ARTICLE |
Correspondence to: Jean-Guy LeHoux, Dept. of Biochemistry, Faculty of Medicine, U. of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4.
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Summary |
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We used an anti-rat adrenal cytochrome P450C17 (P450C17) antibody to perform immunofluorescence and also immunogold electron microscopic studies to determine the zonal and intracellular distribution of P450C17 in hamster adrenals. Because P450C17 activity is regulated mainly by adrenocorticotropin (ACTH), its zonal and intracellular localization was also analyzed after ACTH treatment. The effect of ACTH treatment on protein concentration was also investigated by Western blotting analysis. By immunofluorescence, we found P450C17 to be confined to the zona fasciculata (ZF) in the hamster, in contrast to other small rodents, which do not express P450C17 in their adrenals. After treatment with ACTH, the thickness of the ZF remained unchanged compared to that of control animals, whereas a marked increase in fluorescence intensity was observed. In addition, dispersed cells in the zona reticularis (ZR) showed positive staining after ACTH treatment. Immunocytochemistry with colloidal gold showed P450C17 to be localized and importantly increased only in the cytoplasmic areas between the mitochondria of ZF cells of ACTH-treated animals. These areas are predominantly occupied by elements of the endoplasmic reticulum and other unidentified organelles. Immunoblotting analysis of whole glands revealed a single protein band at approximately 55 kD, which reacted with the 450C17 antibody. After stimulation with ACTH injected at 5-hr intervals over a period of 20 hr, P450C17 protein concentrations were considerably greater than in control animals. In conclusion, P450C17 is located not over mitochondria but probably in the endoplasmic reticulum of the ZF cells in hamster adrenals. Treatment with ACTH induced expression of cytochrome P450C17 in ZF cells, increasing its production in these cells without stimulating cell proliferation. (J Histochem Cytochem 45:1409-1416, 1997)
Key Words:
hamster, adrenal, cytochrome P450C17, 17-hydroxylase, 17,20-lyase, CYP17, ACTH, immunofluorescence, immunocytochemistry
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Introduction |
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Adrenal steroidogenesis involves many complex enzymatic conversions mediated by specific cytochrome P450s. One enzyme is the expression product of CYP17 gene, the microsomal cytochrome P450C17 (P450C17), which intervenes at a key branch point in steroidogenesis, mediating glucocorticoid and androgen biosynthesis. P450C17 catalyzes two reactions, i.e., the 17-hydroxylation and the cleavage of the C17-C20 bond of C21 steroids. The 17
-hydroxylation reaction is an essential step in the biosynthesis of cortisol, and the 17,20-lyase reaction is necessary for the synthesis of androgens.
The adult mammalian adrenal gland is composed of an outer cortex and a thin inner medulla. Histologically, the cortex is subdivided into three distinct zones: the outer zona glomerulosa (ZG), the middle zona fasciculata (ZF), and the inner zona reticularis (ZR) directly apposed to the medulla. The ZG immediately beneath the capsule is responsible for the secretion of aldosterone (
Understanding the functional roles of each of the three layers of the adrenal cortex is important to fully comprehend adrenal steroidogenesis. Immunohisto-chemical methods have proved to be highly useful for investigation of the zonal distribution of the adrenal enzymes. The zonal distribution of P450C17 has been studied in the adrenal cortex of the guinea pig (
Hamsters differ from other rodents in that they express adrenal P450C17 (-hydroxylase activity were shown to be increased after administration of ACTH (
In this study we investigated for the first time the zonal and intracellular localization of the hamster P450C17 in the adrenal gland by immunofluorescence and immunogold electron microscopy. The effects of ACTH on the zonal distribution and protein levels of P450C17 were also examined by Western blotting analysis.
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Materials and Methods |
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Animals
Adult male Syrian golden hamsters (120 ± 10 g) were purchased from Charles River Canada (St Constant, Quebec, Canada). Purina rat chow and tapwater were available ad libitum. Before experimentation the animals were kept for 3 weeks in an isolated room with a controlled light-dark cycle (lights on 0600-1800 hr). The animals were always sacrificed by decapitation between 0800-1000 hr, in accordance with the ethical standards of the institutional review committee.
Treatment
In the immunoblotting experiment, groups of hamsters received an intramuscular (IM) injection of ACTH (1 IU Synacten Depot/100 g bw) at 5-hr intervals for 20 hr. These animals were sacrificed at 0, 5, 10, 15, or 24 hr after the first injection. In the experiments using immunofluorescence and immunocytochemistry, hamsters were injected IM with 1 IU ACTH (1 IU Synacten/100 g bw) at 5-hr intervals for 20 hr. These animals were then sacrificed after 24 hr. ACTH (Synacten and Synacten Depot) was obtained from Ciba Pharmaceuticals, Division of Ciba-Geigy Canada (Mississauga, Ontario, Canada). Controls were injected with 0.15 M NaCl.
Immunoblotting
Homogenates of hamster adrenals were analyzed by immunoblotting as previously described (
Immunofluorescence
Adrenal glands were excised from three different animals in each experimental group to localize P450C17 with the rabbit anti-rat adrenal P450C17 antibody. The glands were bisected and one half was fixed in buffered neutral formalin solution for 24 hr. The fixed tissues were dehydrated in graded alcohols, cleared in toluene, and embedded in paraffin. Five to seven 5-µm-thick sections were prepared according to the usual histological procedure. Sections were deparaffinized, hydrated to water, and treated with NH4Cl in 50 mM PBS (20 min) to block aldehydes. After two washes, tissue sections were incubated for 2 hr at room temperature (RT) with the rabbit anti-rat adrenal P450C17 antibody (diluted 1:100 in PBS containing 1% BSA) and then washed twice. They were next incubated for 30 min with a fluorescein-conjugated goat anti-rabbit IgG (Boehringer; Mannheim, Germany) diluted 1:50, washed in PBS for 5 min, and then mounted in glycerol-PBS (9:1) containing 0.1% phenylenediamine (
Immunocytochemistry
The other halves of the glands were cut into pieces and were fixed in ice-cold 2% paraformaldehyde-0.1% glutaraldehyde for 2 hr, and then embedded in Lowicryl K4M (
Morphometry
Electron micrographs (x 50,000 or x 65,000) of two to four different fields were taken of the ZF for three controls and three ACTH-treated hamsters. The number of colloidal gold particles was determined on mitochondria vs cytoplasm in four to six surface areas equivalent to a grid placed over each electron micrograph. These were expressed as the number of grains per µm2. The relative surfaces of mitochondria and the remaining cytoplasm were evaluated from the weight of the corresponding areas cut out from the electron micrograph.
Statistical Analysis
Results are expressed as mean ± SEM and the statistical significance of the difference of the mean was determined by Student's t-test. The level of significance was fixed at p<0.05.
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Results |
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Western Blotting Analysis
ACTH was administered to hamsters over a 20-hr period to evaluate the effect of ACTH on the concentration of P450C17 protein. Western blotting analysis of hamster adrenal preparations when the anti-rat adrenal P450C17 antibody was used revealed a unique protein band at 55 kD (Figure 1). In control animals at time 0, faint protein bands were observed, whereas the intensity of these protein bands considerably increased as the ACTH treatment was prolonged. A single ACTH injection at time 5 hr produced a faint elevation in P450C17 protein concentration, whereas sustained ACTH stimulation for 10, 15, and 24 hr produced increasing amounts of P450C17 protein in hamster adrenals, reaching a plateau after 15 hr.
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Immunolocalization
The anti-rat adrenal P450C17 antibody was used to determine the glandular distribution of P450C17. The protein was found to be preferentially localized in the ZF. With paraffin sections processed for indirect immunofluorescence (Figure 2A) the fluorescein-conjugated antibody was exclusively confined to the ZF in control animals. The zone was approximately 12-15 cells thick and irregularly stained. Some groups of cells appeared negative or faintly positive, but the majority displayed uniform green cytoplasmic staining. The fibroconnective capsule, the ZG, the ZR, and the medulla were not stained. The bright structures in or near the capsule were artifacts and did not exhibit green fluorescence.
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When hamsters were treated with ACTH, the ZF thickness appeared unchanged (Figure 2B) but the fluorescence intensity was considerably increased compared with that of control animals. The fluorescent P450C17 antibody was evenly distributed in the cell cytoplasm but was absent over nuclei and plasma membranes. Unlike the control animals, all the cells in the ZF appeared to be stained. In addition, some intensely stained cells appeared to be detached from the ZF and were dispersed among the ZR cell population. P450C17 was not detected in the ZG.
To determine the precise intracellular localization of P450C17 and to possibly identify the cytoplasmic organelles responsible for the observed fluorescence, the distribution of gold-labeled P450C17 antibody was examined by electron microscopy. In the ZG of control animals, only a few gold particles were randomly dispersed over the cytoplasm, corresponding to background or nonspecific labeling. In the ZF (Figure 3A), mitochondria were not stained, except for a few randomly dispersed grains here and there, whereas intermitochondrial spaces were positively labeled with gold particles. In immunocytochemistry, morphological integrity has to be sacrificed to preserve antigenic sites.
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The detection of P450C17 in hamsters injected with ACTH was limited to the ZF when the labeled second antibody was used (Figure 3B). Unfortunately, at the electron microscopic level it was not possible to localize and examine the isolated positive cells invading the ZR that were observed in paraffin sections. In the ZF cells, gold particles were mostly confined to cytoplasmic regions between mitochondria and, according to a semiquantitative evaluation, their number was much greater than in the ZF cells of control adrenals. All control sections incubated with the second antibody only were negative.
To obtain quantitative data to support our observations, the number of colloidal gold particles was determined on mitochondria and cytoplasmic areas of control and ACTH-treated animals. As shown in Table 1, the number of particles on mitochondria and cytoplasmic structures was comparable in control animals, but the difference became significant (p<0.012) in ACTH-treated hamsters. In fact, the number of particles in the cytoplasm almost doubled in ACTH-treated hamsters compared to that of controls, whereas it did not significantly change in the mitochondria. These results indicate that the P450C17 increase is important in the ZF intermitochondrial areas after ACTH stimulation. However, although important in absolute values, the difference in particle numbers between controls and treated animals (12.81 vs 22.53, or 76%) remained nonsignificant (p<0.056, or 94.4%) probably due to the rather elevated intragroup variability.
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As shown in Table 2, the P450C17 increase in the cytoplasm of treated animals is real and does not result from changes in relative surfaces of mitochondrial and cytoplasmic structures.
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Discussion |
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We used immunofluorescence and immunocytochemistry to examine the zonal and intracellular distribution of P450C17 in the hamster adrenal, both under normal conditions and after administration of ACTH.
We used an antiserum to a 15 amino acid peptide, which represented the carboxy terminus of the rat testicular P450C17 (
TVRQAWKDAQAEVNT: rat peptide
TVRQAWMDAQAEVST: hamster peptide
This close homology in amino acid sequence between peptides of hamster and rat presumably confers the specificity of the P450C17 antibody against the hamster adrenal P450C17. The specificity of the P450C17 antibody used was further confirmed by Western blotting analysis on different hamster tissue preparations, i.e., the gonads, adrenals, brain, and mesentery of both sexes. Only gonads and adrenals from male and female gave positive signals (
These are original results and they demonstrate that P450C17 is localized in the ZF of the hamster adrenal cortex in control animals (Figure 2). In agreement with our data, -hydroxylated steroids and androgens than that of those other animal species that also express adrenal P450C17, i.e., swine and guinea pigs.
As for the pig or the guinea pig, no cells in the hamster ZG became immunofluorescent even after treatment with ACTH. The absence of P450C17 in the ZG cells is not surprising because 17-hydroxylation or 17,20-lyase action are not required steps in the biosynthesis of aldosterone, which takes place exclusively in the ZG cells.
Le Goascogne et al. (1991) reported immunoreactive P450C17 in rat gonads but not in the adrenals, thus confirming the absence of expression of this cytochrome in this species. In the mouse,
Using immunogold electron microscopy with an anti-rat adrenal P450C17 antibody, we demonstrated that the intermitochondrial spaces in the ZF cells, filled with elements of the ER and unidentified cytoplasmic organelles, are positively stained by P450C17 (Figure 3). This intracellular localization is not surprising because adrenal 17-hydroxylase activity has been found in microsomal preparations in hamsters (
Immunoblotting analysis using the same P450C17 antibody revealed a unique protein band at 55 kD corresponding to hamster P450C17 (Figure 1). In this figure the results were obtained from whole homogenate preparations. However, when P450C17 was analyzed in microsomal preparations (after a first centrifugation at 9500 times g, the supernatant was centrifuged at 105,000 times g for 60 min and the pellet was used as microsomes) (
Hamsters differ from other rodents, such as the rat and the mouse, in that they express adrenal cytochrome P450C17 and, like humans, cortisol is their major glucocorticoid. The hamster adrenal cytochrome P450C17 cDNA was recently cloned in our laboratory and expressed in COS 1 cells, and the expressed product was shown to metabolize pregnenolone into dehydroepiandrosterone and progesterone into androstenedione, both at high rates (
In conclusion, in this study we have found that P450C17 is expressed in the hamster adrenal and that its expression is mediated by ACTH. P450C17 was located exclusively in the ZF and the intensity of P450C17 increased in the ZF after stimulation by ACTH, with some cells in the ZR also becoming positive. The increase in P450C17 observed by immunofluorescence after administration of ACTH was confirmed by immunoblotting analysis. Furthermore, P450C17 was shown by immunogold electron microscopy to be increased only in the intermitochondrial areas of treated hamsters, occupied by unidentified organelles and elements of the ER. Although this increase is not significant because of elevated intragroup variability, as also reported in another morphometric study of the adrenal gland (
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Acknowledgments |
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Supported by a grant from the Medical Research Council of Canada (MT10983) and by the Heart and Stroke Foundation of Canada.
We thank Dr D.C. Johnson, Department of Gynecology and Obstetrics, The University of Kansas Medical School, for his generous gift of the anti-rat P450C17 antibody. The help of Dr D. Shapcott, L. Ducharme, and A. Mathieu is also acknowledged.
Received for publication October 2, 1996; accepted May 15, 1997.
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