Ontogenic corticosteroidogenesis of the domestic fowl: response of isolated adrenocortical cells

Ontogenic adrenocortical function of the domestic was investigated using adrenocortical cells isolated from embryonic chicks (18, 19, 20, and 21 days old) and male and female posthatch birds (1 day, 1 week, and 3 weeks old). Production of the predominant corticosteroids secreted by the chicken adrenal gland, corticosterone, cortisol, and aldosterone, was measured by radioimmunoassay after 2-hr incubation of cells with or without steroidogenic agents. Approaching hatch, basal and maximal ACTH-(1-24) (ACTH)-induced corticosteroid production increased steadily and peaked around 1 day posthatch (5-18 times and 3-9 times, respectively, the production values at 18 days embryonic life). Thereafter, corticosteroid production values decreased steadily to 3 weeks posthatch. Corticosterone predominated over the ages studied: Maximal ACTH-induced corticosterone production averaged 52 and 115 times the production values of aldosterone and cortisol, respectively. In addition, maximal ACTH-induced aldosterone production was roughly 2.2 times greater than cortisol production over the ages studied except for a short-lived, disproportionately greater aldosterone production at 1 day posthatch. In addition to perihatch and age-related differences in cellular corticosteroid production, there were also differences in cellular sensitivity to steroidogenic agents as indicated by the differences in half-maximal steroidogenic concentration values (ED50 values) of the steroidogenic agents. Sensitivity to ACTH increased 2.7 times from Day 18 of embryonic life to 1 day posthatch and then decreased steadily to 3 weeks posthatch. In addition, sensitivity to 8-bromo-cAMP (8-Br-cAMP) increased abruptly at 1 day posthatch (nearly 3 times) but then remained constant thereafter. However, a consistent change in cellular sensitivity to 25-hydroxycholesterol was not observed until 3 weeks posthatch (an increase in sensitivity of 3 times that at Day 18 of embryonic life). These data of cellular sensitivity suggest that there were distinct development and maturational alterations in the cellular loci at which ACTH, 8-Br-cAMP, and 25-hydroxycholesterol acted. Thus, during the transition from embryonic to postembryonic life of the domestic fowl, there are alterations in adrenocortical cell steroidogenic capacity and in the function of some cellular loci comprising the corticosteroidogenic pathway.     

Isolated adrenocortical cells of the domestic fowl (Gallus domesticus): steroidogenic and ultrastructural properties

Isolated adrenocortical cells from White Leghorn chickens (Gallus domesticus) were compared to those from rats (Rattus norvegicus). Cells were prepared from collagenase-dispersed adrenal glands of sexually mature male animals. Corticosterone was measured by radioimmunoassay after incubation for 2 h with steroidogenic agents. Of the four ACTH analogues used, three were 6-17 times more potent with rat cells than with fowl cells (potencies were indicated by half-maximal steroidogenic concentrations). However, 9-tryptophan (O-nitrophenylsulfenyl) ACTH was 8 times more potent with fowl cells than with rat cells, thus suggesting that ACTH receptor differences exist between the two cell types. In addition, cAMP analogues were 10 times more potent with rat cells than with fowl cells suggesting that fowl corticosteroidogenesis is less dependent on cAMP than is rat corticosteroidogenesis. At equal cell concentrations, rat cells secreted 20-40 times more corticosterone than did chicken cells when they were maximally stimulated. Although rat cells converted 8 times more pregnenolone to corticosterone than did fowl cells, the half-maximal steroidogenic concentration for pregnenolone-supported corticosterone synthesis was the same for both cell types (about 5 microM). This suggests that fowl cells have lower steroidogenic enzyme content rather than lower steroidogenic enzyme activity. An unusual feature seen in the isolated fowl adrenocortical cells was an abundance of intracellular filaments.     

ACTH sensitivity of isolated human pathological adrenocortical cells: variability of responses in aldosterone, corticosterone, deoxycorticosterone and cortisol production

In vitro aldosterone, deoxycorticosterone, corticosterone and cortisol production of human adrenocortical cells derived from adenomas (Conn's syndrome, Cushing's syndrome), from hyperplastic adrenals (Cushing's syndrome) and from adrenals surrounding aldosteronoma are described. Cells from adenomas causing either Cushing's syndrome or Conn's syndrome harboured the highest basal and ACTH-stimulated corticosteroid production. Adrenocortical cells derived from micronodular hyperplasia causing Cushing's syndrome and cells from cortisol producing adenoma displayed predominantly cortisol and corticosterone secretion both under basal conditions and following stimulation with ACTH. Aldosteronoma cells showed highly variable aldosterone, deoxycorticosterone, corticosterone and cortisol response to ACTH. However, in aldosteronoma cell suspensions, the basal and ACTH-stimulated ratios of aldosterone to cortisol were increased when compared to ratios of steroids produced by cells from other adrenal tissues. Chronic treatment with spironolactone of patients with Conn's syndrome before surgery was associated with a decreased ratio of aldosterone to corticosterone, revealing that 18-hydroxylase in aldosteronoma cells may be inhibited during long-term therapy. Non-tumorous cells isolated from adrenals surrounding aldosteronoma displayed less aldosterone prior to and after stimulation with ACTH than aldosteronoma cells.     

Taste sensitivity in the embryo of the domestic fowl

Two experiments were carried out to investigate the sense of taste in embryos of the domestic fowl. In the first, four taste substances; NaCl, HCl, glucose and SOA were diluted with distilled water and the response was compared with that to distilled water alone. No significant effects of taste were found. In the second experiment five taste substances: HCl, fructose, NaCl, KCl and quinine were diluted with fluids normally imbibed by the embryo: amniotic and/or allantoic fluid taken from other eggs. These solutions and also distilled water were compared with egg-fluid alone. A highly significant effect of the five solutions was found showing that the taste system becomes functional before the time of hatching. Distilled water produced on an unexpectedly large response in the embryo; possible reasons for this are discussed.     

Catecholamine-containing pancreatic islet cells of the domestic fowl

Summary In an attempt to identify pancreatic islet cells emitting formaldehyde-induced fluorescence (FIF), the pancreatic islets of the domestic fowl were studied by combined fluorescence, ultrastructural, silver-impregnation and immunohistochemical methods in the same section or in consecutive semi-thin and ultra-thin sections. The results indicate that islet cells emitting intense FIF exhibit a strongly argyrophil reaction with the Grimelius' silver method and also immunohistochemical reaction with anti-glucagon serum, but not with anti-5-HT serum. Therefore, the fowl islet A cell, a peptide hormone-producing cell, stores simultaneously catecholamine as biogenic amine. The islet B and D cells did not display any FIF, any argyrophil reaction with the Grimelius' silver method, or any immunoreactivity with anti-glucagon or anti-5-HT sera. The fluorescent but non-argyrophil cells dispersed in the exocrine acinus may well be PP cells.     

Long-term trophic effect of ACTH on rat adrenocortical cells

Summary The changes occurring in rat adrenocortical cells (zona fasciculata) during an 8 day period of treatment with ACTH, were investigated by morphometric and autoradiographic methods.The most important ultrastructural change consists in a conspicuous increase in the smooth endoplasmic reticulum, that accounts for about 50% of the total increase of cellular volume. Also the mitochondrial fraction shows a significant increase, which is found to be due both to the increment in the number of mitochondria per cell and to the increase in the mean volume of organelles themselves.The quantitative autoradiographic data, indicating an increment in the incorporation of 3H-orotate and 3H-leucine into adrenocortical cells of the treated animals, allow us to conclude that the ACTH-induced ultrastructural changes are the morphological expression of a stimulation of the cellular protein synthesis.Since mitochondria are largely autonomous in the synthesis of their enzymes and structural proteins, it is possible to hypothesize that ACTH also intervenes in the regulation of the mitochondrial protein synthesis.The authors wish to express their sincere appreciation to Mr. G. Gottardo for his excellent technical assistance.     

Cytochemical and functional evaluation of ACTH responsive isolated rat adrenocortical cells. The maintenance of sex differences

The critical evaluation of isolation methods for obtaining the adrenocortical cell suspension due to trypsin or collagenase digestion was done. Some collagenase advantages were indicated by morphological observations on the staining smears as well as by ACTH stimulation test. The cytochemical reactions for enzyme activities had the limited applications for those purposes. It also appeared that commonly applied dye exclusion tests were inadequate for characterization of cell suspension. The possible role of the adrenocortical cell debris in the basal corticosterone production was pointed out. The maintenance of the sex dimorphism and the functional differences in the adrenocortical cells isolated from male and female rats have been observed.     

Gluconeogenic and lipogenic properties of isolated kidney tubules from the domestic fowl (Gallus domesticus)

Isolated kidney tubules synthesize glucose actively from fructose, lactate, glycerol and pyruvate and, to a lesser extent, from a variety of amino acids. Ethanol stimulated gluconeogenesis from pyruvate and inhibited it from lactate. The aminotransferase inhibitor, aminooxyacetate, greatly reduced synthesis from lactate but not from pyruvate. Quinolinate inhibited gluconeogenesis from both precursors, indicating an active role for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenic pathway. Incorporation of lactate or glucose into triglycerides was relatively low, and since no fatty acid synthase (FAS) activity could be detected, probably represented chain elongation or reesterification.     

Effect of ACTH on mitochondrial RNA synthesis of rat adrenocortical cells

Summary The incorporation of 3H-thymidine and 3H-uridine into adrenocortical cells of intact and ACTH-treated rats was investigated by high-resolution autoradiography. The quantitative analysis of autoradiographs shows no effect of ACTH on the incorporation of 3H-thymidine, at least in our experimental conditions. On the contrary, ACTH was found to enhance the incorporation of 3H-uridine into both adrenocortical nuclei and mitochondria. These findings are discussed in relation to numerous biochemical and morphological data, indicating that ACTH stimulates the synthesis of enzymes and structural proteins of adrenocortical cells.It is suggested that the mechanism of action of ACTH on adrenal cortex, consists in an integrated stimulation of both nuclear and mitochondrial DNA-dependent RNA synthesis.The authors wish to express their sincere appreciation to Mrs. L. Rebonato and Mr. G. Gottardo for skilled technical assistance.     

Proliferation and distribution of adrenocortical cells in ACTH treated female hamsters

The aim of the study was the analysis, by means of combined stereologic methods and metaphase arrest technique of the adrenocortical growth of ACTH treated hamster. Adult female hamsters were treated daily with 50 micrograms ACTH (Synacthen Depot, Ciba) for 3, 6 and 9 days. Between days 3 and 9 nearly linear increase in adrenal gland weight was observed, mainly due to the enlargement of the fasciculata and reticularis zones. Throughout the experiment, there was no marked change in the number of the zona glomerulosa cells while from the day 3 a notable increase in the number of fasciculata and reticularis cells occurred. Prolonged ACTH treatment resulted in a significant increase in 3H-thymidine incorporation with the highest value on day 6 and subsequent drop on day 9. Also metaphase index of adrenocortical cells notably increased due to ACTH administration and again the highest value was found on day 6 of experiment. From stereologic data and from metaphase index, adrenocortical cell birth-rates in individual adrenocortical zones and in the entire cortex were calculated. There were great differences between the values calculated from metaphase index and those obtained from stereologic data, with the former significantly lower. ACTH enhances centripetal migration of parenchymal cells in the hamster adrenal cortex and causes their accumulation in the zona reticularis.     

ACTH diazotized to agarose: effects on isolated adrenal cells

ACTH coupled in an azo linkage to an agarose support induces steroidogenesis in free adrenal cells in the same manner as does free ACTH. Observation of incubates of adrenal cells and ACTH-agarose indicates that 1) agarose-ACTH is not adherent to the cell surface, 2) entrance of ACTH into the cell may not be a prerequisite to the initiation of steroidogenesis, 3) the continuous presence of ACTH is not necessary to maintain maximal steroid production in cellular incubates, and 4) induction does not alter the functional integrity of the bound corticotropin.     

Cinemicrographic observations of cultured adrenocortical tumor cells. Dynamic responses to ACTH and cytochalasin B

ACTH increases the basal steroidogenic activity of cultured adrenocortical tumor cells, whereas moderate-high doses of cytochalasin B (CB) inhibit both basal and ACTH-induced steroidogenesis. Previous ultrastructural studies have revealed that ACTH rearranges microfilaments in these adrenal cells, whereas CB causes microfilaments to aggregate into felt-like masses. It has been postulated that the ACTH effects may facilitate organelle motility and increase organelle interactions that are required for steroid biosynthesis, and that the CB-created "foci" may impede or prevent the organelle meetings. To shed light on these possibilities, we have employed 16 mm cinemicrography of unstimulated adrenal tumor cells and cells incubated for 1-2 h with ACTH (10 mU/ml), or low (10 micrograms/ml), or high (50 micrograms/ml) doses of CB. ACTH caused initial increases in membrane ruffling and a "flurry" of particle (organelle) activity above that seen in unstimulated cells. The stimulated cells then retracted from each other and began their characteristic "rounding up" in response to the hormone. Particles appeared to move towards the nucleus, and in fully-rounded cells were extremely congested. Steroid production rose several fold above basal levels. CB10 produced slight-marked cell convexities, nearly stopped particle motility and inhibited steroid production moderately. CB50 produced an asymmetrical, spidery cell form, stopped membrane ruffling and particle motility and abolished steroidogenesis. After a washout of CB50, particle motility resumed nearly immediately. Our CB data indicate that associations between particles, presumably between mitochondria and various sources of cholesterol, are prerequisite for basal steroidogenesis in the adrenal tumor cells. In ACTH-stimulated cells, increases in steroid output correspond with increased opportunities for particle associations. These opportunities appear to arise directly or indirectly from ACTH effects on microfilaments. The responses of microfilaments to the hormone may be particularly intense in tumorous forms. By these means, the cells may express their differentiated function, although their cytoplasm has a distinctly unspecialized appearance.     

Ultrastructural changes induced by ACTH in normal adrenocortical cells in culture

The effects of ACTH, its o-nitrophenyl sulfenyl derivative (NPS-ACTH) and dibutyryl cyclic AMP (dbc AMP) on the ultrastructural morphology of adrenocortical cells of adult rats in monolayer culture have been investigated. NPS-ACTH, which has previously been shown to stimulate steroidogenesis but not cAMP synthesis in adrenal cells, induced the same characteristic transformation of mitochondrial architecture as produced by ACTH or high concentrations of dbcAMP. All three agents caused the disappearance of electron-opaque granules present in the mitochondria of unstimulated cells. It was found that these granules could be extracted with EGTA (ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetate). These results are discussed in the light of the known importance of calcium ions in the actions of ACTH.     

Effects of gonadotrophins on progesterone production by isolated granulosa cells of the adenohypophysectomized domestic fowl (Gallus domesticus)

Ovine LH and ovine FSH stimulated progesterone production in granulosa cells isolated from the F1, F2 and F3 follicles of hypophysectomized and control (sham-operation) hens when they were collected 6 h after operation, but the steroidogenic response to LH was greater for granulosa cells from hypophysectomized hens. At 15 h after operation progesterone production by granulosa cells was stimulated by LH in all 3 follicle types of control hens, but only in the F1 follicles of hypophysectomized hens. The response to FSH at 15 h was similar for control and hypophysectomized hens. The time after hypophysectomy therefore appears to affect the LH-stimulated progesterone production by granulosa cells of the F2 and F3 follicles.     

Role of chain termini in selective steroidogenic effect of ACTH/MSH(4-10) on isolated adrenocortical cells

To investigate the role of charged chain ends in the corticosteroidogenic effect of ACTH/MSH(4-10), acetyl and amide derivatives of ACTH/MSH(4-10) were synthesized and tested in isolated zona glomerulosa and zona fasciculata cells. ACTH/MSH(4-10)-NH2, Ac-ACTH/MSH(4-10) and Ac-ACTH/MSH(4-10)-NH2 (10 microM to 1 mM) stimulated the aldosterone production of zona glomerulosa cells, whereas these peptides did not stimulate the corticosterone production of zona fasciculata cells, even at 1 mM concentration. As ACTH/MSH(4-10) has been shown to have a steroidogenic effect on both types of adrenocortical cells, both charged chain termini seem to be essential for triggering of the corticosterone production of zona fasciculata cells, but for aldosterone production their presence appears not to be important.     

Transforming growth factor beta treatment decreases ACTH receptors on ovine adrenocortical cells

Transforming growth factor beta (TGF beta) is a potent inhibitor of adrenocortical cell differentiated functions, whereas corticotropin (ACTH) is the main physiological hormone which acts positively on these functions. We have studied the effects of both TGF beta and ACTH on ovine adrenocortical cell ACTH receptors. Ovine adrenocortical cells contained specific high affinity (Kd = 2.7 +/- 1.6 x 10(-10) M) and low capacity (1190 +/- 120 sites/cell) ACTH receptors. Pretreatment of cells with TGF beta resulted in a time- and dose-dependent (ED50 = 50 pg/ml) decrease of 125I-ACTH1-39 binding. The observed decrease in ACTH binding was due to a 2-3-fold decrease in the number of binding sites without modification of the binding affinity. On the contrary, pretreatment of cells with ACTH caused a 4-4.5-fold increase in the number of ACTH binding sites without an effect on the Kd. When cells were pretreated with both ACTH and TGF beta, TGF beta blocked completely the positive trophic effect of ACTH on its own receptors. The variations in ACTH receptor number were associated with parallel changes on acute ACTH-induced cyclic AMP production. Thus, the effects of TGF beta on ACTH receptor content are likely another important negative action of this peptide on adrenocortical cell differentiation. Moreover, these results suggest that regulation of ACTH receptor number may be one mechanism by which hormones and growth factors control adrenocortical differentiation.