Glucocorticoid receptors in chick embryos: properties and ontogeny of the nuclear renal receptor

A putative nuclear receptor for glucocorticoids was identified in the kidney of chick embryo. This receptor was a thermolabile protein which was readily digested by proteolytic enzymes. Its sedimentation coefficient on sucrose density gradient was 3.5S and its MW approximated, according to the Svedberg formula, at 98,500 Da. Binding assays, performed with crude or purified nuclei, and nuclear extracts showed that the latter preparation was the most suitable for the binding studies since it yielded a Bmax of 8-11% with a very low nonspecific binding (1% or less). Scatchard plots performed at various days of embryogenesis revealed a single class of binding sites with an association constant (Ka) of 0.12 +/- 0.06 X 10(9) M-1 (mean +/- SD; n = 5) and a maximal binding capacity (Nmax) that rose from 3.9 +/- 1.2 fmol/micrograms DNA at Day 13 of age to 13.2 +/- 2.2 fmol/micrograms DNA at Day 16 and then rapidly fell to 1.8 +/- 1.1 fmol/micrograms DNA before hatching (means +/- SD; n = 5). Competition studies with various steroids showed that only glucocorticoids and, to a lesser degree, progesterone had an affinity for the receptor. These results demonstrate that this nuclear-binding protein had physiochemical properties similar to those attributed to other glucocorticoid receptors in target cells.     

Demonstration of glucocorticoid receptors in the adrenal cortex: evidence for a direct dexamethasone suppressive effect on the rat adrenal gland

The adrenal cortex was evaluated for the presence of glucocorticoid receptors and functions. Substantial binding of [3H]dexamethasone was observed in aminoglutethimide-treated, hypophysectomized, and intact rats. Further studies demonstrated binding in cultured bovine adrenocortical cells and in Y-1 cells, a cloned murine cell line of adrenal cortical origin. Scatchard analysis of specific binding data in cytosol from hypophysectomized rats revealed an apparent Kd of approximately 15 nM and a receptor content (Nmax) of 123 fmol/mg cytosol protein. Analysis of Y-1 cell cytosol showed a Kd of approximately 17 nM and Nmax of 190 fmol/mg protein. The binding site in hypophysectomized rats had the following steroid specificities: high affinity for dexamethasone, corticosterone, and progesterone; moderate affinity for 11 beta-cortisol, and low affinity for testosterone, estradiol, pregnenolone, and 11 alpha-cortisol. Sedimentation in sucrose density gradients revealed 8S binding peaks in cytosols prepared from intact rat adrenal glands, Y-1 cells, and cultured bovine adrenocortical cells. Time- and temperature-dependent nuclear uptake of [3H]dexamethasone in Y-1 cells was demonstrated. In vivo treatment of hypophysectomized rats with dexamethasone significantly enhanced the rate of adrenal atrophy. ACTH stimulation tests in hypophysectomized rats showed a decreased corticosterone response in dexamethasone-treated rats compared to that in control animals. However, in vitro, there was no evidence for an effect of dexamethasone on ACTH-stimulated corticosterone production. The data indicate that the adrenal cortex possesses a high affinity binding site that fulfills the criteria for a glucocorticoid receptor. Glucocorticoid administration enhances adrenal atrophy and impairs adrenal function. We speculate that this action contributes to the suppressive effect of glucocorticoids on the pituitary-adrenal axis.     

Immunocytochemical detection of 1,25-dihydroxyvitamin D receptors in normal human tissues

We developed an immunocytochemical technique to visualize the receptors for 1,25-dihydroxyvitamin D [1,25-(OH)2D receptor] in cryostat sections of normal human tissues, using a rat monoclonal antibody (9A7 gamma) to the chick intestinal receptor, which has been found to react with mammalian 1,25-(OH)2D receptors. Localization of the antigen was predominantly nuclear, with little cytoplasmic immunoreactivity. Specific staining was seen in the nuclei of many normal epithelial tissues, including liver, kidney, thyroid, adrenal, gastrointestinal tract, breast, and skin. No nuclear staining was seen when tissue sections were incubated with normal rat immunoglobulin G or when the monoclonal antibody was preincubated with a receptor-enriched chick intestinal cytosol preparation. Our results demonstrate that the receptor for 1,25-(OH)2D is localized in the nucleus and widely distributed in normal human tissues.     

The bursa of fabricius is required for normal endocrine development in chicken

The influence of the bursa of Fabricius on endocrine functions was evaluated in 68-hr chick embryos surgically bursectomized by tail bud ablation. Bursectomized embryos were bursa grafted on the chorioallantoic membrane at 9.5 days of incubation. Endocrine parameters were investigated at 17.5 days of embryonic life and at 24 hr after hatching. In bursectomized chickens testosterone production by the testis in vitro increased, whereas corticosterone production by the adrenal glands in vitro was diminished. The weight and protein content of the oviduct was decreased markedly. The alterations related to early bursectomy were prevented in embryos with bursa grafted on the chorioallantoic membrane on Day 9.5 of development. Accordingly, the bursa of Fabricious appears to produce factors that influence steroid-secreting glands during chick embryo development.     

Corticosteroid receptors in the kidney of chick embryo. III. Nature, properties, and ontogeny of aldosterone receptor

An aldosterone receptor in the cytosol from kidney of chick embryos which had a sedimentation coefficient of 8.2 S and a molecular weight higher than 100,000 was identified. Kinetic analysis at 4 degrees revealed a rapid association of the hormone to the receptor that followed second-order reaction kinetics and a dissociation of pseudo-first-order reaction kinetics. The association (ka) and dissociation (kd) rate constants were, respectively, 4.94 X 10(5) M-1 sec-1 and 8.33 X 10(-6) sec-1. From their ratio a KA value of 5.9 X 10(10) M-1 was calculated. In a series of experiments performed with kidneys of 17-day-old embryos, the KA at equilibrium, obtained from the Scatchard plot, was 3.1 +/- 1.2 X 10(8) M-1, whereas the Nmax was 172 +/- 14 fmol/mg protein. Competition studies with various steroids demonstrated that corticosterone had an affinity for the receptor close to that of aldosterone, thus suggesting a degree of resemblance of the mineralo- and glucocorticoid receptors in the chick embryo. However, the profiles of the binding affinities and capacities during the embryogenesis showed that the aldosterone-binding sites had a pattern completely different from that of the glucocorticoid receptor, indicating that the two receptors are most likely separate entities.     

Characterization of rat brain aldosterone receptors reveals high affinity for corticosterone

The two [3H]aldosterone-binding proteins of rat brain cytosol were characterized by a dextran-coated charcoal method. With molybdate present to stabilize receptors, the affinities of the two sites for [3H]aldosterone in adrenalectomized perfused rat brain cytosols were 0.28 and 18.0 nM at 4 C. High affinity sites comprised 15% of the total receptor number. A small contamination of perfused brain cytosol preparations with corticosteroid-binding globulin (CBG) was found. However, due to the very high affinity of CBG for corticosterone at 4 C, this slight contamination resulted in significant alterations in the apparent affinity of steroids competing for aldosterone-binding sites. Selective precipitation of cytosol receptors with 36% (NH4)2SO4 reduced CBG concentrations to negligible levels. After blockade of low affinity sites with a highly selective glucocorticoid (RU 26988), the order of steroids in competing for the high affinity receptor was desoxycorticosterone greater than fludrocortisone greater than corticosterone greater than aldosterone greater than progesterone greater than dexamethasone. Readdition of a small quantity of dialyzed serum to cytosol preparations yielded a profile of steroid binding similar to that of the kidney mineralocorticoid receptor (aldosterone greater than desoxycorticosterone greater than corticosterone). The distribution of both receptors in brain regions of adrenalectomized rats was determined. Both receptors were at greatest density in the hippocampus and lowest density in the hypothalamus. The high affinity site was at greatest density in limbic regions, whereas the low affinity receptor, apparently identical to the glucocorticoid type II receptor, was at greatest density in cortex and cerebellum. It is concluded that the high affinity aldosterone receptor of rat brain, which had been identified in preliminary studies as a mineralocorticoid receptor, may bind either corticosterone or aldosterone in vivo.     

Effects of hypophysectomy and corticosterone acetate treatment on hepatic lipid composition in the chick (Gallus domesticus) embryo

Lipid is transferred from yolk to liver and to other tissues at an accelerated rate after 16 days of incubation in the intact chicken embryo. Hypophysectomy (partial decapitation) at 36 hr of incubation reduced liver lipid content on Days 16 and 18. The greatest losses on Day 18 were among cholesterol esters but decreased unesterified cholesterol and phospholipid were also observed. Treatment of hypophysectomized embryos with corticosterone acetate (300 μg per day on Days 13, 14, 15) partially restored total hepatic lipid content to control values on Day 16, with an even greater effect on 18 days of incubation. Most of the increase in lipid occurred in the triglyceride fraction. Liver cholesterol content also increased in response to hormone but neither cholesterol esters nor phospholipids were elevated by corticosterone acetate. Hypophysectomy interferes with the absorption of lipids from yolk and with fat deposition in embryonic liver. However, treatment of hypophysectomized embryos with corticosterone acetate did not correct the major defects in hepatic lipid content. Therefore, fat metabolism and transport of yolk lipids to the embryo during the last week of incubation does not depend exclusively on adrenal glucocorticoids.     

The fetal adrenals of the rat: correlations between growth, cytology, and hormonal activity, with and without ACTH deficiency.

Growth, ultrastructure, and corticosterone content were studied in the adrenals of rat fetuses in late pregnancy, between Days 19 and 21. In the fetuses of intact mothers, the hypophysial corticostimulating activity decreased between Days 19 and 21. Ablation of the fetal hypothalamus by total removal of the brain, leaving the hypophysis in situ (encephalectomy), induced atrophy of the adrenals, changes in adrenal ultrastructure, and decrease in the adrenal corticosterone content. Such modifications were also observed in response to the ablation of the pituitary gland by decapitation. The hypothalamus was involved in the control of ACTH release in late pregnancy. When mothers were adrenalectomized on Day 14, in the absence of hypothalamic tissue, the pituitary gland of the encephalectomized fetuses showed some corticostimulating activity; indeed, the adrenals of these fetuses were heavier on Day 21 than those of the decapitated fetuses but smaller than those of the littermate controls, and the adrenal corticosterone content was also greater than in the decapitated but less than in the controls. Encephalectomy induced modifications in adrenal cytology less marked than those induced by decapitation. These data suggest that the pituitary gland and the hypothalamo-hypophysial complex are the sites of the corticosteroid-induced inhibition of the corticostimulating activity of the fetal hypophysis.     

FSH- and TSH-binding cells in the ovary of the developing chick embryo

The numerical density (Nv) and location of thyroid stimulating hormone (TSH)- and follicle stimulating hormone (FSH)-binding cells in the left ovary of the chick embryo were determined on Days 6.5 through 19.5 of incubation. From Days 6.5 to 11.5, TSH- and FSH-positive cells were located in the medullary cords and there was no statistically significant difference in the Nv of these two cellular populations. However, beginning on Day 12.5 and continuing through Day 19.5, TSH and FSH were bound principally to cells of the cortical cords and there was a significant difference (P less than 0.05) between the Nv of TSH-positive and FSH-positive cells. Results are discussed in terms of a prevalent hypothesis that throughout chick embryo development the left ovary lacks specific receptors for TSH and FSH.     

Ontogenesis of immunocytochemically demonstrable somatotrophs in the adenohypophyseal pars distalis of the developing chick embryo

Growth hormone (GH)-containing cells in the hypophyseal pars distalis of the chick embryo were identified immunohistochemically using anti-chicken GH sera. GH cells are first demonstrable in Rathke's Pouch as early as Day 4.5 of incubation. By Day 9.5, when the two lobes (rostral and caudal) of the pars distalis are easily recognized, GH cells are confined exclusively to the caudal lobe. Although the number of GH cells increases gradually during embryonic development, there is a statistically significant difference between Days 10.5 and 12.5 in both the cellular density and the percentage of somatotrophs. GH cells, which contain coarse granules evenly distributed throughout the cytoplasm, varied in diameter from 9.3 micron on Day 4.5 to 11.8 micron on Day 20.5, while the nuclear diameter of these cells increased from 2.8 micron on Day 4.5 to 4.9 micron on Day 20.5. There was a statistically significant difference in the nuclear/cytoplasmic ratio between Days 4.5 and 13.5 and between Days 13.5 and 20.5 of incubation. Aspects of the regulation of growth hormone synthesis and secretion in the chick embryo are discussed.     

Plasma 17β-estradiol levels in the chick embryo

Plasma 17β-estradiol (E₂) concentrations in male and female chick embryos were determined by radioimmunoassay (RIA) on Days 7.5, 9.5, 11.5, 12.5, 13.5, 14.5, 15.5, and 17.5 of development. E₂ levels in female embryos increased gradually from Days 7.5 (889.5 pg/ml) to 12.5 (1003.8 pg/ml), followed by a marked increase from Days 13.5 (1103.3 pg/ml) to 17.5 (1360.2 pg/ml). In male embryos, E₂ concentrations remained at a relatively constant level throughout the embryonic period studied (Day 7.5: 639.9 pg/ml; Day 17.5: 747.8 pg/ml) and were significantly lower than female E₂ levels of the same age (P<0.001). A correspondence in embryonic time was observed between the two phases of the female plasma E₂ profile (Days 7.5–12.5 and 13.5–17.5) and the biphasic growth and differentiation pattern of the female left Müllerian duct (Days 8.0–12.0 and 14.0–18.0). Results are also discussed in terms of other E₂-dependent events which occur during chick embryo development.     

Immunohistochemical localization of insulin in the chick embryo during development

Since an extrapancreatic source of insulin has been postulated in the chick embryo, insulin was sought by immunohistochemistry in the embryo before and after development of the pancreas and other tissues. Using for the first time an antiserum specific for chicken insulin, localization of insulin was investigated in whole embryos (Days 1-8) and in brain, kidney, liver, and pancreas (Days 9-21 and 30). Results indicate the presence of immunoreactive insulin in pancreatic islets from Day 5 onward with an increase in staining intensity during development. Insulin was not detected in other tissues of the early developing embryo or in brain, liver, or kidney in later development. These data suggest that although extrapancreatic insulin has been reported, concentrations are not high enough in tissues to be detected with a specific, homologous antiserum and sensitive immunohistochemical techniques.     

Progesterone and its reductive metabolism in steroidogenic tissues of the developing hen embryo

Progesterone contents of adrenals and ovaries and the--mainly reductive--metabolism of [3H]progesterone by these organs and liver were investigated in hen embryos between Days 13 and 21 (hatching). Progesterone contents are similar in adrenals and ovaries on Day 13 (approx 3.5 ng/mg) but descend in characteristic manners toward Day 17 and rise steeply, only in the ovaries, then descend in these organs toward hatching. [3H]Progesterone is converted by the adrenals toward 12 main metabolites, the main glucocorticoid being corticosterone (B), and the main reduced metabolite, 5 beta-pregnane-3,20 dione (5 beta-P). On day 13, 5 beta-P is five times as important as B, but both steroids evolve in a symmetric fashion, so that at hatching this proportion is reversed. In all tissues at all stages, except the liver on Day 13, the yields of 5 alpha-pregnane-3,20 dione (5 alpha-P) are one order of magnitude below those of 5 beta-P. Both diones exhibit maxima on Day 17, probably extending until Day 19. Concomitantly, [3H]progesterone disappearance is maximal on Day 17. Both ovaries differ in the shape of their 5 alpha-P/5 beta-P curves in that the left ovary exhibits for this curve a function ascending continuously toward hatching.     

Pharmacological characterization of central and peripheral type I and type II adrenal steroid receptors in the prairie vole, a glucocorticoid-resistant rodent.

The prairie vole (Microtus ochrogaster) has recently been shown to be glucocorticoid resistant; that is, the prairie vole adrenal axis is refractory to dexamethasone challenge, and highly elevated basal corticosterone titers occur without apparent pathophysiology. This study investigates the physiological correlates of glucocorticoid resistance in the prairie vole. We provide a detailed pharmacological characterization of intracellular type I and type II adrenal steroid receptors in peripheral tissues and the hippocampus of the prairie vole and the Sprague Dawley rat, a corticosensitive rodent. Adrenalectomy markedly reduces, but does not eliminate, circulating glucocorticoids in the prairie vole. Nonetheless, molecular, cellular, and physiological assays indicate adrenal insufficiency; salt appetite and dentate gyrus granule cell death are increased after adrenalectomy, suggesting vacancy of the high affinity type I subtype of central adrenal steroid receptor. Analysis of adrenal steroid receptor binding constants and selectivity for endogenous and synthetic steroids in the vole and rat indicated that the vole type I receptor is nearly identical to that of the rat in brain and periphery. However, voles demonstrated a 2-fold lower type I receptor binding density in colon and hippocampus compared with that in rats. The vole type II receptor bound the endogenous glucocorticoid corticosterone with an 8- to 10-fold lower affinity than the rat type II receptor and was expressed in lower densities in thymus and hippocampus. These data indicate physiological adaptations in the prairie vole adrenal axis consistent with other glucocorticoid-resistant species, such as the guinea pig and squirrel monkey.     

11β-Hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress

Glucocorticoid hormones, acting via nuclear receptors, regulate many metabolic processes, including hepatic gluconeogenesis. It recently has been recognized that intracellular glucocorticoid concentrations are determined not only by plasma hormone levels, but also by intracellular 11β-hydroxysteroid dehydrogenases (11β-HSDs), which interconvert active corticosterone (cortisol in humans) and inert 11-dehydrocorticosterone (cortisone in humans). 11β-HSD type 2, a dehydrogenase, thus excludes glucocorticoids from otherwise nonselective mineralocorticoid receptors in the kidney. Recent data suggest the type 1 isozyme (11β-HSD-1) may function as an 11β-reductase, regenerating active glucocorticoids from circulating inert 11-keto forms in specific tissues, notably the liver. To examine the importance of this enzyme isoform in vivo, mice were produced with targeted disruption of the 11β-HSD-1 gene. These mice were unable to convert inert 11-dehydrocorticosterone to corticosterone in vivo. Despite compensatory adrenal hyperplasia and increased adrenal secretion of corticosterone, on starvation homozygous mutants had attenuated activation of the key hepatic gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, presumably, because of relative intrahepatic glucocorticoid deficiency. The 11β-HSD-1 −/− mice were found to resist hyperglycamia provoked by obesity or stress. Attenuation of hepatic 11β-HSD-1 may provide a novel approach to the regulation of gluconeogenesis.     

Hypothalamo-adenohypophyseal-thyroid interrelationships in the chick embryo. II. Effects of thiourea treatment on plasma total thyroxine levels and thyroidal 125I uptake

After thiourea administration to chick embryos on Day 5.5, total plasma thyroxine concentrations were significantly decreased on Days 7.5, 9.5, 10.5, and 11.5 of incubation. Also, radioiodide uptake in normal and thiourea-treated embryos was measured at 1.0, 3.0, and 5.0 hr post-treatment on all days studied. There was no statistically significant difference in radioiodide uptake between normal and goitrogen-treated embryos on Days 7.5 and 9.5. However, on Days 10.5 and 11.5, a statistically significant increase in radioiodide uptake was observed in thiourea-treated individuals as compared to nontreated embryos. Thyroid wet weights were compared between normal and thiourea-treated embryos on all days studied. There were no statistically significant differences in wet weights between normal and goitrogen-treated individuals on the days assayed. The data indicate that on Day 10.5 of incubation, the pituitary and/or hypothalamus of the chick embryo first responds to decreases in circulating T₄ levels. Thus, the maturation of the hypothalamo-adenohypophyseal-thyroid axis during Days 10.0–13.0 of development in the chick embryo may be similar to that found in sheep and humans at midgestation.     

Development of the adrenal gland in the tropical lizard Calotes versicolor

The development of the adrenal gland in the lizard Calotes versicolor was studied histologically and histochemically from the day of oviposition (stage 27) to 60 days after hatching. At stage 27, the adrenocortical cells are found in association with the genital ridge (primordial gonad). The separation of adrenocortical cells from the gonad takes place at stage 31. Organization of adrenocortical cells into cords takes place at stage 34. The catecholamine-secreting chromaffin cells can be seen distinctly on the dorsal region of the adrenal at stage 36, indicating the presence of biologically active catecholamines; the noradrenaline-secreting chromaffin cells appear first at stage 36 and the adrenaline-secreting cells appear later at stage 41. The cortico-medullary ratio of 6:1 during early embryonic development decreases with the increase in age and is 3:1 in posthatching lizards. The histochemical localization of Delta(5)-3beta-hydroxysteroid dehydrogenase (3beta-HSD) and glucose-6-phosphate dehydrogenase in the adrenocortical cells as early as at stage 27 (prior to the gonadal differentiation) indicates the capability of these cells to synthesize steroids. The intensity of the enzyme activity is maximum on the day of hatching and remains more or less the same in the posthatching lizards. The localization of 17beta-HSD enzyme activity observed in the adrenocortical cells at stage 34 is suggestive of their ability to synthesize sex steroids during embryonic life. The intense 3beta-HSD activity on the day of hatching in C. versicolor suggests high production of steroids which may be corticoids. The results of the present work also suggest that the onset of steroid secretion occurs prior to catecholamine secretion during embryogenesis of the adrenal gland in C. versicolor. In addition, there is a significant relationship between ontogenic steroidogenesis of the adrenal gland and sexual differentiation of the gonad.     

Self-inhibition of steroid secretion by amphibian adrenocortical cells is not mediated through glucocorticoid receptors

To investigate a possible direct action of glucocorticoids on adrenal steroidogenesis, the effect of corticosterone on the conversion of pregnenolone into various metabolites by frog adrenal tissue was examined. Frog interrenal slices were incubated with [3H]pregnenolone (1 mCi/ml) and the various labelled metabolites analysed by reverse-phase high-performance liquid chromatography. With the methanol gradient used, five identified steroids were resolved: progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone and aldosterone. Corticosterone (10 micrograms/ml) induced a 45-80% decrease in all steroids synthesized from [3H]pregnenolone. In contrast, the glucocorticoid agonist dexamethasone did not reduce the rate of conversion of pregnenolone into its metabolites. In addition, the inhibitory effect of corticosterone was not reversed by the specific glucocorticoid antagonist RU 43044. These results show that corticosterone exerts a direct inhibitory effect on adrenal steroid secretion. In addition, our data indicate that the ultra-short regulation induced by corticosterone is not mediated through glucocorticoid receptors.     

Prenatal Exposure to Ethanol Alters the Ontogeny of the β-Endorphin Response to Stress

To determine whether prenatal exposure to ethanol alters the response of the β-endorphin (β-EP) system to stress, the effect of two types of stressful stimuli, ether and cold, was examined in the offspring of rats which during pregnancy were: (a) fed with an ethanol-containing diet; (b) pair-fed with an isocaloric sucrose diet; and (c) fed ad libitum with standard lab chow (basic control group). The effect of stress on the content of β-EP in the serum, pituitary gland and hypothalamus, as well as on the serum corticosterone and hypothalamic corticotropin-releasing factor (CRF) content was examined. Pups prenatally exposed to ethanol had significantly higher serum β-EP levels on Day 1 and higher serum corticosterone levels on Days 1-3 when compared to their pair-fed or basic controls. On all days tested pituitary β-EP content was lower in the offspring of the ethanol-treated rats than in the control groups. There was no difference in the total hypothalamic β-EP content between the three treatment groups; however, during the first 10 days of life a higher concentration (ng/mg protein) of β-EP was observed in the hypo-thalami of the ethanol and the pair-fed group when compared to the basic control pups. Hypothalamic CRF levels, though significantly lower in the pups exposed to ethanol in utero than in the control groups on Day 3, increased significantly in the ethanol group between Days 14 and 22, while no significant change was observed during this period in either of the control groups. From Days 1 to 10 significant elevations in serum corticosterone, levels were observed following both types of stress in the pair-fed and the basic control group, while no significant response, to either type of stressful stimuli, was observed prior to Day 14 in the pups exposed to ethanol prenatally. On Day 14 cold stress induced small increases in the serum β-EP and corticosterone levels of the offspring treated prenatally with ethanol which, however, were less pronounced than the elevations observed in both of the control groups. Furthermore, exposure to ether stress on Day 14 elicited no response by the β-EP system of the ethanol-exposed offspring. In contrast, the 22-day-old offspring of the ethanol-treated rats exhibited greater elevations in serum β-EP and corticosterone levels, following stress, than the offspring of the pair-fed and basic control groups. The elevations of serum β-EP levels following stress were associated with small decreases in the pituitary β-EP and hypothalamic CRF contents. Thus, the response of the β-EP system to stress was modified by prenatal exposure to ethanol, being suppressed at early postnatal life and enhanced at later stages of development. The transition from reduced to enhanced responsiveness seemed to occur during the third week of life.