Immunohistochemical Demonstration of Adrenodoxin Reductase in Bovine and Human Adrenals1
H. Sasano and N. Sasano
Department of Pathology, Tohoku University School of Medicine, Sendai, Japan
M. Okamoto
Department of Molecular Physiology, Osaka University Medical School, Osaka, Japan
Y. Nonaka
Department of Biochemistry, Osaka University Medical School, Osaka, Japan
SUMMARY
Adrenodoxin reductase (ADR) was purified from bovine adrenocortical mitochondria and specific antibody was raised in rabbits. Immunohistochemical analysis of ADR was per- formed in the bovine and human adrenals. ADR was present in all of the zones in both bovine and non-pathological human adrenal cortex. In non-pathological human adrenals, the immunoreactivity was particularly prominent in the zona glomerulosa (ZG) and reti- cularis (ZR). Intensive immunoreactivity was observed in the ZG and some cells of the outer fasciculata and the ZR in the adrenal glands with idiopathic hyperaldosteronism. In adrenal glands with Cushing’s disease, immunoreactivity was present in the compact cells of cortical micronodules. In all cases, sites of immunoreactivity correspond to sites of increased steroidogenesis. In aldosteronoma and cortical adenoma with Cushing’s syn- drome, the immunoreactivity was generally marked in compact cells but not in large cells with clear cytoplasm, ADR was present in the ZG and the ZR, and the ZG in the non- neoplastic adrenal glands attached to aldosteronoma and Cushing’s adenoma, respecti- vely. ADR was present in the compact cells in adrenocortical carcinoma clinically mani- festing Cushing’s syndrome.
Introduction
Adrenodoxin reductase (adrenal ferredoxin: NADP+ oxidoreductase, EC.1.18.1.2) is a monomeric flavoprotein with a molecular weight of 54.0001. The enzyme is a com- ponent of the cytochrome P-450-linked monooxygenase system of the mitochondrial inner membrane type and is
essential to the electron transfer chain in steroid hydroxyl- ase of adrenocortical mitochondria2,3. Adrenodoxin reductase (ADR) catalyzes electron transport from NADPH to adrenodoxin, which subsequently reduces cytochromes P-450scc (cholesterol side-chain cleavage) and P-450118 (11ß hydroxylase)2,4. It has been purified from adrenocortical mitochondria and its physicochemical characteristics have been extensively studied1,4-7. The authors recently reported a crystallographic investigation of ADR8 and isolated full-length cDNA corresponding to bovine adrenocortical ADR9.
1 A part of this manuscript was presented at Eighth Interna- tional Congress of Histochemistry and Cytochemistry, August, 1988 in Washington D.C., USA
@ 1989 by Gustav Fischer Verlag, Stuttgart
In addition to physical and biochemical studies, it is important to know the localization of the enzymes involved in steroidogenesis in various tissues to obtain a better understanding of steroidogenesis. This is most im- portant in adrenocortical disorders with excessive gluco- and/or mineralocorticoid production. Toward this pur- pose, immunohistochemical studies of steroidogenic en- zymes employing specific antibodies are useful. The authors recently reported immunohistochemical analysis of cytochrome P-450 specific for C-21-hydroxylase in the bovine adrenals and kidneys10 as well as the human adrenal cortex and its disorders11. In order to understand the role of ADR in adrenocortical steroidogenesis, we have purified ADR, raised a specific antibody against it in rab- bits and used this in immunohistochemical studies of the bovine and human adrenals.
Material and Methods
Preparation of adrenodoxin reductase
Adrenodoxin reductase was purified from bovine adrenocorti- cal mitochondria by a modified method of Sugiyama and Yamano6. The details were already described by Nonaka et al.8. Electrophoresis of the purified ADR (0.3 ug) and the bovine adrenocortical mitochondrial protein (2.8 µg) was performed on sodium dodecyl sulfate (SDS)-polyacrylamide gel12 stained with Coomassie brilliant blue R.250.
Preparation of antisera
A rabbit was immunized with 100 µg of purified ADR emul- sified with complete Freund adjuvant. 50 µg of ADR were injected four weeks after the initial injection. One week after the reinjection, sera were obtained from an immunized rabbit and the IgG fraction was produced after ammonium sulfate precipita- tions.
Immunoblot analysis
0.3 µg of purified ADR and 2.8 ug of bovine adrenocortical mitochondrial protein were separated on SDS polyacrylamide gel. Then, the proteins on a slab gel were electrophoretically transferred to a nitrocellulose membrane. The electrotransfer was performed at 75 V for 5 hours in a buffer containing 25 mM Tris-HCI, 192 mM glycine, 20% methanol and 0.05% SDS pH 8.313. Specific antigenic proteins on the nitrocellulose mem- brane were detected by a modified enzyme-immunostaining method of Domin et al.14. The nitrocellulose membrane was incu- bated with 5 µg/ml of anti-ADR for 15 minutes at 25 ℃ and then incubated with goat antirabbit IgG peroxidase conjugate (TAGO Inc, Cal, USA), diluted at 1 : 1000 of 333 units/ml for 15 minutes at 25 ℃. The membrane was subsequently stained with 4-chloro- 1-naphthol and hydrogen peroxide. Non-specific binding on the membrane was blocked with 3% skim milk. Staining of the mem- brane by the peroxidase reaction revealed a band corresponding to the antigen recognized by the antibody. The apparent relative molecular weight of the immunochemically stained protein was estimated by comparing its mobility with those of standard pro- teins which were electrophoresed on the same gel and stained with Coomassie brilliant blue R.250.
Adrenal glands
The bovine adrenal glands were obtained immediately after the animals had been killed at a local slaughter house. All the human adrenals used in this study were obtained from surgical material. Morphologically normal adrenal glands were obtained from 3 patients who underwent bilateral adrenalectomy for advanced breast cancer and 2 patients with radical nephrectomy for renal cell carcinoma. No hormonal adrenocortical abnormalities were detected preoperatively in those patients. Surgical specimens of bilateral adrenocortical hyperplasia were obtained from 5 patients with idiopathic hyper-aldosteronism (IHA) and 6 patients with Cushing’s disease. The latter cases were adrenalec- tomized in the years before trans-sphenoidal hypophysectomy was available. Well-circumscribed adrenocortical tumors were resected from 7 patients with hyper-aldosteronism with sup- pressed plasma renin activity (aldosteronoma) as well as from 10 patients with Cushing’s syndrome. Adrenocortical carcinoma were obtained from 5 patients including 4 cases with evidence of metastasis. Clinical signs of glucocorticoid excess, i.e. Cushing’s syndrome, were present in 3 cases and those of deoxycortico- sterone excess in one case and the remaining single case did not show any clinical hormonal abnormality.
Preparation of tissues
After removing surrounding adipose and connective tissues, the glands were cut into small pieces (approximately 0.2 to 0.3 cm thickness). The bovine adrenal glands were fixed immedi- ately in-4% paraformaldehyde, or periodate-lysine-paraformal- dehyde (PLP) solution (containing 0.01 sodium periodate, 0.075 M lysine and 2% paraformaldehyde) buffered at pH 7.4 for 24 to 48 hours at 4℃, 100% methanol or 10% neutral formalin for 48 to 72 hours at 23 ℃. After fixation the specimens were embedded in paraffin, cut into 2.5 um thick sections and mounted on regular glass slides. A portion of the specimens was frozen, sectioned in a cryostat at 6 um and mounted on albumin- coated glass slides.
Human materials fixed in 10% neutral formalin solution were embedded in paraffin and sectioned 2.5 um thick mounted on regular glass slides.
Immunostaining
Sections were routinely deparaffinized and immersed in methanol with 0.3% hydrogen peroxidase for 30 minutes to block the endogenous peroxidase activity. Frozen sections were put into 0.01 M PBS pH 7.2 with 0.3% hydrogen peroxide for 10 minutes. They were washed in three changes of 0.01 M PBS for five minutes each and treated with 1% normal goat serum for 30 minutes at room temperature. After washing, the sections were incubated with primary antibody, 1:50 to 1:500 dilution, diluted with 0.01 M PBS containing 0.5% bovine serum albumin (BSA) for 18 hours at 4℃ in a humidified chamber.
Biotin-StreptAvidin (B-SA) amplified method (StrAviGen B- SA, Biogenic Laboratories, Dublin, CA, USA) was employed for immunostaining in this study. After washing in 0.01 M PBS, the sections were incubated with biotinylated anti-rabbit immuno- globulin and peroxidase conjugated streptavidin. Each was incu- bated for 30 minutes at room temperature in a humidified chamber with washing in 0.01 M PBS between incubations. A final wash was followed by immersion of the reacted sections for 5 to 10 minutes in a solution containing 0.66 mM 3.3’- diaminobenzidine and 2 mM hydrogen peroxide in 0.05% Tris- HCI buffered at pH 7.6. Specific staining was identified by the presence of brown reaction products. The sections were finally
counterstained with 1% methyl green and mounted with a gly- cerol-gelatin water soluble medium. To establish the specificities of immunohistochemical staining with anti-ADR, control sec- tions were incubated with anti-sera preincubated with 5 to 100 µM of purified ADR for 18 hours at 4℃, normal rabbit IgG and 0.01 M PBS containing 0.5% BSA instead of primary anti- body.
Results
Characterization of anti-ADR
As shown in Fig. 1, purified ADR formed a single band on SDS-PAGE. The anti-ADR produced recognized the purified adrenocortical ADR and a protein in bovine adrenocortical mitochondria which had the same molecu- lar weight as ADR (Mr. 54.000).
A
B
C
D
O
G
8
F
C
R
M
b
Bovine adrenal glands
The immunoreactivity to ADR was present in adrenocortical parenchymal cells in all the layers but not in the capsule, adrenal medulla or sinusoidal cells (Fig. 2). The distribution of immunoreactivity was not homogene- ous and intrazonal variance was observed particularly in the zona fasciculata (ZF), while no significant interzonal differences were present. No significant difference in localization of immunoreactivity were detected among different fixatives or between paraffin-embedded and fro- zen sections. No immunoreactivity was observed in adrenocortical parenchymal cells in the control sections.
Human adrenal glands
Non-pathological adrenal glands
ADR was present in adrenocortical parenchymal cells. Relatively more intensive immunoreactivity was observed in some cortical cells in the zona glomerulosa (ZG) and reticularis (ZR) than in the bovine adrenals (Fig. 3). No immunoreactivity was present in adrenocortical parenchy- mal cells in the control sections.
IHA
Marked immunoreactivity was observed in adrenocorti- cal cells in the hyperplastic ZG and outer fasciculata
(Fig. 4). In three cases, the intensity of immunoreactivity in the zona glomerulosa was much stronger than that of the zona fasciculata and reticularis in the adrenal cortex.
Cushing’s disease
Marked immunoreactivity was present in cells of the cortical micronodules (Fig. 5), less so in the hyperplastic ZF and reticularis. The immunoreactivity in cells of the zona glomerulose was faint.
Aldosteronoma
In tumors, well-stained cells were generally small, com- pact and contained little lipid in the cytoplasm (Fig. 6).
M
C
G
Large tumor cells with abundant clear cytoplasm showed faint immunoreactivity. In the adjacent non-neoplastic adrenal tissue, ADR was present in cortical cells, particu- larly in the ZG and ZR.
Cushing’s adenoma
In tumors, the immunoreactivity was intense in small and compact cells forming nests or cords (Fig. 7) as well as in tumor cells around myelolipomatous lesions. In the
adjacent non-neoplastic adrenals, ADR was exclusively present in ZG in all the cases examined and faintly in ZF and ZR.
Adrenocortical carcinoma
Distinctive immunoreactivity was observed in three cases of adrenocortical carcinoma producing excessive glucocorticoid, most marked in one case with small and compact cells forming the nest (Fig. 8). Large carcinoma cells with abundant clear cytoplasm showed a faint immunoreactivity. Only a small number of carcinoma cells was positive in the cases with excessive deoxycortico- sterone production or with no endocrine abnormalities.
Discussion
The cytochrome P-450-linked mixed function oxidase system of adrenocortical inner mitochondrial membranes is involved in the side-chain cleavage reaction of choles- terol and the 11ß-, 18-, and 19-hydroxylation of steroid hormones. The system comprises three components: NADPH-adrenodoxin reductase, adrenodoxin and cyto- chrome P-450. Side-chain cleavage of cholesterol or the conversion of cholesterol to pregnenolone is the first and rate-limiting step of corticosteroidogenesis. This is cataly- zed by one mitochondrial cytochrome P-450 (P-450scc)15. 11ß-, 18- and 19-hydroxylation, which is required in mineralo- and glucocorticoid synthesis, is catalyzed by another mitochondrial cytochrome P-450 (P-450118)16, 17. Both of these reactions require NADPH-adrenodoxin reductase as the mediator of electron transfer2,4. Thus, ADR is biochemically closely associated with adrenodox- in, P-450scc and P-450118.
In order to obtain a better understanding of adrenocor- tical steroidogenesis, it is important to know the distribu- tion and localization of steroidogenic enzymes in the adrenal cortex, particularly in cases of adrenocortical hor- monal abnormalities. Immunohistochemical studies of ADR have been reported in the bovine18 and rat19 adrenal glands but not in the normal human adrenal and in cases of hypercorticism. In our current investigation, there were no significant intracortical differences in immunoreactivity of ADR in the bovine adrenal glands, regardless of the methods of fixation and preparation of the specimen. Some of the cortical cells in the ZG and the ZR of the normal human adrenal glands exhibited intense immunoreactivity. In previous reports of the bovine18 and rat19 adrenal glands, ADR was demonstrated mostly in the ZF and the ZR, while immunoreactivity in the ZG was faint. However, in an earlier study, no intracortical differ- ences of the immunoreactivity to adrenodoxin had been reported in the bovine adrenal cortex20. In a more recent study of the bovine adrenal cortex, the specific content of cytochrome P-450 in the mitochondria has been reported to be higher in the ZF and the ZR than in the ZG21. We have recently demonstrated that the immunoreactivity of P-450scc22 and P-45011823 was less intense in the ZG than in the ZF and the ZR. These findings may suggest that
there are discrepancies in the intracortical distribution of each component of the adrenocortical mitochondrial elec- tron transfer system involved in corticosteroidogenesis, but it awaits further investigations to clarify this point.
In hyperfunctioning adrenocortical hyperplasia, the immunoreactivity was relatively intense in the cells with morphological features of hypercorticism, i.e. zona glomerulosa cells, some of outer fasciculata cells in IHA, and cells of cortical micronodules in Cushing’s disease. In adrenocortical adenoma, the immunoreactivity in small and compact cells was generally more intense than that in large clear tumor cells, whether they produce gluco- or mineralocorticoids. This is consistent with biochemical studies24, morphological studies24, 25, immunohistochemi- cal distribution of cytochromes P-450c2111, P-450scc22 and P-45011823, and the results of lectin histochemistry of adrenocortical adenomas with excessive production of mineralo- and glucocorticoids26. The distinctive presence of ADR in adrenocortical carcinoma manifesting Cush- ing’s syndrome may be consistent with the ultrastructural observations that most of the characteristics of normal adrenocortical cells including mitochondria were retained to varying degrees24,25. The exclusive presence of ADR in zona glomerulosa cells of the non-neoplastic adrenal glands attached to Cushing’s adenoma, that of P-450C2111, P-450scc22, P-450118 as well as ultrastructural observa- tions27 corroborates with the persistence of aldosterone synthesis in the ZG. There was atrophy of the ZF and the ZR because of suppressed ACTH secretion consequent upon excessive glucocorticoids produced by the tumor. The immunoreactivity of ADR in the non-neoplastic adrenal tissue adjacent to aldosteronoma suggests cor- ticosteroid production.
The immunohistochemical localization of steroidogenic enzymes imparts important information about the patho- physiology of adrenocortical steroidogenesis.
Acknowledgements
The authors appreciate Dr. Dermot Hughes, M. B., B. C. H. Department of Pathology, Belfast City Hospital, Northern Ire- land, United Kingdom for critical comments of the manuscript.
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Received August 30, 1988 . Accepted November 17, 1988
Key words: Adrenodoxin reductase - Adrenal cortex - Adrenocortical disorders - Steroidogenesis - Cushing’s syndrome
Prof. em. Nobuaki Sasano, Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryomachi, Sendai 980, Japan