone cells became morphologically indistinguishable. The patterns of steroid production and, in particular, the relative production of delta 4,3-ketosteroids and delta 5,3 beta-hydroxysteroids were similar in both preparations, as were their dose-response relationships during incubation with alpha ACTH-(1-24). Although considerable variability in total steroid production was observed between cells from different adrenal glands, in no specimen was any evidence for functional zonation of the fetal adrenal cortex observed in vitro. The results suggest that the apparently unique histological appearance and function of the fetal adrenal cortex may only reflect intense stimulation by ACTH secondary to the combined influences of a rapid cortisol MCR and of some inhibitor of fetal adrenal 3 beta-hydroxysteroid dehydrogenase activity.     

Identification of definitive and fetal zone markers in the human fetal adrenal gland reveals putative developmental genes

Organogenesis is a coordinated process involving cell replication, differentiation, adhesion, and migration. We seek to understand the complex developmental signals involved in the ontogeny of the human fetal adrenal gland. The gland is comprised initially of two zones, the definitive and fetal zones. A third zone, the transitional zone, develops between them after midgestation. We have suggested that the definitive zone is comprised of a pool of progenitor cells that proliferate and differentiate into cells of the transitional and fetal zones. However, it has not been possible to demonstrate that definitive zone cells have this capacity because of the absence of protein markers unique to these cells; thus, they could not be purified or positively identified. We sought to identify definitive and fetal zone markers to facilitate cell sorting and identify molecules of biological interest in adrenal development. We performed subtractive hybridization, in situ hybridization, and immunofluorescence to identify unique markers of definitive zone cells. NovH and metallopanstimulin were identified by subtraction hybridization, primarily in the definitive zone. P-Glycoprotein, also principally on definitive zone cells, and the low density lipoprotein (LDL) receptor, predominantly on fetal zone cells, were identified by immunofluorescence. Identification of cellular markers unique to each zone of the fetal adrenal gland will enhance the ability to characterize the proliferative potential of definitive zone cells and assess their capacity to differentiate into cells of the transitional and fetal zones. Purified cells also will permit detailed molecular and mechanistic studies of regulation of human fetal adrenal development.     

Lipoprotein utilization and cholesterol synthesis by the human fetal adrenal gland

A model proposed for regulation of steroidogenesis, lipoprotein utilization and cholesterol metabolism in HFA tissue is presented in Fig 17. We envision that the role of ACTH and cAMP in steroidogenesis and cholesterol metabolism is as follows. ACTH binds to specific receptors on the surface of the cells of the HFA gland and as a consequence, adenylate cyclase is activated, leading to increased formation of cAMP. cAMP causes activation of protein kinase that leads, presumably, to phosphorylation of specific proteins. This leads to the initiation of reactions that give rise to increased activity of key enzymes and levels of proteins involved in adrenal cholesterol metabolism. Presumably, the action of ACTH causes an increase in the activity of cholesterol side chain cleavage, the rate-limiting step in the conversion of cholesterol to steroid hormones. We suggest that once the mitochondrial cholesterol side-chain cleavage system is fully activated by ACTH, the supply of cholesterol to the mitochondria becomes rate-limiting for steroidogenesis. To meet this demand for cholesterol, a further action of ACTH results in an increase in the number of LDL receptors. LDL binds to specific receptors on the cell surface that are localized in coated pits. LDL is internalized by a process of adsorptive endocytosis and the internalized vesicles fuse with lysosomes and the protein component of LDL is hydrolyzed by lysosomal proteolytic enzymes to amino acids. The cholesteryl esters of LDL also are hydrolyzed to give rise to fatty acids and cholesterol. The liberated cholesterol is available for utilization in the biosynthesis of steroid hormones and other cellular processes. In addition, ACTH stimulates the activity of HMG CoA reductase and, thus, the rate of de novo cholesterol biosynthesis. In this way sufficient cholesterol is obtained to provide for precursor cholesterol to maintain the high rate of steroid synthesis by the HFA. HDL is not utilized as a source of cholesterol by the HFA. Because of the rapid rate of utilization of LDL by the HFA, fetal plasma levels of LDL are low and the activity of the HFA is a primary determinant of these levels. Thus, in the case of anencephaly, in which the activity of the adrenal is very low, plasma levels of LDL are 2--3 times higher than in normal fetuses, whereas plasma HDL levels are similar. In addition, in the normal neonate plasma LDL levels rise rapidly after birth, and this event is coincident with the involution of the fetal zone of the adrenal. The fetal liver is likely to be the major source ultimately of the LDL-cholesterol utilized by the HFA. Consequently, factors that regulate cholesterol and lipoprotein synthesis in the fetal liver may, in turn, affect the steroidogenic activity of the HFA through regulation of the supply of cholesterol precursor. Thus, if trophic factors for the HFA other than ACTH exist, an important site of their action might be the fetal liver, rather than a direct action to influence the rate of synthesis of steroids by the fetal adrenal.     

Protein kinase-C in the human fetal adrenal gland

The fetal zone (FZ) of the human fetal adrenal gland undergoes rapid growth and exhibits a high rate of steroidogenesis throughout fetal life. In addition to cAMP-dependent processes regulating steroidogenesis and possibly growth of the FZ, evidence is accumulating that cAMP-independent mechanisms are also involved. The purpose of this study was to determine if the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of protein kinase-C activity, stimulates steroidogenesis in FZ cells and to characterize protein kinase-C activity in FZ, neocortex zone, and anencephalic adrenal tissues. Adrenal glands were obtained from first and second trimester abortions and two anencephalic fetuses. The FZ was dissected from the neocortex. In some experiments, dispersed FZ cells were incubated in the presence and absence of ACTH and TPA for 3 h. TPA and ACTH stimulated steroidogenesis 2- and 5-fold, respectively. In other experiments, the separated zones and anencephalic adrenal tissues were homogenized, and the homogenates were subjected to DEAE-cellulose column chromatography. A single peak with phospholipid- and calcium-dependent activity was found. Subcellular distribution studies demonstrated greatest activity in the cytosolic fraction. The specific activity of protein kinase-C was significantly greater in FZ than neocortex zone, whether expressed per mg protein or per microgram DNA content. The activity in anencephalic tissue was low. In addition, protein kinase-C (80,000-dalton molecular size protein) was detected in adrenal tissues after electrophoresis and immunoblotting using an antibody directed against protein kinase-C. Greater amounts of protein kinase-C were detected in FZ tissue than in NC or anencephalic adrenal tissue. These results indicate that the lower activities of protein kinase-C in neocortex and anencephalic adrenal tissues were due to low amounts of enzyme rather than inactive enzyme. In summary, TPA-stimulated steroidogenesis in fetal zone cells and fetal zone cells contained greater activity and a greater amount of protein kinase-C than neocortex cells. Minimal activity and enzyme protein were found in anencephalic tissues. These results suggest that cAMP-independent mechanisms may play a role in fetal adrenal steroidogenesis.     

Low density lipoprotein binding and de novo synthesis of cholesterol in the neocortex and fetal zones of the human fetal adrenal gland

The binding of low density lipoprotein (LDL) and the de novo synthesis of cholesterol in separated zones of human fetal adrenal (HFA) tissues were investigated. The number of LDL-binding sites was 2-fold greater in membrane fractions prepared from fresh fetal zone tissue than in those from neocortex tissue. The binding capacity for LDL in fetal zone and neocortex membrane preparations of HFA tissues maintained in culture in the presence of ACTH was 2-fold greater than that in membrane fractions of control tissues. The rates of de novo synthesis of cholesterol also were determined in separated zones of HFA tissue by measuring the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in microsomal fractions prepared from HFA tissues and by determining the rate of incorporation of tritium from [3H]water into cholesterol in HFA tissue fragments. The rate of de novo synthesis of cholesterol in fresh fetal zone tissue was twice that in neocortex tissue as estimated by these methods. When separated zones of HFA tissue were maintained in culture in the presence or absence of ACTH, the rates of de novo synthesis, as determined by the rate of incorporation of tritium from [3H]water into cholesterol, were stimulated to a similar extent by ACTH in both fetal zone and neocortex tissues. However, the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase was increased to a greater extent by ACTH pretreatment in neocortex tissues than in fetal zone tissues. In summary, fetal zone tissues of the HFA gland have a larger number of LDL-binding sites and higher rates of de novo synthesis of cholesterol than do neocortex tissues, and ACTH stimulates LDL binding and de novo synthesis of cholesterol in both zones of the HFA gland.     

Steroid hydroxylase gene expression in the ovine fetal adrenal gland following ACTH infusion

Between 90 and 120 days of gestation (term = 147 +/- 5), when plasma cortisol concentrations in the fetus are at a minimum, levels of mRNA encoding the steroidogenic enzymes 17 alpha-hydroxylase (P-450(17 alpha] and cholesterol side-chain cleavage (P-450scc) are also very low. Over the following 30 days, P-450(17 alpha) and P-450scc gene expression increases concurrent with increasing fetal cortisol concentration. The hypothesis tested in this study was that cortisol biosynthesis is minimal in the period 90-120 days because of insufficient ACTH. Fetuses were cannulated between 98-102 days of gestation. Following recovery, 7 fetuses received 24-h ACTH infusions (12 micrograms/24 h) and 5 fetuses received 24-h vehicle infusions; 4 ACTH-infused and 4-vehicle-infused fetuses were then sacrificed immediately after cessation of the infusion. The other fetuses were left in utero for 3 days prior to sacrifice. Fetal blood samples were analysed for ACTH and cortisol and the adrenals processed for hybridization histochemistry and Northern blot analysis. ACTH, but not vehicle, induced significant increases in the width of the adrenal cortex and in the levels of P-450(17 alpha) and P-450scc mRNA. Concurrently, fetal plasma ACTH and cortisol concentrations also increased significantly. In adrenals from fetuses left in utero for 3 days after cessation of the ACTH infusion, P-450(17 alpha) and P-450scc mRNA levels returned to control levels. Plasma ACTH and cortisol levels also approximated basal values. P-450c21 mRNA levels did not vary significantly at any time with the treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of hCG in regulation of the fetal zone of the human fetal adrenal gland.

It has been suggested that hCG is a trophic hormone for the fetal zone of the human fetal adrenal gland. To test this hypothesis, the isolated fetal zones of adrenals from eight fetuses (12-17-week gestation age) were superfused in the presence or absence of hCG. Dehydroepiandrosterone sulfate (DHAS) was measured in the superfusion effluent. A significant increase in DHAS production was observed in the presence of hCG. DHAS secretion decreased during the first 60 min in the control and experimental superfusions from 83 +/- 10.0 (mean +/- SE) to 71 +/- 8.0, and from 90 +/- 9.0 to 70 +/- 6.0 ng/100 mg/ml, respectively. In the presence of hCG (250 ng/ml), DHAS secretion increased significantly (P less than 0.01) over the controls to 116 +/- 12.0 at 120 min, and remained above the controls thereafter. These results support the hypothesis that hCG is one of the regulators of DHAS production by the human fetal adrenal gland early in gestation. As we found that ACTh stimulated DHAS secretion in a previous study and as there is indirect evidence for a role of ACTH in DHAS regulation late in pregnancy, these observations suggest dual regulation by hCG and ACTH early in pregnancy, and a possible transition to ACTH regulation of the fetal zone of the human fetal adrenal after midgestation.     

Prostaglandin biosynthesis in the human fetal adrenal gland: Regulation by glucocorticosteroids

Human fetal adrenal (HFA) tissue was maintained in organ culture to evaluate the biosynthesis of prostaglandins and hormonal regulation of prostaglandin formation by this tissue. The HFA tissue secreted substantial amounts of prostaglandin E(2), prostaglandin F(2alpha), 13,14-dihydro-15-ketoprostaglandin F(2alpha), 6-ketoprostaglandin F(1alpha), and thromboxane B(2); secretion of prostaglandin D(2) could not be demonstrated. Prostaglandin biosynthesis in HFA tissue was inhibited in a time-dependent manner by corticotropin (ACTH; 0.4 muM); by the fourth day of culture, the extent of inhibition of biosynthesis of each prostaglandin was 60-90%. Progesterone (1 muM), cortisol (1 muM), and dexamethasone (1 muM) inhibited prostaglandin biosynthesis whereas estradiol (1 muM) did not. Of the compounds tested for inhibitory activity, dexamethasone was the most potent. An inhibitor of 11beta-hydroxylase activity (metyrapone; 0.1 mM) effectively eliminated the inhibition of prostaglandin biosynthesis caused by corticotropin and progesterone. Metyrapone treatment alone caused a 3-fold increase in prostaglandin biosynthesis by fetal adrenal tissues. Similar stimulatory effects resulted from treatment with inhibitors of (i) 3beta-hydroxysteroid dehydrogenase (cyanoketone; 15 muM), (ii) steroid 17alpha-hydroxylase (SU 10603; 19 muM), and (iii) cholesterol side-chain cleavage (aminoglutethimide; 1 mM). Inhibition of prostaglandin biosynthesis by dexamethasone in the presence or absence of metyrapone was concentration dependent and 50% inhibition could be demonstrated at 1 nM. A competitive inhibitor of the binding of glucocorticosteroids to cytoplasmic receptors (cortisol 21-mesylate; 1 muM) significantly reduced the inhibition of prostaglandin biosynthesis effected by dexamethasone (10 nM). These findings suggest that prostaglandin biosynthesis in the HFA gland is regulated by endogenously synthesized glucocorticosteroids, the actions of which are mediated by a glucocorticosteroid receptor. Such glucocorticosteroids induce the synthesis of a substance that inhibits prostaglandin biosynthesis.     

Midkine, a heparin-binding growth factor, selectively stimulates proliferation of definitive zone cells of the human fetal adrenal gland

CONTEXT: In the human fetal adrenal gland (HFA), the inner fetal zone (FZ) secretes dehydroepiandrosterone sulfate. The function of the outer definitive zone (DZ) is less clear; however, the DZ phenotype is that of a reservoir of progenitor cells, many of which are mitotically active. Midkine (MK) is a heparin-binding growth factor with various bioactivities. OBJECTIVE: The objective of this study was to investigate expression, proliferative effects, and ACTH regulation of MK in the HFA. DESIGN AND SETTING: RNA, cryosections, and primary cell cultures from HFAs (14-24 wk) and adult adrenal RNA were used. MAIN OUTCOME MEASURES: The main outcome measures were MK mRNA levels (measured by quantitative real-time RT-PCR); MK localization (measured by immunostaining); MK proliferative effects and mechanism (measured by proliferation assays, flow cytometry, pharmacological interventions); and ACTH regulation (measured by quantitative real-time RT-PCR). RESULTS: HFA MK mRNA levels were 4-fold higher than in adult adrenals (P < 0.05) and were comparable to levels in fetal and adult brains (positive controls). MK immunoreactivity was abundant throughout the HFA. Exogenous MK caused proliferation of isolated DZ cells but not FZ cells (72 h, P < 0.05). In contrast, basic fibroblast growth factor induced proliferation of cells from both zones. Pharmacological interventions indicated that MK-induced DZ cell proliferation may be mediated by phosphatidylinositol 3-kinase, MAPK kinase, and Src family kinases. ACTH (1 nm) increased MK mRNA by 3.5-fold (48 h, P < 0.01) in isolated FZ cells. CONCLUSIONS: MK likely plays a key role in HFA development. MK's selective in vitro mitotic effects on DZ cells may provide insights into the mechanism underlying the distinct in vivo differences in mitotic activity between the DZ and FZ.     

The role of calmodulin antagonists on steroidogenesis by fetal zone cells of the human fetal adrenal gland

The adrenal gland of the human fetus (HFA) is relatively large compared to that of the adult and exhibits an extremely high rate of steroidogenesis both in vivo and in vitro. The fetal zone cells make up 80-85% of the volume of the HFA and are the major site of steroid production during fetal development. We have recently demonstrated that calcium is involved in the regulation of steroidogenesis in fetal zone cells of the HFA. There is considerable evidence that many actions of calcium within cells are mediated by the calcium-binding protein calmodulin. The purpose of the present investigation was to determine if calmodulin also plays a role in HFA steroidogenesis. To investigate this possibility, the fetal zone was dissected from fetal adrenals of first and second trimester human abortuses. After collagenase digestion of the tissue, dispersed fetal zone cells were maintained in a Krebs-Ringers medium at 37 C for a 3-h incubation. Cells were incubated with and without ACTH (10(-8) M) in the presence of the calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ), and calmidazolium (CAL) at concentrations of 5-100 microM. The media were assayed for contents of dehydroepiandrosterone sulfate (DS), cortisol (F), pregnenolone, and cAMP by RIA. The addition of ACTH stimulated F secretion 5- to 10-fold compared to that in control fetal zone cells. DS secretion increased up to 5-fold and pregnenolone about 2-fold in the presence of ACTH compared to values in control cells. ACTH also stimulated cAMP secretion by 10-fold compared to that in control cells. The addition of TFP, CPZ, and CAL significantly inhibited ACTH-stimulated DS, F, and pregnenolone secretion in a dose-related fashion to near-control levels. We observed that TFP, CPZ, and CAL inhibited cAMP accumulation as well as Bu2cAMP-stimulated steroid secretion. The metabolism of 22R-hydroxycholesterol to pregnenolone was inhibited by TFP and CPZ, but not by CAL. These studies suggest that calmodulin plays a role in regulating steroidogenesis in fetal zone cells of the HFA.     

Localization of G protein alpha-subunits in the human fetal adrenal gland

The aim of the present study was to investigate the presence and localization of the main G protein alpha-subunits in the human fetal adrenal gland during the second trimester of gestation. Immunofluorescence studies conducted on sections from frozen glands obtained immediately after therapeutic abortion indicated that the alpha s subunit of the heterotrimeric Gs protein was detected in all adrenal cell types, except for endothelial cells. The other alpha-subunits had a more specific pattern of distribution. Indeed, the alpha il-2 protein was restricted to the definitive zone, whereas alpha i3 labeling was mainly expressed in the fetal zone. The alpha q protein subunit was localized in vascular endothelial cells at the periphery of the adrenal gland and in fetal cells at the center. Finally, chromaffin cells expressed alpha s, alpha q, and alpha o1, but not alpha o2 nor alpha i. Altogether, these results indicate that the human fetal adrenal gland is not only unique in its particular morphology and expression of steroidogenic enzymes, but also by the differential expression of G protein alpha-subunits. Such cell specific distribution in glands from midgestational fetuses may account for the absence or the different responses to stimuli, when compared with the adult adrenal gland.     

Adenylate cyclase activity in neocortex and fetal zone membrane fractions of the human fetal adrenal gland

Whether peptide hormones other than ACTH may be responsible for the difference in size or rate and pattern of steroidogenesis of the fetal zone (FZ) compared to those of the neocortex (NC) of the human fetal adrenal gland is controversial. In the present investigation, the activity of adenylate cyclase in membrane fractions of separated zones of the human fetal adrenal gland was determined. Basal adenylate cyclase activity was 2- to 3-fold greater in NC than in FZ membrane fractions. The addition of ACTH-(1-24) stimulated adenylate cyclase activity in both zones, but the activity was more sensitive to ACTH (10(-10) M) in NC fractions than in FZ fractions (10(-7) M). In addition to ACTH-(1-24), the effect of other ACTH-related peptides on the activity of adenylate cyclase in the separated zones of the adrenal gland was investigated. 16K fragments 2-36, gamma 3MSH, alpha MSH, beta-endorphin, leu-enkephalin, and met-enkephalin, as well as hCG, FSH, prostaglandin E2, prostaglandin F2 alpha, epinephrine, and norepinephrine did not stimulate adenylate cyclase activity in either zone. It is concluded that basal and ACTH-(1-24)-stimulated adenylate cyclase activities are greater in NC than in FZ membrane fractions. In addition, the results of the present investigation do not support the concept that other ACTH-related peptides or peptide or protein hormones increase steroidogenesis by stimulating adenylate cyclase activity in the human fetal adrenal gland.     

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

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