Development of the human adrenal zona reticularis: morphometric and immunohistochemical studies from birth to adolescence.

Age-related morphologic development of human adrenal zona reticularis (ZR) has not been well examined. Therefore, in this study, 44 human young adrenal autopsy specimens retrieved from large archival files (n=252) were examined for immunohistochemical and morphometric analyses. Results demonstrated that ZR became discernible around 4 years of age, and both thickness and ratio per total cortex of ZR increased in an age-dependent fashion thereafter, although there was no significant increment in total thickness of developing adrenal cortex. We further evaluated immunoreactivity of both KI67 and BCL2 in order to clarify the equilibrium between cell proliferation and apoptosis in the homeostasis of developing human adrenals. Results demonstrated that proliferative adrenocortical cells were predominantly detected in the zona glomerulosa and partly in outer zona fasciculata (ZF) before 4 years of age and in ZR after 4 years of age, but the number of these cells markedly decreased around 20 years of age. The number of BCL2-positive cells increased in ZR and decreased in ZF during development. Adrenal androgen synthesizing type 5 17beta-hydroxysteroid dehydrogenase (HSD17B5 or AKR1C3 as listed in the Hugo Database) was almost confined to ZR of human adrenals throughout development. HSD17B5 immunoreactivity in ZR became discernible and increased from around 9 years of age. Results of our present study support the theory of age-dependent adrenocortical cell migration and also indicated that ZR development is not only associated with adrenarche, but may play important roles in an initiation of puberty.

Coregulatory protein-orphan nuclear receptor interactions in the human adrenal cortex.

The capacity of the adrenal to produce steroids is controlled in part through the transcriptional regulation of steroid enzymes. The orphan nuclear receptor steroidogenic factor 1 (SF-1) is central to the transcriptional regulation of all steroid hydroxylase enzymes, whereas nur77 can preferentially regulate steroid enzyme genes relevant to cortisol production. We hypothesised that, in the presence of secretagogues, SF-1 and nur77 may differentially interact with coregulatory proteins in the human adrenal cortex. Both coregulatory proteins, steroid receptor coactivator (SRC-1) and silencing mediator for retinoid and thyroid hormones (SMRT), were found to be expressed in the zona fasciculata and reticularis in the human adrenal cortex, but were largely absent from the zona glomerulosa. Both coregulatory proteins were colocalised with SF-1 and nur77. In the H295R adrenal tumour cell line, SF-1 and nur77 transcripts were increased in cells in the presence of forskolin, whereas nur77 mRNA was also induced with angiotensin II (AII). The coactivator SRC-1 mRNA was increased in the presence of both forskolin and AII. Forskolin induced recruitment of SRC-1 to the SF-1 response element and induced SRC-1-SF-1 interactions, whereas AII increased recruitment of SRC-1 to the nur77 response element and induced SRC-1-nur77 interactions. The corepressor SMRT interacted with SF-1 in the presence of AII and with nur77 in cells treated with forskolin. Orphan nuclear receptor-coregulatory protein interactions may have consequences for the regulation of key steroidogenic enzymes in the human adrenal cortex.

Expression of activin/inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis.

Activins and inhibins are structurally related glycoprotein hormones modulating pituitary FSH secretion and gonadal steroidogenesis. Activins and inhibins are also produced in the adrenal cortex where their physiological role is poorly known. Hormonally active human adrenocortical tumors express and secrete inhibins, while in mice adrenal inhibins may function as tumor suppressors. To clarify the significance of adrenal activins and inhibins we investigated the localization of activin/inhibin signaling components in the adrenal gland, and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis. Activin receptor type II/IIB and IB, activin signal transduction proteins Smad2/3, and inhibin receptor betaglycan were expressed throughout the adrenal cortex, whereas Smad4 expression was seen mainly in the zona reticularis and the innermost zona fasciculata as evaluated by immunohistochemistry. Treatment of cultured adrenocortical carcinoma NCI-H295R cells with activin A inhibited steroidogenic acute regulatory protein and 17alpha-hydroxylase/17,20-lyase mRNA accumulation as evaluated by the Northern blot technique, and decreased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate secretion as determined by specific enzyme immunoassays. Activin A increased apoptosis as measured by a terminal deoxynucleotidyl transferase in situ apoptosis detection method. Inhibins had no effect on steroidogenesis or apoptosis. In summary, activin/inhibin signaling components are coexpressed in the zona reticularis and the innermost zona fasciculata indicating full signaling potential for adrenal activins and inhibins in these layers. Activin inhibits steroidogenic enzyme gene expression and steroid secretion, and increases apoptosis in human adrenocortical cells. Thus, the activin-inhibin system may have a significant role in the regulation of glucocorticoid and androgen production and apoptotic cell death in the human adrenal cortex.

Different immunohistochemical patterns of TGF-beta1 expression in benign and malignant adrenocortical tumours.

OBJECTIVE: Transforming growth-factor beta1 (TGF-beta1) influences a number of specific functions of adrenocortical cells in several animal species. The aim of our study was to evaluate by immunohistochemical analysis the presence and distribution of TGF-beta1 in normal adrenal tissue and in different adrenal tumours. PATIENTS: We analysed 8 functioning (5 adenomas and 3 carcinomas) and 15 non functioning (6 adenomas and 9 carcinomas) adrenal tumours and 6 normal adrenal glands. RESULTS: In normal adrenal glands, the glomerulosa and the reticularis zones displayed diffuse cytoplasmic staining, while the fasciculata zone was almost completely negative. Functioning adenomas displayed cytoplasmic staining restricted to compact cells while in nonfunctioning adenomas, prevalently composed by clear cells, no staining was observed. Overall, adrenal carcinomas were characterized by the lack of cytoplasmic positivity and by sporadic positive cells around vessels both in functioning and in nonfunctioning tumours. CONCLUSIONS: TGF-beta1 expression is associated with active steroid secretion in normal adrenal tissue, as well as in benign cortical adenomas, while this relationship is lost in primary adrenal malignancies. These data provide indirect evidence for a regulatory role played by TGF-beta1 on steroid secretory pathways.

Immunohistochemical study of cytochrome b5 in human adrenal gland and in adrenocortical adenomas from patients with Cushing's syndrome.

Cytochrome b5, a component of the electron transfer system increases the relative activity of 17,20-lyase to 17alpha-hydroxylase of P450c17 in vitro. In the present study, immunohistochemical analysis of cytochrome b5 was performed in the human adrenal gland and in adrenocortical adenomas from patients with Cushing's syndrome. In the human adrenal gland, cytochrome b5 was stained in all three adrenocortical layers but the staining was most remarkable in the zona reticularis. All of the adenomas were composed mainly of compact cells, which exhibited immunoreactive staining for cytochrome b5 as well as for P450c17 and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). The distribution of b5 in the adenomas was correlated with that of P450c17 rather than with that of 3beta-HSD. The immunoreactive staining for cytochrome b5 appeared to be more prominent in the two adenomas that produced relatively high concentrations of adrenal androgens than in adenomas that produced low concentrations of adrenal androgens. These results immunohistochemically support the functional association of b5 with androgen production through interaction with P450c17 and the previous finding that higher concentrations of cytochrome b5 are associated with greater production of adrenal androgens in adrenocortical adenomas from patients with Cushing's syndrome.

Expression of inositol 1,4,5-trisphosphate receptors in rat adrenocortical zones.

Previously we demonstrated the presence of InsP3R-I, -II and -III subtypes in the zona glomerulosa. Now we have examined the expression of different subtypes of inositol 1,4,5-trisphosphate receptor (InsP3R) in the inner zones of rat adrenal cortex. RNA extracted from decapsulated adrenal tissue (zonae fasciculata-reticularis and the medulla) or from fasciculata-reticularis cells was reverse transcribed. Subsequent polymerase chain reaction revealed the presence of InsP3R-I, -II and -III subtypes in decapsulated tissue but failed to demonstrate the expression of any known subtypes of InsP3R in fasciculata-reticularis cells. Accordingly, InsP3 receptors expressed in the decapsulated tissue are of medullary origin.

Regulation of ACTH receptor mRNA and binding sites by ACTH and angiotensin II in cultured human and bovine adrenal fasciculata cells.

Human (HAC) and bovine (BAC) adrenal fasciculata cells express ACTH and angiotensin-II (A-II) receptors. In the present work, we have studied the effects of both hormones on ACTH receptor (ACTH-R) mRNA and binding sites. Both HAC and BAC expressed several ACTH-R transcripts. Although in both cell types, ACTH and A-II increased ACTH-R transcripts in a time- and dose-dependent manner, the maximal effects were different. Thus, ACTH at 10(-9) M enhanced 21- and 5-fold the level of ACTH-R mRNA and binding sites in HAC, whereas in BAC both parameters were enhanced only 3-fold. A-II at 10(-7) M increased 17- and 3.5-fold ACTH-R mRNA and binding sites in HAC, whereas in BAC, it caused only a 2-fold increase in ACTH-R mRNA and a small decrease in receptor number. In HAC, the stimulatory effects of both hormones on ACTH-R mRNA are mainly transcriptional, whereas in BAC they are mainly post-transcriptional, by decreasing the rate of degradation of ACTH-R mRNA. The stimulatory effects of ACTH on ACTH-R in both HAC and BAC were associated with an enhanced steroidogenic response to further hormonal stimulation. In contrast, specific species differences were observed with A-II. Thus, in HAC A-II increased ACTH-R mRNA and binding sites and the ACTH-induced cortisol production, whereas in BAC, A-II caused a slight decrease of ACTH binding sites and steroidogenic desensitization.

Distribution of parvalbumin immunoreactivity in the human brain.

The cellular distribution of parvalbumin-like immunoreactivity (PA-LI) in the human brain was investigated by peroxidase-antiperoxidase methods using antiserum to rat skeletal muscle parvalbumin. PA-LI was present in non-pyramidal neurons of the cerebral cortices, stellate cells, basket cells and Purkinje cells in cerebellar cortices, and neurons of some nuclei in human brain stem; the distribution of PA-LI in human brain was very similar to that in rat brain. These results indicate that PA-LI is widely distributed in a specific subpopulation of neurons of the human brain.

Beta-endorphin-like and adrenocorticotropin-like materials in turtle heart and intestine.

1. Turtle heart and intestine acetone powders were extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to gel filtration on Sephadex G-25 and ion exchange chromatography on CM-cellulose. 2. In turtle heart, corticotropin-like bioactivity was distributed among chromatographic fractions (derived from material unretarded on Sephadex G-25) unadsorbed and adsorbed on CM-cellulose. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were adsorbed on CM-cellulose. 3. In turtle intestine, corticotropin-like bioactivity was absent. Opiate receptor binding activity was present in fractions unretarded as well as in fractions retarded on Sephadex G-25, indicating a molecular weight of greater and smaller than 5000 respectively. 4. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were found in a fraction adsorbed on CM-cellulose.