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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0017
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 7 4357-4361
Copyright © 2005 by The Endocrine Society

Effects of Aging on Cytochrome B5 Expression in the Human Adrenal Gland

Sejal Dharia, Audry Slane, Ming Jian, Michael Conner, Alan J. Conley, Robert M. Brissie and C. Richard Parker, Jr.

Departments of Obstetrics and Gynecology (S.D., A.S., M.J., C.R.P.) and Pathology (M.C., R.M.B.), University of Alabama at Birmingham, Birmingham, Alabama 35249; and Department of Population Health and Reproduction (A.J.C.), University of California Davis, Davis, California 95616

Address all correspondence and requests for reprints to: C. Richard Parker, Jr., Ph.D., Professor, Department of Obstetrics and Gynecology, University of Alabama at Birmingham, 618 South 20th Street, 360 Old Hillman Building, Birmingham, Alabama 35294-7333. E-mail: crparker{at}uab.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Aging in humans is characterized by a selective decline in circulating levels of adrenal androgens. The results of in vivo studies are suggestive of reduced adrenal 17,20-lyase activity in aging men and women.

Objective: We sought to determine whether there are changes in the distribution and/or expression of cytochrome B5 (CytB5), an accessory protein important in the regulation of 17,20-lyase activity, in the adrenals of aging humans.

Design: Comparison between younger and older adrenal glands.

Setting: The study was conducted in a University Center.

Patients or Other Participants: Adrenal glands obtained at autopsy after sudden death as a result of trauma from 46 young (age 20–40 yr) and 26 older (age 50–91 yr) humans were obtained and fixed within 24 h postmortem.

Interventions: Paraffin sections were stained with hematoxylin and eosin and also were subjected to immunohistochemical staining for CytB5. All sections were quantitatively evaluated using an image capture and analysis program and qualitatively evaluated with respect to staining intensity.

Main Outcome Measures: To determine whether there are any changes in CytB5 distribution in the adult human adrenal cortex during the aging process using qualitative and quantitative analysis with respect to age, gender, race, and postmortem interval.

Results: CytB5 immunoreactivity was found in cells that corresponded to those of the zona reticularis. The percentage of the adrenal cortex immunoreactive for CytB5 decreased with aging (38.6 ± 7.6% for young and 30.1 ± 5.9% for older, mean ± SD; P < 0.0001) as did the percentage of adrenocortical tissue comprising the zona reticularis (36.8 ± 10.8% for young and 27.2 ± 5.9% for older; P < 0.001). However, there was no apparent change in the staining intensity of CytB5 among those cells that were immunopositive for this factor with aging.

Conclusions: There appears to be a reduction in the proportion of the adrenal cortex that expresses CytB5 with aging, and this likely corresponds to a shrinkage of the zona reticularis. The mechanism and cause for this cortical regression are unknown.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ONE CHARACTERISTIC OF the aging process in humans is the dramatic decline in circulating levels of adrenal androgens, especially dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) (1, 2, 3, 4, 5). Peak levels of DHEA/DHEAS occur in the third decade of life and then decline gradually thereafter (2, 5). The age-related changes in DHEA/DHEAS are not paralleled by changes in basal or ACTH-stimulated levels of cortisol (5, 6), suggesting that the effect of aging on production of DHEA/DHEAS is unique. Reductions in adrenal androgen production likely are clinically relevant because androgens have been shown to have a role in maintenance in cognitive ability (7), bone mineral density (8, 9), and libido (10).

The production of DHEA/DHEAS is determined in part by cytochrome P450c17 (P450c17), an enzyme that catalyzes both 17-hydroxylase and 17,20-lyase activities (11). The hydroxylase reaction is important in the formation of glucocorticoids and of intermediates in androgen synthesis. On the other hand, 17,20-lyase activity is required for androgen production (12). There are many situations in which the dual activities of P450c17 appear to be dissociated, such as in trauma, aging, adrenal insufficiency, and puberty (13, 14, 15, 16, 17). Further evidence for selective regulation of 17,20-lyase activity is provided by studies on mutations of the P450c17 gene (18), located on chromosome 10q24.3 (19). These mutations are characterized by a clinical spectrum ranging from ambiguous genitalia to delayed puberty with normal glucocorticoid production (12).

The modulation of 17,20-lyase activity has been linked to many factors: P450 oxidoreductase, phosphorylation of serine and threonine residues on P450c17, and an accessory protein, cytochrome B5 (CytB5). P450 oxidoreductase, which acts through a transfer of electrons, facilitates both 17,20 lyase and 17-hydroxylase (20, 21). A second mechanism that may modify 17,20-lyase activity occurs through phosphorylation of serine and threonine residues of P450c17; however, the precise mechanism by which phosphorylation selectively promotes 17,20-lyase selectively has not been definitively identified (22). Additionally, 17,20-lyase activity can be modulated by CytB5, an accessory protein that alters the conformation of P450c17 and enhances 17,20-lyase independently of 17-hydroxylase activity, which subsequently increases adrenal androgen production (21, 23, 24). We have previously reported that CytB5 and P450c17 are colocalized in many cell types of the human known to synthesize androgens, i.e. theca interna cells, theca lutein cells, testicular Leydig cells, the fetal zone of the fetal adrenal, and the zona reticularis of the adult adrenal (25). In addition, other authors have found CytB5 to be localized to the zona reticularis of the human adrenal cortex (26, 27).

The production of DHEA/DHEAS occurs principally in cortical cells of the adrenal zona reticularis; it is unlikely that the zona fasciculata produces a significant amount of these B5, C19 steroids because of the presence of 3ß-hydroxy- steroid dehydrogenase (28). Using an image analysis system, we have previously found in men that aging is associated with a decrease in the width of the zona reticularis but no change in the overall width of the adrenal cortex (29). This implies that the aging adrenal cortex does not undergo global cortical atrophy but a selective alteration in the zona reticularis with aging. Whether such changes in the histological characteristics of the adrenal cortex with aging are reflective of an alteration in the androgen biosynthetic pathway is unclear.

Therefore, we sought to determine whether there are any changes in CytB5 distribution in the adult human adrenal cortex during the aging process to further elucidate the mechanism of decline in adrenal androgen production with aging. We found that the percentage of the adrenal cortex composed of cells immunoreactive for CytB5 decreased with aging, as did the percentage of adrenocortical tissue identifiable as zona reticularis. These findings are suggestive of a selective loss of cells (zona reticularis) capable of androgen biosynthesis in the adrenal cortex during aging.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human adult adrenal glands were obtained at autopsy from 39 men and 33 women who suffered a sudden, traumatic death. Of these tissue specimens, 20 were obtained from young women (age 21–40 yr), 13 from older women (age 50–91 yr), 26 from young men (age 21–40 yr), and 13 from older men (age 54–90 yr). All tissues were fixed in buffered formalin within 2–24 h after death and embedded in paraffin. Each tissue sample chosen for analysis contained either a full-thickness cross-section of the cortex extending from the adrenal capsule to the medulla and/or a full-thickness section of the adrenal alae. The Institutional Review Board of the University of Alabama at Birmingham approved these studies.

Immunohistochemistry

Thin sections (5 µm) from each adrenocortical sample were processed for routine hematoxylin and eosin (H&E) staining and immunohistochemistry for CytB5. Immunohistochemical staining was performed by use of a rabbit polyclonal antiserum (diluted 1/25,000) directed against human CytB5. The CytB5 antiserum was developed by Dr. Conley against purified recombinant human CytB5, which was kindly provided by Ron Estabrook and Manju Shet (University of Texas Southwestern Medical Center, Dallas TX). When used for Western immunoblots of human ovarian extracts, the CytB5 antiserum cross-reacts with a single protein band that has the same molecular size (16 kDa) as that of recombinant human CytB5. By use of this antiserum, we previously have found it to immunostain cells in the human known to synthesize androgens and that also are immunopositive for P450c17 (ovarian theca interna cells, ovarian theca lutein cells, testicular Leydig cells, fetal zone cells of the fetal adrenal, and cells of the adult zona reticularis) (25). Immunohistochemistry was conducted as follows. After deparaffinization and hydration, the endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Then, sections were rinsed in PBS and incubated in 5% goat serum for 30 min at 25 C. The sections were drained and incubated for 1 h at 25 C with the antihuman CytB5 antiserum. Sections were then incubated with antirabbit biotinylated second antibody followed by treatment with streptavidin-peroxidase (Biogenix, San Ramon, CA). Immunoreactivity was detected by incubation of the tissue sections in 3',3'-diaminobenzidine as the chromagen (Biogenix). Hematoxylin was used as a nuclear counterstain. The observed immunostaining patterns were described according to their correspondence to cortical zones. To evaluate the possible presence of nonspecific staining, we incubated tissue sections with nonimmune serum rather than the usual primary antisera (negative controls) and then completed the immunostaining procedure as outlined above; there was no immunostaining evident in such specimens.

Qualitative and quantitative image analyses

Quantitative analyses of histological and immunohistochemical staining patterns of the adrenal specimens were conducted with the Image-Pro Plus computerized image capture and analysis system (Image-Pro Plus 3.0; Media Cybernetics, L.P., Silver Spring, MD). All H&E-stained slides and CytB5 slides were examined at x4 magnification, and representative areas in which the full thickness of cortex extending from the capsule to the medulla or an alae with the full thickness of the cortex extending from the capsule to the capsule without intervening medulla were captured through the use of a digital color camera mounted to our Nikon microscope. To avoid any bias, all measurements were conducted on coded sections in which the age and sex of the source were not apparent. The images of the H&E-stained sections were analyzed by measuring the width of the zona reticularis, recognized by standard histological features, and the width of the total cortex. For the immunoreactive distribution of CytB5, the width of the cortex positive for CytB5 was measured. Three separate areas of each adrenal section were quantitatively analyzed in the aforementioned manner, and the average values were used. The data for the width of the zona reticularis and for the cortex immunopositive for CytB5 were normalized by expressing the width of each as a percentage of the total cortical width. Because the original tissue specimens had not been cut in a uniform manner, no attempt was made to compare absolute cortical dimensions between age, sex, or racial categories.

Qualitative analyses of immunostaining results were performed by enlisting three independent observers to grade the intensity of immunostaining for CytB5 on a scale of 0 (no staining), +1 (light staining), +2 (moderate staining), or +3 (dark staining) for the entire slide simultaneously; all observers were blinded as to the sex or age of the tissue source.

Statistical analyses used {chi}2, Fisher exact testing, and regression analysis using the JMP Statistical Program (JMP Software for MacIntosh; SAS Institute Inc., Cary, NC).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The cortical area that was immunopositive for CytB5 corresponded well to the area histologically identifiable as the zona reticularis as shown in Fig. 1Go, A and C. In many younger subjects, the CytB5 staining consistently encompassed the entire zona reticularis as shown for such adrenals in Figs. 1Go, A and C, and 2A. In H&E-stained sections of adrenals from young adults, the zona reticularis consisted of cells that were prominently eosinophilic and was usually found to have clearly distinguishable and smooth borders with the medulla and the adjacent zona fasciculata. However, in adrenals from older subjects, cells that were immunopositive for CytB5 and that appeared to represent zona reticularis cells on H&E-stained sections occasionally were found in scattered clusters rather than as a continuous zone or band of cells, such as shown for an extreme case of zonal irregularity seen in the adrenal of an elderly adult (Fig. 2BGo). There was evidence for blurring of the border between the zona reticularis (CytB5-containing cells) and the zona fasciculata (which we find to be negative for CytB5) in aging. Our findings of irregularities in histological zonation of the adrenal in aging are consistent with the results of previous studies (30).



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  		FIG. 1. Immunolocalization of CytB5 and demonstration of adrenocortical zonation in the adrenal of a young and elderly adult. The pattern of immunohistochemical staining for CytB5 and histology of an adrenal from a young adult female (age 24 yr) (A and C) and an adrenal from an elderly adult male (age 66 yr) (B and D) are shown. Tissues were processed as described in Materials and Methods, and slides were photographed at x10 magnification. The percentage of the cortex composed of CytB5-positive cells was 45% for the young adult and 30% for the elderly adult.

 


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  		FIG. 2. Qualitative differences in the immunolocalization of CytB5 in the adrenal of a young and elderly adult. The pattern of staining for CytB5 from a young adult male (age 34 yr) (A) and an adrenal from an elderly adult male (age 74 yr) (B) are shown. Tissues were processed as described in Materials and Methods, and slides were photographed at x4 magnification. In the adrenal of the young adult there is uniformity in the border of the cortical zone immunopositive for CytB5. In contrast, in the adrenal from the older adult, which represents an extreme case, CytB5 expression was diffuse, and there was evidence for significantly irregular zonal borders.

 
Among our study population, there were no significant differences in the mean age as a function of gender in older subjects (females, 74.7 yr; males, 70 yr) or younger subjects (females, 28.5 yr; males, 28 yr). In addition, there were no differences in the interval between death and fixation of the tissue at autopsy (postmortem interval) or race as a function of age group or gender (Table 1Go).


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  		TABLE 1. Main outcome measures as a function of age and gender

 
The percentage of the cortex composed of cells (mean ± SD) identifiable as zona reticularis decreased significantly with aging (36.8 ± 10.8% for young and 27.2 ± 10.9% for older; P < 0.001). In addition, the percentage of the total cortex made up by immunoreactive CytB5 cells diminished significantly with aging (38.6 ± 7.6% for young and 30.1 ± 5.9% for older; P < 0.0001) (Fig. 3Go). This decrease in immunoreactive CytB5 distribution during aging was independent of gender; similar changes were noted to occur in both men (36.3 ± 7.8% for young and 28.9 ± 5.0% for older; P < 0.005) and women (41.6 ± 6.4% for young and 30.1 ± 5.9% for older; P < 0.0001) as shown in Fig. 4Go. The percentage of the cortex composed of the zona reticularis and made up by immunoreactive CytB5 cells also was not different as a function of race in either age group. In addition, no correlation was found between the percentage of the cortex either composed of the zona reticularis or immunopositive for CytB5 and postmortem interval.



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  		FIG. 3. Changes in the percentage of the cortex composed of CytB5-positive cells and cells identifiable as zona reticularis with aging.

 


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  		FIG. 4. Differences during aging in the percentage of the cortex composed of CytB5-positive cells as a function of gender.

 
With respect to qualitative aspects of CytB5 immunostaining, we found no difference in CytB5 intensity by age group (2.2 ± 0.70 arbitrary units for young and 2.2 ± 0.64 arbitrary units for older, mean ± SD), gender, or race. There also was no correlation between staining intensity and postmortem interval.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Reductions in adrenal androgen production during aging are clinically relevant because of age-associated changes in many physiological systems that are regulated to varying degrees by androgens. Adrenal androgens may affect the immune system (31) and cognitive ability (7). Also, there may be a role of DHEAS/DHEA in age-related physiological and pathophysiological declines in bone mass density (8) and libido (10).

In a preliminary study of adrenals from men, we found evidence for relative reductions in the percentage of adrenal cortex that consisted of the zona reticularis during aging (29). The selective reduction in the zona reticularis as a percentage of the total cortex contrasted with the findings of another group, who found no correlation between the width of the zona reticularis and aging (32). However, they subjectively evaluated the overall prominence of the zona reticularis but did not use an image analysis system to quantify changes, and therefore, their findings are not directly comparable with our data.

Zona reticularis cells of human and nonhuman primates have high levels of CytB5 and DHEA sulfotransferase (DST) (25, 26, 27, 33, 34, 35). Previously, we found a decrease in the distribution of cells immunoreactive for DST with aging (36). Consistent with conclusions reached previously, our findings in the current study of a decrease in the relative width of the cortex immunopositive for CytB5 with aging parallels the changes in DST with aging. These findings are suggestive of a decline in the mass of the zona reticularis in aging, which would impair DHEA/DHEAS synthesis relative to young adults.

One potential limitation of our study is attributable to the type of tissue we obtained for analysis. We used autopsy specimens from subjects who died of sudden trauma that were collected and fixed within 2–24 h postmortem. Although not expected, there could be cellular changes in adrenal histology or enzyme levels that occur at or after death that are not reflective of the in vivo situation. Although this is possible, we are reassured by the work of Didenko et al. (37) that suggests that the pattern of markers of DNA damage (p21, P53, and DNA fragmentation) found in postoperative or postmortem tissues is the result of the underlying pathophysiological process and do not appear to be the result of artifactual changes. Additionally, our data on the localization of CytB5 in autopsy cases from trauma victims is consistent with the localization of CytB5 in the zona reticularis noted by Yanase et al. (38) in surgical adrenal specimens obtained at time of nephrectomy. This supports the view that there are no functional immunohistochemical differences in adrenal tissues obtained within the first 24 h after death compared with those from surgical extirpation.

In this study, we detected an age-dependent reduction in the percentage of the adrenal gland immunoreactive for CytB5 that parallels a reduction in the width of the zona reticularis. We believe that the reduction in the apparent mass of the zona reticularis and the accompanying reduction in the distribution of this important factor in androgen synthesis play a role in the reduced adrenal secretion of DHEA and DHEAS in aging (1, 2, 3, 4, 5, 6). Because, however, the extent of cortical change (~25%) noted in this study during aging is somewhat less than the reduction noted for adrenal androgen production in aging (ranging from 33–75%), involution of the zona reticularis may not be the only factor responsible for the reduction in adrenal capacity. Nevertheless, we propose that these changes are likely to contribute to the etiology of diminished adrenal production of DHEA and DHEAS during aging. Additional investigation into the mechanism for selective loss in aging of cells having the histological and enzymatic properties characteristic of zona reticularis cells is warranted.


   Footnotes
 
These studies were supported from grants from the Office of Naval Research, the National Institute on Aging, and American College of Obstetricians and Gynecologists/Ortho-McNeil.

First Published Online May 3, 2005

Abbreviations: CytB5, Cytochrome B5; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; DST, DHEA sulfotransferase; H&E, hematoxylin and eosin; P450c17, cytochrome P450c17.

Received January 5, 2005.

Accepted April 27, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Parker LN, Odell WD 1978 Decline of adrenal androgen production as measured by radioimmunoassay of urinary unconjugated dehydroepiandrosterone. J Clin Endocrinol Metab 47:600–602[Abstract]
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  25. Dharia SP, Slane A, Jian M, Conner M, Conley AJ, Parker Jr CR 2004 Co-localization of P450c17 and cytochrome b5 in androgen synthesizing tissues of the human. Biol Reprod 71:83–88[Abstract/Free Full Text]
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