Hormonal regulation of the adrenocortical cell

Monolayer cultures of bovine and human adrenocortical cells have been used to study regulation of growth and function. Homogeneous bovine adrenocortical cells exhibit a finite life span of ～60 generations in culture. Full maintenance of differentiated function (steroid hormone synthesis) requires an inducer such as ACTH and antioxidizing conditions. Full induction of differentiated function occurs only when cellular hypertrophy is stimulated by growth factors such as fibroblast growth factor and serum. ACTH and other agents that increase cellular cAMP inhibit replication but do not block growth factor-induced cellular hypertrophy. ACTH and growth factors together result in a hypertrophied, hyperfunctional cell. Replication ensues only when desensitization to the growth inhibitory effects of ACTH occurs. Cultures of the definitive and fetal zones of the human fetal adrenal cortex synthesize the steroids characteristic of the two zones in vivo. ACTH stimulates production of dehydroepiandrosterone (DHA), the major steroid product of the fetal zone, and of cortisol, the characteristic steroid product of the definitive zone. Prolonged ACTH treatment of fetal zone cultures results in a preferential increase in cortisol production so that the pattern of steroid synthesis becomes that of the definitive zone. The preferential increase in cortisol production by fetal zone cultures results from induction of 3β-hydroxysteroid dehydrogenase, Δ^4,5 isomerase activity, which is limiting in fetal zone cells. ACTH thus causes a phenotypic change in fetal zone cells to that of definitive zone cells. In both bovine and human adrenocortical cells, the principal effect of ACTH is to induce full expression of differentiated function. This occurs only under conditions where growth substances and nutrients permit full amplication.     

3 Beta-hydroxysteroid dehydrogenase-isomerase activity in bovine adrenocortical cells in culture: lack of response to ACTH treatment

Primary cultures of bovine adrenocortical cells (BAC) were used to determine whether the adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase complex (3 beta-HSD), like the 17 alpha-hydroxylase (17-OHase), responded to ACTH treatment with an increase in activity. Both enzymes influence the steroidogenic path leading to 17 alpha-hydroxyprogesterone formation and thus could affect adrenal androgen biosynthesis. 3 beta-HSD Activity in postmitochondrial supernatant fluid, homogenates or cell monolayers remained unchanged after cells had been maintained in 1 microM ACTH up to 48 h. Since ACTH exposure led to a marked increase in 17-OHase activity over the same time period, it is concluded that, under the conditions used, the 3 beta-HSD-isomerase complex in BAC is nonresponsive to tropic hormone treatment.     

Maintenance of cell proliferation and steroidogenesis in cultured human fetal adrenal cells chronically exposed to adrenocorticotropic hormone: rationalization of in vitro and in vivo findings

Previous studies indicated that acute exposure of adrenal cells to adrenocorticotropic hormone (ACTH) markedly stimulates steroidogenic capacity in vitro but also inhibits cell proliferation. However, in vivo, ACTH is known to stimulate adrenal cell growth. To address this discrepancy, we determined the effect of long-term (9-11 days) continuous or intermittent exposure to ACTH on human fetal adrenal cell proliferation and steroidogenesis. Adrenal glands from fetuses 18-22 wk gestation were studied. Fetal zone cells were plated either on plastic or on an extracellular matrix (ECM) in the presence and absence of basic fibroblast growth factor (bFGF) (0.5 ng/ml) and 1 or 10 nM ACTH. As determined by cell counting, bFGF stimulated cell proliferation during 9 days in culture. In the presence of bFGF, the average doubling time decreased from 44 to 30 h on plastic and from 37 to 26 h on ECM. Under these conditions, ACTH did not inhibit cell proliferation. Proliferation of fetal adrenal corticosteroid-producing cells in the ACTH-treated cultures also was assessed by histochemical staining for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). The number of positive cells increased more than 4-fold between Days 5 and 9 in culture. Continuous treatment with 1 nM ACTH increased dehydroepiandrosterone sulfate (DHAS) production 5- to 10-fold during the first 5 days in culture. Thereafter, the stimulated hormone production decreased over time, although there was still a difference of almost 100-fold between the control and ACTH-treated cultures at the end of 9 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of corticosterone on adrenal 5-ene-3 beta-hydroxysteroid dehydrogenase in estrogen-treated rats

Treatment of adult male rats with estradiol-17 beta for 7 days results in adrenal hyperplasia, increased level of serum ACTH along with reduction in serum level of alpha 2u-globulin and inhibition of adrenal 5-ene-3 beta-hydroxysteroid dehydrogenase (5-ene-3 beta-HSD) activity. Administration of corticosterone in estrogen-treated rats reverses the effects of estrogen while in normal rats corticosterone treatment reduces adrenal weight, serum ACTH and adrenal 5-ene-3 beta-HSD activity. In vitro experiments show that alpha 2u-globulin fails to change adrenal 5-ene-3 beta-HSD activity in corticosterone pretreated rats while in normal and estrogen pretreated rats alpha 2u-globulin increases 5-ene-3 beta-HSD activity.     

Response of adrenal 3beta-hydroxy-delta5-steroid dehydrogenase to adrenocorticotropin treatment in thiouracil-fed male mice

A number of parallels can be drawn between the reported endocrine status of thiouracil-fed young rodents and that of aged animals, particularly with regard to the hypothalamus-pituitary-adrenal axis. Since the activity of the adrenal steroidogenic enzyme 3beta-hydroxy-delta5-steroid dehydrogenase (3beta-HSD) has been shown to be depressed in aged rats and mice, the present study was done to determine whether exposure of young mice to thiouracil had a similar effect on adrenal 3beta-HSD activity. Feeding the goitrogen thiouracil at 0.25% (w/w) of the maternal diet from conception, and keeping it 0.25% of the offsprings' diet after weaning, significantly elevated activity of 3beta-HSD per gram of adrenal gland above control levels in 4-month-old mice, perhaps to compensate for depressed adrenal mass. Daily subcutaneous injections of physiological saline (0.9%) for 4 days was sufficient to increase 3beta-HSD activity per gram of adrenal tissue in euthyroid (P less than 0.05) but not in thiouracil-fed mice. Subcutaneous administration of ACTH (2 IU daily for 4 days) significantly increased adrenal 3beta-HSD activity to comparable levels in thiouracil-fed and euthyroid animals. Thus, thiouracil enhances the activity of 3beta-HSD per gram of adrenal tissue and does not prevent response of enzyme activity to exogenous ACTH.     

Functional capacity of fetal zone cells of the baboon fetal adrenal gland: a major source of alpha-inhibin.

We have shown that ACTH receptor mRNA expression and steroidogenesis were increased in the transitional zone and decreased in the fetal zone of the baboon fetal adrenal in the second half of gestation. Thus, we proposed that there is a divergence in ACTH receptor-mediated zone-specific steroidogenesis within the fetal adrenal during mid to late gestation. We have also demonstrated that fetal serum alpha-inhibin levels decline with advancing development. It is possible, therefore, that the alpha subunit of inhibin provides a good marker of fetal zone cellular function and that the changes in circulating fetal alpha-inhibin with advancing pregnancy reflect ontogenetic changes in fetal adrenal cortical zone-specific cell function. However, it remains to be determined whether the fetal adrenal is a major source of circulating alpha-inhibin in the fetus and whether alpha-inhibin is expressed in the fetal, definitive, and/or transitional zones. Therefore, the current study compared fetal serum alpha-inhibin levels with immunocytochemical localization of alpha-inhibin in baboon fetal adrenals obtained on Days 60 (early), 100 (mid), and 165 or 182 (late) of gestation (term averages Day 184) from animals untreated or treated with betamethasone, which we previously demonstrated suppressed fetal pituitary ACTH and adrenal weight. Fetal serum alpha-inhibin levels (mean +/- SE) were greater (p < 0.05) at mid (5863 +/- 730 microliter eq/ml) than at late (3246 +/- 379) gestation and were reduced (p < 0. 05) by betamethasone. The inhibin alpha subunit was expressed in abundant quantities in the fetal adrenal cortex, but not in medulla, throughout gestation. At mid and late gestation, alpha-inhibin was expressed throughout the fetal adrenal cortex but most intensely in the innermost area of fetal zone cells. By late gestation, the fetal adrenal exhibited a gradient of alpha-inhibin expression. Thus, the outermost definitive zone cells were devoid of alpha-inhibin, the transitional zone exhibited a relatively low alpha-inhibin content, and fetal zone cells continued to exhibit extensive expression of alpha-inhibin. Betamethasone diminished the intensity of alpha-inhibin expression throughout the fetal adrenal cortex. These results indicate that the fetal adrenal fetal zone is a significant source of circulating alpha-inhibin in the baboon fetus and that alpha-inhibin provides a good marker to study the developmental regulation of fetal zone-specific adrenocortical function.     

Characterization and modulation of sex steroid metabolizing activity in normal human keratinocytes in primary culture and HaCaT cells

Skin, the largest organ of the human body, synthesizes active sex steroids from adrenal C19 precursor steroids. Normal human breast epidermal keratinocytes in primary culture were used to evaluate the enzymatic activities responsible for the formation and degradation of active androgens and estrogens during keratinocyte differentiation. Enzymatic activities, including 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD), 17beta-hydroxysteroid dehydrogenase (17beta-HSD), 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) were measured using [3H] steroids as substrates. After 10-60 days in culture, no 3beta-HSD activity was detected, but all other activities were measured, demonstrating the ability of keratinocytes to convert androstenedione (4-DIONE) into the potent androgen dihydrotestosterone (DHT). Furthermore, marked changes in enzymatic activity were observed during cell differentiation: 17beta-HSD was first detected during the third week of culture, the level of activity reaching a peak during the fourth week. This peak was followed by a progressive decrease during keratinization. On the other hand, 5alpha-reductase and 3alpha-HSD activities were first detected during the fourth week of culture. The enzymatic activities involved in the formation and degradation of sex steroids were also characterized in the immortalized human keratinocyte cell line HaCaT. It was then found that HaCaT cells possess a pattern of steroid metabolizing enzymes similar to that of human epidermal keratinocytes in culture. Since glucocorticoids are known to exert potent pharmacological effects on the skin, the effect of dexamethasone (DEX) on cell proliferation and enzymatic activities was determined using HaCaT cells. DEX causes a 55% decrease in HaCaT cell proliferation (IC50: 10nM) whereas DEX caused a three- to five-fold stimulation of oxidative 17beta-HSD activity in intact cells in culture (ED50: 30 nM) and this stimulatory effect was competitively blocked by the glucocorticoid antagonist RU486. A four-fold increase in type 2 17beta-HSD mRNA levels was also observed as measured by real-time PCR, correlating with the increase in oxidative activity. No effect of DEX on the other enzymatic activities (3beta-HSD, 5alpha-reductase, and 3alpha-HSD) was observed. Since increased levels of inflammatory cytokines have been detected in some skin diseases then these cytokines might play a role in the differentiation of keratinocytes. In this regard, we found that interleukin-4 (IL-4) induced the expression of 3beta-HSD in HaCaT cells, thus allowing the cells to produce a different set of sex steroids from adrenal C19 precursors. The present data thus indicate that HaCaT cells are a useful model to further study the regulation of the enzymes involved in the metabolism of sex steroids in keratinocytes.     

Distribution of 3 beta-hydroxysteroid dehydrogenase during development of the rat adrenal cortex and capsule

Fibroblasts of the adult adrenal cortex are considered to be nonsteroidogenic connective-tissue cells. However, it has been reported that in response to regenerative stimuli, adrenocorticotropic hormone (ACTH), and transformation to malignancy, these cells acquire characteristics of parenchymal cells, which includes delta 5, 3 beta-hydroxysteroid-dehydrogenase (delta 5, 3 beta-HSD) activity. To determine whether such delta 5, 3 beta-HSD activity in adult adrenocortical fibroblasts was due to the activation or augmentation of gene expression normally occurring during embryogenesis, a histochemical study of adrenocortical development, with particular attention to the connective-tissue capsule, was undertaken. Cryostat sections of rat embryos, from 14-days postconception (PC) to birth, and of adrenal glands 1-8, 44 and 90 days after birth were tested histochemically for delta 5, 3 beta-HSD. The same or adjacent sections were stained for PAS-positive material and reticulin, and with hematoxylin and eosin. delta 5, 3 beta-HSD activity overlapped with fibroblast-like cells and with extracellular connective-tissue components in the periphery of the glands from day-17 PC onward. delta 5, 3 beta-HSD activity over the capsule diminished shortly after birth and was absent in the adult. Appropriate controls showed that the staining within the capsule was specific and not an artifact. 3 beta-HSD activity in the capsule was more intensive when dehydroepiandrosterone (DHEA) was replaced by etiocholan-3 beta-ol-17-one (ETIO) as the steroid substrate. Furthermore, the spatial distribution of 3 beta-HSD activity in the cortex differed depending on the substrate used, and the distribution patterns changed with developmental age. (ABSTRACT TRUNCATED AT 250 WORDS)     

The role of alpha-2u-globulin in corticosteroidogenesis in male rats

Administration of estradiol-17 beta for 7 days to the adult male rat results in adrenal hyperplasia, decreased serum corticosterone along with elevation in serum ACTH and inhibition of adrenal 5-ene-3 beta-hydroxysteroid dehydrogenase activity (5-ene-3 beta-HSD). Treatment with alpha 2u-globulin for following 14 days of estrogen-treated rats reversed the effects of estrogen while in normal rats alpha 2u-globulin treatment increased adrenal 5-ene-3 beta-HSD activity and serum corticosterone level while causing a fall in serum ACTH. It is concluded that alpha 2u-globulin may play a role in ACTH secretion by inducing corticosterone synthesis.     

Adrenocorticotropic hormone does not induce desensitization in human adrenal cells during fetal life

We investigated whether human fetal adrenal cells pretreated with or continuously exposed to adrenocorticotropic hormone (ACTH) would develop refractoriness of the steroidogenic response. Fetal adrenal glands from fetuses of 18-24 wk gestation, were studied. Fetal zone cells were pretreated with increasing doses of ACTH (0-10(-6) M) for 24 h and then restimulated with a single dose of ACTH (10(-6) M) for an additional 24 h. Regardless of the dose of ACTH in the first incubation, the cells responded to the second stimulation with a 2- to 6-fold increase in dehydroepiandrosterone sulfate (DHAS) production. When human fetal adrenal cells were incubated in the continuous presence of 10(-8) M ACTH for 72 h, DHAS production was increased compared to that of the untreated cultures (5-fold at 24 h and 50-fold at 72 h), and the cells remained responsive during the entire experimental period. In contrast, human adult adrenal cells showed a significant decrease of the steroidogenic response after 48 h of ACTH treatment. Twenty-four hours of incubation with increasing doses of ACTH also increased the basal steroidogenic capacity of the fetal adrenal cells. One of the steroidogenic enzymatic steps stimulated by ACTH pretreatment was that of 17 alpha-hydroxylase/17, 20-lyase, since conversion of pregnenolone and 17 alpha-hydroxypregnenolone to DHAS was increased in a dose-dependent manner. These results demonstrate that human fetal adrenal cells, in contrast to those of the adult, do not become desensitized to ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of ACTH synthetic fragments with rat adrenal cortex membranes.

Synthetic peptide, corresponding to the amino acid sequence 11-24 of human adrenocorticotropic hormone (ACTH), was labeled with tritium (specific activity of 22 Ci/mmol). [(3)H]ACTH (11-24) was found to bind to rat adrenal cortex membranes with high affinity and specificity (K(d) = 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) have been synthesized and their ability to inhibit the specific binding of [(3)H]ACTH (11-24) to adrenocortical membranes has been investigated. Unlabeled fragment ACTH 15-18 (KKRR) was found to replace in a concentration-dependent manner [(3)H]ACTH (11-24) in the receptor-ligand complex (K(i) = 2.3 +/- 0.2 nM). ACTH (15-18) was labeled with tritium (specific activity of 20 Ci/mmol). [(3)H]ACTH (15-18) was found to bind to rat adrenal cortex membranes with high affinity (K(d) = 2.1 +/- 0.1 nM). The specific binding of [(3)H]ACTH (15-18) was inhibited by unlabeled ACTH (11-24) (K(i) = 2.2 +/- 0.1 nM). ACTH (15-18) at the concentration range of 1-1000 nM did not affect the adenylate cyclase activity in adrenocortical membranes.     

Identification of two novel ACTH-responsive genes encoding manganese-dependent superoxide dismutase (SOD2) and the zinc finger protein TIS11b [tetradecanoyl phorbol acetate (TPA)-inducible sequence 11b

ACTH is the major trophic factor regulating and maintaining adrenocortical function, affecting such diverse processes as steroidogenesis, cell proliferation, cell migration, and cell survival. We used differential display RT-PCR to identify genes that are rapidly induced by ACTH in the bovine adrenal cortex. Of 42 PCR products differentially amplified from primary cultures of bovine adrenocortical cells treated with 10 nM ACTH, six identified mRNAs that were confirmed by Northern blot analysis to be induced by ACTH. Four of these amplicons encoded noninformative repetitive sequences. Of the other two sequenced amplicons, one encoded a partial sequence for mitochondrial manganese-dependent superoxide dismutase (SOD2), an enzyme that is likely to protect adrenocortical cells from the cytotoxic effects of radical oxygen species generated during steroid biosynthesis. The second was identified as TIS11b (phorbol-12-myristate-13-acetate-inducible sequence 11b)/ERF-1/cMG, a member of the CCCH double-zinc finger protein family. SOD2 induction by ACTH was independent of extracellular steroid concentration or oxidative stress. SOD2 and TIS11b mRNA expressions were rapidly induced by ACTH, reaching a maximal level after 8 h and 3 h of treatment, respectively. These ACTH effects were mimicked by forskolin but appeared independent of cortisol secretion. Upon ACTH treatment, induction of TIS11b expression closely followed the previously characterized peak of vascular endothelial growth factor (VEGF) expression. Transfection of a TIS11b expression plasmid into 3T3 fibroblasts induced a decrease in the expression of a reporter gene placed upstream of the VEGF 3'-untranslated region, indicating that TIS11b may be an important regulator of VEGF expression through interaction with its 3'-untranslated region.     

Vasopressin: a potent growth factor in adrenal glomerulosa cells in culture

Previous studies have shown that vasopressin stimulates the mitotic activity in adrenal zona glomerulosa cells in intact as well as in hypophysectomized rats. (Payet and Isler, Cell and Tissue Res. 172, 1976; Payet and Lehoux, J. steroid Biochem. 12, 1980). We now report that this effect is direct and specific, since vasopressin stimulates the mitotic activity of rat adrenal zona glomerulosa cells in primary cultures. These cells were prepared by dissociation with collagenase in the culture medium MEM-d-Valine. Isolated cells were placed in 3.5 diameter petri dishes in MEM-d-valine medium containing 15% fetal calf serum and antibiotics for two days and 5% fetal calf serum for subsequent cultures. The medium was changed at 24 hr intervals. The hormones were added 3 days after the culture was started. The mitogenic effect of vasopressin was found to be dependent both on time and hormone concentrations. Vasopressin (10(-11) M) stimulated thymidine incorporation 4.8 +/- 0.6-fold after 2 days of treatment and 5.3 +/- 1.6-fold after 8 days. When ACTH (10(-11) M) was added together with vasopressin (10(-11) M) the mitogenic effect was enhanced at 6.5 +/- 1.9-fold after 2 days and 12.9 +/- 6.9-fold after 8 days of treatment. The aldosterone and corticosterone outputs were also stimulated by the combined presence of vasopressin and ACTH in the incubation medium; a maximal effect was observed between 6 and 8 days of treatment. Vasopressin (10(-11) M) + ACTH (10(-11) M) stimulated the aldosterone output 7-fold and that of corticosterone by 18-fold. When added alone, vasopressin, as well as ACTH alone had only a small effect on the aldosterone output. However, ACTH alone stimulated the corticosterone output 10-fold. In conclusion, vasopressin is an important and specific growth factor of the adrenal zona glomerulosa cells. In addition, together with ACTH vasopressin stimulates the aldosterone and corticosterone output both in vivo and in vitro in primary cell cultures.     

The stimulation by ACTH of 17 alpha-hydroxylation in cultures or rabbit adrenal cells

Adrenal cells from control rabbits (control-cells) and from rabbits that had been injected twice daily for 3 days prior to sacrifice with 25 IU ACTH (ACTH-cells) were cultured both in the absence and presence of 100 mIU ACTH. Culture durations varied from 24 to 120 h in 24 h increments. The culture medium was changed daily and fresh ACTH added to appropriate vessels. At the time of the final media change 0.1 muCi [4-14C]pregnenolone was added. Twenty-four hours later the cultures were terminated and the products formed from the pregnenolone were isolated, quantified and identified by solvent extraction, chromatography and crystallization to constant specific activity. After 72 h ACTH-cells cultured in the presence of ACTH converted 18.5% of the pregnenolone substrate to 17-hydroxycorticosteroids (cortisol plus 11-deoxycortisol) while ACTH-cells cultured in the absence of ACTH converted a maximum of 1.6%. A similar but smaller difference, 10.9 vs 2.1%, was recorded with control cells cultured in the presence and absence of ACTH. Corticosterone production from [4-14C]pregnenolone in the 72-h cultures was increased to a lesser degree by ACTH exposure. In ACTH-cells the conversion climbed from 5.9 to 10.5% and from 9.6 to 12.0% in control cells. Microscopic examination of parallel cultures showed no significant differences in cell density between cells cultured in the presence or absence of ACTH. 17 alpha-Hydroxylase activity arising from the in vivo stimulation did not survive cell division in culture, but required the continual presence of ACTH. In conclusion, the data show that ACTH is capable of stimulating 17-hydroxycorticoid formation in rabbit adrenal cell cultures.     

Adenylate cyclase activity and cyclic AMP production in the outer and inner zones of the adrenal cortex

It has been reported that cells isolated from the inner zone of the guinea pig adrenal cortex fail to have a steroidogenic response to ACTH. To further explore this, adenylate cyclase activity of membrane particles and cAMP production by cells prepared from the inner and outer adrenocortical zones were determined. The cAMP response to ACTH and forskolin was similar for cells from both zones. Basal adenylate cyclase activity was significantly higher in the inner zone; and while absolute responses to ACTH, GppNHp, GTP, NaF, and forskolin were greater for the inner zone, relative responses were similar for the two zones. These observations suggest that the inner zone of the guinea pig adrenal cortex may have a defect in ACTH action at a step(s) beyond cAMP formation.     

Effect of placental factors on growth and function of the human fetal adrenal in vitro

Conditioned medium from human placental monolayer cultures (PM) had a marked stimulatory effect on proliferation (3H-thymidine uptake) of human fetal zone adrenal cells in primary monolayer culture, even in the absence of serum. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) also significantly stimulated fetal adrenal cell growth. However, the effects of PM differed from those of EGF and FGF in several respects: 1) maximal response to PM was 2-5 times greater; 2) mitogenic effects of EGF and FGF were suppressed by adrenocorticotropic hormone (ACTH), whereas that of 50% PM was not; 3) PM inhibited ACTH-stimulated steroidogenesis (dehydroepiandrosterone sulfate and cortisol), but EGF and FGF did not. Preliminary characterization studies have indicated that approximately half of the placental growth-promoting activity is heat resistant and sensitive to bacterial proteases, and that 50-60% of the activity is lost after dialysis with membranes having a molecular weight cutoff of 3500. These findings suggest a role for the placenta in the growth and differentiated function of the human fetal adrenal gland.     

Placental-derived mitogenic factor for human fetal adrenocortical cell cultures

Summary The human fetal adrenal cortex is one of the largest fetal organs and synthesizes precursors for placental estrogen production as part of the feto-placental unit. The factors controlling the rapid growth of the human fetal adrenal cortex during the second and third trimesters are not known. Placental regulation of the growth of human fetal adrenocortical cell cultures from second trimester fetuses was studied. A placental-derived mitogenic factor (PDMF) was detected in tissue homogenates of 14 to 22 week human placentas and stimulated adrenocortical cell number and [³H]thymidine incorporation into DNA 5–8 fold. PDMF has been partially purified by ammonium sulfate precipitation and anion exchange chromatography. PDMF is a heat sensitive protein with disulfide bonds required for activity. The growth stimulation by PDMF was significantly greater than that for basic or acidic fibroblast growth factor by 25–50% and epidermal growth factor by 3–4 fold. The placental hormones, progesterone, estriol, estradiol, placental lactogen and chorionic gonadotropin, either alone or in combination did not stimulate fetal adrenocortical cell growth, except for a 41% cell number increase by progesterone. Platelet-derived growth factor and insulin-like growth factors I and II were not mitogenic for these cells. These results show that the placenta contains a potent growth factor for human fetal adrenocortical cell cultures. This implies a direct role for the placenta in control of this fetal organ’s growth, which would make the human feto-placental unit a bi-directional relationship. This research was supported by Grants HD15882 and HD21798 from the National Institutes of Health, Bethesda, MD. This work was presented in part at the 68th Annual Meeting of The Endocrine Society, Anaheim, CA, June 1986. Editor’s Statement This report describes a potentially new relationship between placenta and the regulation of growth of fetal adrenalcortical cells. Since these cells produce several of the essential hormones influencing fetal development, characterization of this factor(s) could provide important insights into the process of differentiation. David A. Sirbasku