ARTICLE

Immunolocalization and Biochemical Determination of Cytochrome P450C17 in Adrenals of Hamsters Treated with ACTH

Correspondence to: Jean-Guy LeHoux, Dept. of Biochemistry, Faculty of Medicine, U. of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4.

	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

	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 (Giroud et al. 1956 ) and is primarily regulated by the renin-angiotensin system. The thickest layer of the cortex, the ZF, is composed of irregular large cuboidal or polyhedral cells arranged in long radial cords usually one or two cells wide. The cells contain abundant smooth endoplasmic reticulum (ER) and many mitochondria. Both the ZF and the ZR are under the control of ACTH. The ZF is mainly responsible for the synthesis of glucocorticoids, whereas the ZR is responsible for the biosynthesis of androgens (Sandor et al. 1976 ).

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 (Shinzawa et al. 1988 ; Le Goascogne et al. 1991 ; Colby et al. 1993 ), rat (Le Goascogne et al. 1991 ), pig (Sasano et al. 1989 ), Rhesus monkey (Mesiano et al. 1993 ), and the human fetus (Mesiano et al. 1993 ; Breault et al.. 1996 ). The results obtained in the guinea pig and the pig demonstrated that P450C17 was present only in the zonae fasciculatae (ZF) and reticularis, with intense immunoreactivity in cells of the outer fasciculata zone and decreasing intensity towards the reticularis cells (Shinzawa et al. 1988 ; Le Goascogne et al. 1991 ; Colby et al. 1993 ). In the adrenal cortex of the rat, however, no immunoreactivity could be detected, which is in accordance with the fact that their adrenal glands do not express P450C17 (Van Weerden et al. 1992 ). In the fetal human (Mesiano et al. 1993 ; Breault et al. 1996 ) and Rhesus monkey adrenals (Mesiano et al. 1993 ), staining for P450C17 was detected only in transitional and fetal zone cells.

Hamsters differ from other rodents in that they express adrenal P450C17 (Cloutier et al. 1997 ) and produce cortisol as their major corticosteroid (Schindler and Knigge 1959 ; Whitehouse and Vinson 1971 ; LeHoux and Lefebvre 1980 ; LeHoux et al. 1992 ). Studies of P450C17 have demonstrated that it is mainly regulated by ACTH in a cAMP-dependent manner (Zuber et al. 1986 ; DiBlasio et al. 1987). Moreover, the levels of hamster adrenal P450C17 mRNA, protein and 17-hydroxylase activity were shown to be increased after administration of ACTH (LeHoux et al. 1992 ).

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.

	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 (LeHoux et al. 1992 ), using a rabbit polyclonal anti-rat P450C17 antibody (kindly provided by Dr. D.C. Johnson, University of Kansas Medical Center). Two protein concentrations (6 and 10 µg) were used to ensure linearity of assays. [¹²⁵I]-Protein A (Dupont Canada; Mississauga, Ontario, Canada) and autoradiography were used to analyze antibody-antigen complexes.

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 (Calvert et al. 1994 ). The tissue sections were studied with a Reichert Polyvar 2 microscope equipped for epifluorescence.

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 (Katsumoto et al. 1993 ). Three or four thin sections from each gland were deposited on two or three coated grids and were treated for 15 min with PBS-BSA containing 0.5% fish gelatin. The sections were then incubated for 2 hr at RT with the anti-rat adrenal P450C17 antibody diluted 1:100 and washed three times in PBS and PBS-BSA. They were next incubated for 30 min with 1:20 gold-conjugated (Stirling 1990 ) goat anti-rabbit IgG (10-nm gold particles; Cedarlane Laboratories, Hornby, Ontario, Canada), washed in bidistilled water, stained with uranyl acetate and lead citrate, and examined in a JEOL JEM-100 CX electron microsope.

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.

	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.

View larger version (67K): [in this window] [in a new window]   Figure 1. A time study of the effects of ACTH on the level of hamster adrenal P450C17 protein. Animals were sacrificed as indicated after the initial injection. Controls were injected with 0.15 M NaCl. A single band of P450C17 protein is observed in the 55-kD area. The intensity of this protein band increases after ACTH administration. STD, standard.

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.

View larger version (148K): [in this window] [in a new window]   Figure 2. Indirect immunofluorescence micrograph of paraffin sections of hamster adrenals at (A) 0 and (B) 24 hr after ACTH stimulation. In control animals (A), the specific fluorescein-conjugated antibody detected P450C17 in the zona fasciculata (ZF) only. After ACTH treatment (B), the intensity of the fluorescent signal was increased in the ZF. Some highly fluorescent cells are observed in the zona reticularis (ZR). ZG, zona glomerulosa. Bars = 100 µm.

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.

View larger version (135K): [in this window] [in a new window]   Figure 3. Colloidal gold electron microscopic studies of thin adrenal sections. (A) In control hamsters, cytochrome P450C17 was detected in intermitochondrial areas of ZF cells. (B) After ACTH treatment, the number of gold particles appeared to increase in the intermitochondrial spaces occupied by unidentified organelles and elements of the endoplasmic reticulum (arrow). Bars = 0.1 µm.

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.

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.

	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 (Johnson 1992 ) and which had been used to study the immunolocalization of P450C17 in the immature ovaries of hypophysectomized rats (Johnson and Crane 1995 ). This antiserum was also shown to be specific for the hamster P450C17, a result that is not surprising since 13/15 amino acid residues of the carboxy terminus of the hamster cytochrome P450C17 are identical to that of the rat enzyme:

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 (Cloutier et al. 1997 ) due to the presence of P450C17, thus confirming that the anti-rat P450C17 was also suitable to analyze P450C17 from hamster tissues.

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, Suzuki et al. 1992 obtained hybridization signals in the ZF cells of swine adrenals by immunocytochemistry, employing an anti-P450C17, although such signals were also found in the ZR. The same authors also found co-localization of P450C17 protein and mRNA in the same tissue section. When bovine cell cultures prepared from adrenal zona fasciculata-reticularis were analyzed for the presence of P450C17 by immunofluorescence and hybridization in situ, those cells that were positive for immunofluorescence were also positive for hybridization (Ryan et al. 1989 ), indicating that the P450C17 mRNA was effectively translated into protein. A decrease in immunofluorescent staining from the ZF to the ZR for P450C17 was reported for the guinea pig (Shinzawa et al. 1988 ) and pig (Sasano et al. 1989 ) adrenal cortices. In contrast, as mentioned above for the hamster, ZR cells were unstained in control animals. However, after injection of hamsters with ACTH the ZF cells became more intensely immunofluorescent, and some positive cells were also present in the ZR (Figure 2B). The presence of a few positively stained cells in the hamster ZR only after ACTH stimulation suggests that the hamster ZR is less active in synthesizing 17-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, Keeney et al. 1995 reported clusters of fetal adrenal cells that hybridize specifically with murine P450C17 cRNA. However, P450C17 was not expressed in adult mouse adrenal glands. This suggests that the high ratios of cortisol to corticosterone reflect a functionally immature adrenal cortex in the fetal adrenal glands of the mouse and rabbit, which also secrete cortisol (Hirose 1977 ),

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 (LeHoux et al. 1992 ).

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) (Kramer et al. 1979 ), a single band of immunoreactive protein was also observed at the same location as for homogenates (results not shown), thus confirming that P450C17 was located in the microsomal fraction. The apparent molecular weight of the hamster adrenal preparations is in agreement with results reported by Chouinard and Fevold 1990 , who determined that the molecular weight of rabbit and guinea pig adrenal P450C17 corresponded to a single protein band at 52 kD. The observed increase in P450C17 protein concentration after ACTH stimulation correlates with an increase in enzyme activity, as shown in our previous study (LeHoux et al. 1992 ). In the latter work, using a different antibody, the reported molecular weight of P450C17 was 51 kD. A similar increase in P450C17 protein concentration after ACTH stimulation was reported for the rabbit by Chouinard and Fevold 1990 .

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 (Cloutier et al. 1997 ). These data and those of the present study show that the hamster is a potentially useful animal model for the study of adrenal steroidogenesis in relation to human biochemistry and physiology.

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 (Rocco et al. 1994 ), the increase was nevertheless rather important (76%). These quantitative data support the immunofluorescence observations.

	Acknowledgments

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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