Ultrastructural Mechanisms of Impaired Aldosterone Synthesis in Rats Exposed to DDT during Prenatal and Postnatal Development.

The study is aimed at elucidation of ultrastructural mechanisms underlying impaired aldosterone synthesis by glomerulosa cells in Wistar rats exposed to low doses of endocrine disrupter DDT during prenatal and postnatal development. Analysis of rat zona glomerulosa histology and function during the pubertal and postpubertal periods showed that exposure to endocrine disrupter DDT disturbs its development and reduced the production of aldosterone. Electron microscopy showed that changes in the aldosterone synthesis are related to impaired reorganization of the mitochondrial apparatus, one of the leading factors in the regulation of steroidogenesis, in glomerulosa cells in DDT-exposed rats during puberty.

ß-Catenin and FGFR2 regulate postnatal rosette-based adrenocortical morphogenesis.

Rosettes are widely used in epithelial morphogenesis during embryonic development and organogenesis. However, their role in postnatal development and adult tissue maintenance remains largely unknown. Here, we show zona glomerulosa cells in the adult adrenal cortex organize into rosettes through adherens junction-mediated constriction, and that rosette formation underlies the maturation of adrenal glomerular structure postnatally. Using genetic mouse models, we show loss of ß-catenin results in disrupted adherens junctions, reduced rosette number, and dysmorphic glomeruli, whereas ß-catenin stabilization leads to increased adherens junction abundance, more rosettes, and glomerular expansion. Furthermore, we uncover numerous known regulators of epithelial morphogenesis enriched in ß-catenin-stabilized adrenals. Among these genes, we show Fgfr2 is required for adrenal rosette formation by regulating adherens junction abundance and aggregation. Together, our data provide an example of rosette-mediated postnatal tissue morphogenesis and a framework for studying the role of rosettes in adult zona glomerulosa tissue maintenance and function.

In vitro culture on Matrigel favors the long-term maintenance of rat zona glomerulosa-cell differentiated phenotype.

Zona glomerulosa (ZG) cells cultured on plastic within few days dedifferentiate losing their capacity to secrete aldosterone (ALDO) in appreciable amounts. Evidence indicates that extracellular matrix modulates the secretory behavior of adrenocortical cells cultured in vitro. Hence, we compared the morphology and function of rat ZG cells grown on plastic and Matrigel basement membrane matrix (hereinafter Matrigel) for up to 12 days. At day 3, no significant differences were observed between cells cultured on plastic and Matrigel. Starting from day 6, ZG cells cultured on plastic lost their ultrastructural differentiated features (mitochondria with tubular cristae, smooth endoplasmic reticulum cisternae and lipid droplets), exhibiting a fibroblast-like appearance. The mRNA expression of the main steroidogenic enzymes, as evaluated by real-time polymerase chain reaction, the baseline secretion of ALDO and other post-pregnenolone hormones, as evaluated by high pressure liquid chromatography, and the secretory response to ACTH, angiotensin-II and K(+), as evaluated by radioimmunoassay, displayed a time-dependent decrease. Matrigel was found to maintain unchanged both the ultrastructure and the expresion of steroidogenic enzymes of ZG cells until day 12 of culture. Baseline and agonist-stimulated steroid-hormone secretion decreased with the duration of culture on Matrigel, but was always higher than that of ZG cells grown on plastic. Hence, our study clearly indicates that the culture on Matrigel favors the maintenance of rat ZG-cell differentiated phenotype, allowing the conclusion that this technique is suitable for long-term in vitro investigations.

A model for studying regulation of aldosterone secretion: freshly isolated cells or cultured cells?

Practically all studies relating to zona glomerulosa function have been performed either with freshly isolated cells or with cells used after 2 or 3 days in culture. This study compares the step-by-step response (binding, second messenger production and aldosterone response) of isolated glomerulosa cells vs cells maintained in primary culture to the main stimuli of aldosterone secretion. One day in culture induces a decrease of 77 and 65% in the basal level of corticosterone and aldosterone secretions, compared to that observed in freshly isolated cells. In these conditions, the cells become more sensitive to most of their stimuli, but not all: e.g. important differences are noted in the dose-response of aldosterone secretion to adrenocorticotropin (ACTH), which is often shifted to a lower concentration sensitivity in cultured cells. For example, 0.1 nM ACTH stimulates steroid secretion by three-fold in isolated cells while 1 pM ACTH already induces a 25 and nine-fold increase, respectively, in corticosterone and aldosterone output in cultured cells. Moreover, some stimuli such as isoproterenol do not have any effect in isolated cells but do stimulate steroid secretion in cultured cells. In contrast, other stimuli, such as serotonin or DA (via DA2 receptors) act preferentially in freshly isolated cells. The main observation derived from this study is that glomerulosa cells, under appropriate conditions, are able to respond to their main secretagogues even after 4 days in culture. At this time, glomerulosa cells maintain their ultrastructural characteristics and functional properties and, aside from a few exceptions, demonstrate higher sensitivity to their known stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural evidence for a paracrine regulation of the rat adrenal cortex mediated by the local release of catecholamines from chromaffin cells.

The adrenal glands of perfusion fixed rats were investigated by light and electron microscopy. Within the rat adrenal cortex occurred rays and islets of chromaffin cells which were in close contacts with cortical cells on the electron microscopical level. We achieved to catch the process of exocytosis from a chromaffin cell located within the zona glomerulosa in direct apposition with an adrenocortical cell. The documentation of an exocytotic process from a chromaffin cell neighbouring a cortical cell provides direct evidence in support of a paracrine regulation of the cortex mediated by chromaffin cells.

Regulation of aldosterone synthase cytochrome P-450 in rat adrenals by angiotensin II and potassium.

Changes in the levels of aldosterone synthase cytochrome P-450, a recently identified enzyme in rat adrenals, were studied in response to the renin-angiotensin system and K stimuli. As examined by an immunoblot technique, the zona glomerulosa mitochondria from rats fed on a low Na-normal K diet (8.6 mmol Na+ and 207 mmol K+/kg of diet) or a low Na-high K (0.2 M KCl in drinking water) diet for 4-10 days contained significantly higher amounts of aldosterone synthase cytochrome P-450 than those from rats fed on a normal diet (86 mmol Na+ and 207 mmol K+/kg of diet). Activities of the enzyme were also found to increase by about 10-fold on day 10. In concert with these changes, both plasma renin activity and plasma aldosterone concentration increased, indicating that the renin-angiotensin system was activated in these rats. Feeding with a normal Na-high K diet also induced significantly higher levels of both amount and activity of aldosterone synthase cytochrome P-450 together with an elevated serum K concentration on day 4, though they all decreased to near the control level on the following days. On the other hand, when enalapril malate, an angiotensin I-converting enzyme inhibitor, was administered to the low Na-normal K rats, the increases in the amount and activity of the enzyme as well as in plasma aldosterone concentration were suppressed altogether. However, the enalapril administration to the low Na-high K rats suppressed the increases only partially. These results indicate that the aldosterone synthase cytochrome P-450 is an ultimate target of the regulation of aldosterone biosynthesis by angiotensin II and K.

Hormone-sensitive lipase deficiency in mice causes lipid storage in the adrenal cortex and impaired corticosterone response to corticotropin stimulation.

Hormone-sensitive lipase (HSL, E.C.3.1.1.3, gene designation Lipe) is reportedly the major cholesteryl esterase of adrenal cortex. Because of the potential importance of cholesteryl ester hydrolysis in steroidogenesis, gene-targeted HSL-deficient mice were assessed for adrenal cortical morphology and function. Compared with control animals, HSL deficiency results in a marked accumulation of lipid droplets both in zona glomerulosa and zona fasciculata. In the zona fasciculata, lipid accumulation was observed progressively from the outer to the inner regions, culminating near the corticomedullary junction with the formation of syncytial-lipoid structures having the appearance of degenerative cells. These morphological changes did not significantly alter the basal levels of circulating corticosterone, but following ACTH stimulation, corticosterone levels were decreased (P < 0.001). The observation of normal basal corticosterone and aldosterone levels demonstrates that some free cholesterol for steroid synthesis can be produced independently of HSL. Taken together, these results indicate that HSL-deficient mice accumulate lipid droplets in such a way as to impair acute ACTH stimulation of corticosterone secretion. Such observations are also found in some forms of congenital adrenal hyperplasia. By extension, HSL deficiency may be a cause of hereditary adrenocortical hypofunction in humans.

Association of the G protein alpha(q)/alpha11-subunit with cytoskeleton in adrenal glomerulosa cells: role in receptor-effector coupling.

In 3-day primary cultures of rat glomerulosa cells, a 30-min pre-incubation with either 10 microM colchicine (a microtubule-disrupting agent) or 10 microM cytochalasin B (a microfilament-disrupting agent) decreased angiotensin II (Ang II)-induced inositol phosphate accumulation by 50%. Moreover, both drugs decreased inositol phosphate production induced by fluoroaluminate (a nonspecific activator of all G proteins), indicating that both microtubules and microfilaments are essential for phospholipase C activation. Analysis of microfilament- and microtubule-enriched fractions and immunoprecipitation of actin and tubulin revealed that the alpha(q)/alpha11-subunit of the G(q/11) protein was associated with both structures. Ang II stimulation induced a rapid translocation of alpha(q)/alpha11, microfilaments, and microtubules to the membrane and induced a time-dependent increase in the level of alpha(q)/alpha11 associated with both microfilaments and microtubules. Moreover, double immunofluorescence staining clearly showed a colocalization of the alpha(q)/alpha11-subunit of the G(q/11) coupling protein and microfilament distribution. These associations and plasma membrane redistribution under Ang II stimulation indicate that microfilaments and microtubules are both involved in phospholipase C activation and inositol phosphate production. Moreover, our results indicate that the alpha(q)/alpha11 protein is closely associated with cytoskeletal elements and is found both at the plasma membrane level as well as on intracellular stress fibers.

Zonal differences in adrenocortical lipid peroxidation: role of alpha-tocopherol.

Studies were done to evaluate the relationship between alpha-tocopherol (alpha-T) concentrations and lipid peroxidation (LP) in vitro in microsomal preparations from the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Microsomes were incubated with ferrous ion (Fe2+) to promote free radical production, and alpha-T levels and LP were monitored after various incubation times. alpha-T concentrations were far lower in inner than outer zone preparations and were rapidly depleted from inner zone microsomes by incubation with Fe2+. Coinciding with alpha-T depletion was a large and rapid increase in LP. With outer zone microsomes, alpha-T depletion required more than 30 min, and very little LP was demonstrable during this period. However, once alpha-T depletion occurred, LP was rapidly initiated and reached levels similar to those obtained with inner zone preparations. Inhibition of LP by MnCl2 prevented the Fe(2+)-induced declines in alpha-T in both zones. The results demonstrate the importance of alpha-T as a modulator of adrenal LP and indicate that the zonal differences in LP are largely attributable to the differences in alpha-T concentrations.

Catecholamines released from local adrenergic axon terminals are possibly involved in fine tuning of steroid secretion from zona glomerulosa cells: functional and morphological evidence.

The effect of supramaximal electric field stimulation on [3H]noradrenaline (NA) release and hormone production by rat adrenal capsule-glomerulosa preparations was studied using a microvolume perfusion system. A substantial proportion (about 20%) of nerve endings (varicosities) were observed close to zona glomerulosa cells, and about half of them appeared to be catecholaminergic, as judged by the chromaffin reaction of the synaptic vesicles studied at electron microscopic level. In tissue, preloaded with [3H]NA, the release of NA in response to electrical stimulation was frequency-dependent. Reserpinization, calcium removal or inhibition of Na+ influx by tetrodotoxin completely blocked NA release by field stimulation, indicating that the release resulted from axonal activity and is of vesicular origin. Neither the alpha 2-adrenoceptor agonist xylazine nor the muscarine-receptor agonist oxotremorine affected the stimulation-evoked release of [3H]NA, suggesting that, in contrast with other neurones present in the central nervous system or in the peripheral autonomic nervous system but like those in the median eminence, these axon terminals contained few presynaptic modulatory receptors. The NA (10.20 +/- 1.79 (S.E.M.) micrograms/g, n = 9), adrenaline (24.38 +/- 5.50 micrograms/g, n = 9) and dopamine (0.35 +/- 0.09 micrograms/g, n = 6) contents of the preparations were high, as determined by high-performance liquid chromatography. Our observations that the release and content of NA is high, and that a substantial proportion of catecholaminergic axon terminals lie in close proximity to zona glomerulosa cells (median value of the distance 300 nm) or to smooth muscle cells of the vessels, suggest that NA released from local adrenergic neurones without being presynaptically modulated may play an important role in fine-tuning both steroid production and/or blood flow through the gland, itself a powerful modulator of the adrenocortical response. This local modulating effect of NA may be especially significant when sympathetic activity is enhanced.

The effect of cytoplasmic Ca2+ signal on the redox state of mitochondrial pyridine nucleotides.

As first observed in rat adrenal glomerulosa cells, cytoplasmic Ca(2+) signal, induced by K(+), angiotensin II or vasopressin, evokes an increase in the level of reduced mitochondrial pyridine nucleotides, NADH and NADPH. Prostaglandin F(2)alpha and extracellular ATP exert similar effects in rat ovarian luteal cells. This coupling of cytoplasmic Ca(2+) concentration and mitochondrial metabolism occurs also when the stimuli are applied at physiological concentration and under conditions when no formation of high-Ca(2+) perimitochondrial microdomains may be presumed. We present evidence that low submicromolar Ca(2+) signals in the cytoplasm can increase mitochondrial Ca(2+) concentration and activate mitochondrial dehydrogenation processes. Several observations support the assumption that intramitochondrial Ca(2+) signals play a significant role in the stimulation of steroid hormone production.

Chronic hypoxia induced ultrastructural changes in the rat adrenal zona glomerulosa.

The adrenal cortex plays an important role in adaptation to various forms of stress, including hypoxia. While physiological changes in the aldosterone metabolism during hypoxia have been extensively described, few studies have focused on the morphological changes in the adrenal glands under chronic hypoxia. We studied the ultrastructure of the zona glomerulosa of 6-month-old Wistar rats exposed to chronic normobaric hypoxia. Animals were divided into two groups: control (n=12) and hypoxic (n=12). In this latter group, the animals were kept at 7% O2 concentration after a gradual adaptation (21, 15, 12, 10, 8, 7 vol% O2). The duration of the study was 112 days. In comparison with normoxic rats, body weight and adrenal gland weight of hypoxic animals was significantly reduced by 18.5% (p=0.006) and 14.7% (p=0.001) respectively. The thickness of the zona glomerulosa decreased due to atrophy of cells. The main ultrastructural changes observed were: 1) a decrease in, or complete elimination of, lipid droplet content; 2) a marked increase in lysosome number; and 3) the presence of giant mitochondria. Our findings show that rats fail to adapt to severe chronic hypoxia. The ultrastructural changes in the zona glomerulosa found in the present study could reflect changes in the aldosterone pathway.

An ultrastructural stereologic study of aldosterone secreting adrenal adenomas and of adjacent zona glomerulosa.

The ultrastructure of four aldosterone secreting adenomas and of the adjacent zona glomerulosa has been described by the use of stereological techniques. Adenomatous cells (about 2800 microns 3 in volume) invariably displayed a striking abundance of lipid droplets, which occupied about 30% of the cytoplasm. Mitochondria prevalently contained tubulo-lamellar or lamellar cristae, but some cells exhibited organelles with vesicular cristae. Smooth endoplasmic reticulum (SER) was not very abundant. Small lipofuscin-pigment granules were frequently seen and in a few cells they were exceedingly numerous. Zona glomerulosa cells were smaller (about 950 microns 3 in volume) and possessed mitochondria with typical tubulo-lamellar cristae, a plentiful SER and few lipid droplets. They showed the ultrastructural features of elements actively engaged in steroid synthesis. The possible origin of aldosteronoma cells from the zona glomerulosa is discussed.

Comparative stereological studies on zonation and cellular composition of adrenal glands of normal and anencephalic human fetuses. II. Cellular composition of the gland.

In our previous paper (Bocian-Sobkowska et al., 1997) we demonstrated a striking difference in development of zonation in adrenals of normal and anencephalic human fetuses. The purpose of the present study was to characterize, by means of stereology, the cellular composition of developing adrenals in the same case. Studies were performed on 11 pairs of adrenal glands from normal fetuses and 10 from anencephalic fetuses. In the studied period of development (24 to 39 weeks of intra-uterine life) the average volume of cells in normal glands increased as follows: zona glomerulosa (ZG) from 355 to 870 microns3; zona fasciculata (ZF) from 779 to 1200 microns3; fetal zone (FZ) from 2004 to 2380 microns3: and medulla (M) from 600 to 970 microns3. In anencephalic fetuses, the appropriate values were: ZG-380-680 microns3; ZF-460-680 microns3; FZ-1820-1680 microns3; and M-870-1400 microns3. At the end of the studied period the number of ZG cells in normal fetuses was two fold higher than in anencephalics, ZF cells-6-fold and in FZ-5-fold higher, while in the M the number of cells was nearly equal in both groups. During the whole investigated period of intra-uterine development the total number of adrenocortical cells in normal glands increased ca 2.5-fold, while in anencephalic glands only ca 0.5-fold, reaching at the end ca 40% of normal value. In both normal and anencephalic adrenals the number of ZG and M cells was highly correlated with ZG/M cell ratio, being slightly higher in normal glands. No such relation was demonstrated for cells of the remaining adrenocortical zones.

A clinical pregnancy after a simple method of zona cutting, cryopreservation, and zygote intrafallopian transfer.

A 30-year-old nulliparous woman, who was infertile for greater than 3 years, conceived after the procedures of a simple method of zona cutting, cryopreservation, and ZIFT. This case illustrates: (1) acid medium, chymotrypsin, or sucrose are not needed for the procedure of zona cutting; (2) the zygotes resulting from zona cutting survive through freezing and thawing; and (3) oocyte retrieval can be done concomitant with conservative surgery for endometriosis.

Micromanipulation in a center for reproductive medicine.

Several variations of micromanipulation of the female gamete (zona drilling, zona cracking, ooplasmic sperm injection, partial zona dissection) have been applied recently to human IVF to overcome severe male factor. Of the first 16 cycles attempted using partial zona dissection, one pregnancy resulted leading to a normal term delivery. Careful removal of the coronal cells, as well as stepwise removal of sucrose postpartial zona dissection, will facilitate this procedure and greatly reduce potential damage to the oocyte by pH, mechanical, or thermal injury. Micromanipulation has become a routine service offered in our program in cases where the likelihood of a poor IVF outcome is either known or suspected, and also serves as a replacement for simple reinsemination in cases of failed fertilization.

Adrenomedullin and calcitonin gene-related peptide inhibit aldosterone secretion in rats, acting via a common receptor.

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) did not affect either basal or ACTH-stimulated secretion of a1dosterone and corticosterone by dispersed rat capsular and inner adrenocortical cells, respectively. However, both peptides strongly depressed angiotensin-II (ANG- II)-stimulated a1dosterone production by capsular cells, the minimal effective concentration was 10(-7) M. The inhibitory effect of both ADM and CGRP was reversed by CGRP8-37, a specific CGRP1 receptor antagonist; a complete reversal was obtained with a CGRP8-37 concentration of 10(-6) M. Our findings indicate that ADM and CGRP specifically interfere with the intracellular mechanisms transducing the secretagogue signal of ANG-II, and suggest that the ADM effect is mediated by CGRP receptors

Hormonal modulation of atrial natriuretic factor receptors and effects on adrenal glomerulosa cells of female rats.

This study was done to determine if a decrease in the aldosterone-suppressant effect of atrial natriuretic factor (ANF) by progesterone and an increase by estrogen was caused by modulation of adrenal zona glomerulosa ANF receptors. Freshly dispersed glomerulosa cells from virgin, 13-15 day pregnant, ovariectomized (OVX) estradiol-17 beta-treated and OVX progesterone-treated rats were used. Competitive displacement of specifically bound [125I]ANF1-28 with unlabelled ANF1-28 yielded concentrations of guanylate cyclase-linked ANF-R1 plus ANF-R2 (clearance) receptors and the displacement with unlabelled ANF4-23 yielded ANF-R2 receptors; the difference between the two was treated as the concentration of ANF-R1 receptors. Pregnancy and progesterone decreased and estrogen increased the number of glomerulosa ANF-R1 receptors. ANF produced a significantly greater suppression of potassium-induced aldosterone secretion in cells from OVX estradiol-treated rats than in cells from OVX progesterone-treated animals. These data suggest that the inhibition of the aldosterone-suppressant activity of ANF by progesterone is the result of a downregulation of ANF-R1 receptors.

Age-related changes in the morphology and function of the zona glomerulosa of the rat adrenal cortex.

The age-related changes in the morphology and function of rat adrenal zona glomerulosa (ZG) were investigated by coupled stereological and radioimmunological techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Aging caused a notable lowering in the plasma aldosterone concentration and a marked decrease in both basal and ACTH- or angiotensin II (ANG-II)-stimulated secretion of collagenase-dispersed ZG cells. Plasma renin activity (PRA) underwent an age-dependent decrease, while the plasma level of ACTH displayed a significant rise. ZG and its parenchymal cells did not evidence any age-related morphologically demonstrable alteration in their growth, nor ZG cells showed any marked ultrastructural change, with the exception of a severe lipid-droplet repletion. This last finding is in keeping with the aging-induced decrease in the secretory activity of ZG cells, inasmuch as lipid droplets are the intra-cellular stores of cholesterol esters, the obligate precursors of steroid hormones in rat adrenals. ACTH and ANG-II are well known to be involved in the maintenance of the growth of rat ZG; thus, the combined impairment of ANG-II production (as evidenced by PRA lowering) and increase in ACTH secretion may maintain unchanged ZG growth during aging.

Time-dependent changes in adrenal cortex ultrastructure and corticosterone levels after noise exposure in male rats.

In response to a stressful stimulus, there is a marked activation of the hypothalamic-pituitary-adrenal axis leading to a release of adrenocorticotropic hormone. This, in turn, acts on the zona fasciculata of the adrenal cortex to increase corticosterone plasma levels. Given the frequency of chronic intermittent noise exposure in man, we selected loud noise to evaluate concomitant changes in the ultrastructure of the adrenal cortex and corticosterone release. Following chronic (21 days, 6 h per day) loud white noise exposure (100 dBA, 0-26 KHz), we found the zona fasciculata to be most sensitive to time-dependent ultrastructural changes. These consisted of modifications in cell compartments involved in hormone synthesis and release. On the other hand, we found a progressive increase in corticosterone plasma levels which reached a plateau 9 days after noise exposure. The significance of these changes, in relation to phenomena like sensitization to repetitive stress, are discussed. Furthermore, the present data suggest that chronic loud noise exposure might potentially lead to endocrine dysfunctions.