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Endocrinology Vol. 143, No. 4 1451-1458
Copyright © 2002 by The Endocrine Society

CRH-ACTH-POMC-ADRENAL

Adrenocortical Cytochrome b5 Expression during Fetal Development of the Rhesus Macaque

Samantha Mapes, Alice F. Tarantal, C. Richard Parker, Francisco M. Moran, Janice M. Bahr, Leah Pyter and Alan J. Conley

Department of Population Health and Reproduction (S.M., F.M.M., A.J.C.), California Regional Primate Research Center (A.F.T.), Department of Pediatrics (A.F.T.), University of California, Davis, California 95616; Department of Obstetrics and Gynecology University of Alabama (C.R.P.), Birmingham, Alabama 35233; and Department of Animal Sciences, University of Illinois (J.M.B., L.P.), Urbana, Illinois 61801

Address all correspondence and requests for reprints to: Dr. A. J. Conley, Department of Population Health and Reproduction, University of California School of Veterinary Medicine, Davis, California 95616. E-mail: . ajconley{at}ucdavis.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The developmental expressions of cytochrome b5 (b5), 17{alpha}-hydroxylase/17,20-lyase cytochrome P450 (P450c17), and 3ß-hydroxysteroid dehydrogenase were examined in primate fetal adrenals by immunocytochemistry from 50–160 d gestation. The expression of b5 was evident at 50 d in the developing fetal zone (FZ), but decreased markedly through midgestation, then increased again from 150 d to term. Similar changes in the temporal expression was observed for P450c17. Whereas P450c17 was induced largely in the transitional zone (TZ; outer-most FZ), b5 expression was strongest in FZ cells further from the capsule, although overlap between these regions involved a narrow band of cells beneath the TZ that may represent the developing zona reticularis. Thus, the induction of b5 in the FZ and of P450c17 in the TZ of the fetal adrenal late in gestation coincided temporally with the prepartum rise in dehydroepiandrosterone previously reported. These data are consistent with the proposed role of b5 in supporting 17,20-lyase activity of P450c17. However, the lack of cytochrome b5 and P450c17 expression in the FZ of the developing macaque adrenal cortex for much of the second and third trimesters distinguishes it from the mature zona reticularis seen in adult animals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GROWTH AND differentiation of the adrenal cortex of the human and nonhuman primate fetus are unusual in possessing a very prominent fetal zone (FZ), a morphologically distinct region of large, spongiform cells in the cortex (1, 2). This is surrounded by a narrower band of smaller, more densely packed cells known as the definitive zone (DZ). However, another cell population has been described lying between the DZ and FZ that is especially recognizable in late gestation and the early neonatal period (3), a region that has been referred to as the transitional zone (TZ) by some authors (4, 5, 6). McNulty et al. (3) speculated from their purely morphological assessment that the zona reticularis might develop from the TZ. However, Mesiano et al. (4) suggested that the DZ, TZ, and FZ of the fetal adrenal are analogous to the zona glomerulosa, zona fasciculata, and zona reticularis of the mature adrenal cortex. The functional significance associated with the unusual development and morphology of the human and primate adrenal cortex relates to its capacity to secrete {Delta}5 C19 steroids, specifically dehydroepiandrosterone (DHEA) in free and sulfated forms, from the zona reticularis (7, 8, 9, 10). Like the zona reticularis of the mature adrenal cortex, the FZ of the human and nonhuman primate fetal adrenal cortex is also believed to be responsible for the production of DHEA and DHEA sulfate (DHEAS) (9, 11, 12). Parker et al. (13) demonstrated that plasma concentrations of DHEAS increase steadily in human fetuses throughout gestation in parallel with adrenal mass. By comparison, DHEAS levels in macaque fetuses are much lower for most of gestation, but increase rapidly at term, reaching concentrations comparable with those seen in the human fetus (13, 14, 15). Although this increase in circulating DHEAS concentrations by the FZ presumably is accompanied by rapid adrenal growth that could explain the apparent increase in steroid output, it might also reflect changes in the expression of steroidogenic enzymes or their function.

The dramatic preparturient increase in circulating DHEA levels seen in the rhesus macaque provides an opportunity to examine factors that might be involved in physiological regulation of adrenal androgen secretion. Specifically, evidence is convincing that in humans, the accessory protein cytochrome b5 is capable of modulating catalysis by the enzyme 17{alpha}-hydroxylase/17,20-lyase cytochrome P450 (P450c17) and is minimally required to promote efficient 17,20-lyase activity and thereby C19 steroid synthesis (16, 17, 18, 19, 20, 21). Consistent with this idea, our previous studies identified the zona reticularis as a site of strong and virtually exclusive cytochrome b5 expression within the primate adrenal cortex (22). However, whether it is a factor that limits androgen synthesis and therefore a likely physiological regulator in nonhuman primate tissues at least remains to be determined. The present studies were conducted to investigate the temporal and spatial patterns of cytochrome b5 expression during nonhuman primate fetal adrenal development as an initial step toward understanding the role of this protein in the control of androgen synthesis. Additionally, based on the results of our previous studies of mature adrenal glands, we were interested to examine the pattern of cytochrome b5 expression as a potential marker of the development of the zona reticularis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All animal procedures conformed to the requirements of the Animal Welfare Act, and protocols were approved before implementation by the institutional animal use and care administrative advisory committee at the University of California-Davis. Twenty-two normally cyclic, adult female rhesus monkeys (Macaca mulatta) with a history of prior pregnancy were bred and identified as pregnant using established methods (23). Pregnancy in the rhesus monkey can be divided into trimesters by 55-d increments, with 0–55 d gestation representing the first trimester, 56–110 d gestation representing the second trimester, and 111–165 d gestation representing the third trimester (term, 165 ± 10 d) (24). Complete fetal tissue harvests were performed using standard techniques (25) at the following gestational ages: 50 (n = 2), 80 (n = 4), 100 (n = 1), 115 (n = 1), 130 (n = 5), 145 (n = 2), 150 (n = 4), and 160 (n = 2). Representative sections of all tissues collected including the adrenals were preserved in 10% buffered formalin, embedded within 1 wk of collection, then sectioned at 5–6 µm and stained with hematoxylin and eosin to confirm normal histology. One adrenal gland per fetus was analyzed for the study.

Immunohistochemical localization of the steroidogenic enzymes cytochrome b5, P450c17, and 3ß-hydroxysteroid dehydrogenase (3ßHSD) was conducted using the Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA). Endogenous peroxidase activity was quenched by incubating the sections in 0.3% hydrogen peroxide in methanol for 30 min. Slides were then incubated (20 min) with diluted normal goat serum to decrease nonspecific binding. Sections were subsequently incubated overnight at 4 C with primary antiserum diluted in buffer. Polyclonal antisera raised against human cytochrome b5 (1:3000), human P450c17 (1:1000), and human placenta 3ßHSD (1:400) were used as primary antibodies, and normal rabbit serum (1:1000) was used as a control. The antihuman cytochrome b5 antiserum was raised in rabbits in our laboratory using purified recombinant protein provided by Drs. Ron Estabrook and Manju Shet (Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX). Antihuman P450c17 antibodies were raised in chickens using purified recombinant protein that was generated in our laboratory using an expression plasmid provided by Dr. Mike Waterman (Department of Biochemistry, Vanderbilt University, Nashville, TN). The antihuman 3ßHSD antisera was raised in rabbits and was a gift from Dr. J. Ian Mason (Clinical Biochemistry, University of Edinburgh, Edinburgh, Scotland). Detection of primary antibody binding was conducted the following day by incubation with diluted (1:200) antirabbit or antichicken biotinylated (5 mg/ml) secondary antibody preparations (Sigma, St. Louis, MO). Finally, sections were incubated with avidin-biotin-conjugated peroxidase, developed with 3-amino-9-ethylcabazole (Vector Laboratories, Inc.) for 10 min, and counterstained with Gill 2 hematoxylin. Slides were coverslipped with Paramount (DAKO Corp., Carpinteria, CA). Micrographs were taken using brightfield illumination on a DMRB microscope (Leica Corp., Rockleigh, NJ) that was fitted with a Polaroid (Cambridge, MA) DMC digital camera.

The specificity of all three antisera has been demonstrated in previous immunocytochemical studies (22, 26). The specificity and sensitivity of the antihuman cytochrome b5 and P450c17 antisera were further evaluated by immunoblot analysis. Porcine neonatal testes were homogenized in PBS containing 1% sodium cholate and 0.1% SDS and sonicated for 3 sec. Homogenates and purified recombinant human and bovine P450c17 proteins were subjected to SDS-PAGE (12% gel). Separated proteins were transferred by electroblot onto polyvinylidene difluoride membranes, immunoblotted with antihuman cytochrome b5 and P450c17 primary antisera, followed by the appropriate biotinylated secondary antibody, and developed by chemiluminescence (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The developing fetal adrenal cortex consisted of two easily recognizable and distinct cell layers from the earliest stages examined. Even at 50 d gestation (late first trimester), the earliest stage examined, a DZ was obvious immediately beneath the capsule, consisting of cells distinguishable by their relative lack of cytoplasm, which contrasted with the abundant cytoplasm characteristic of cells in the centrally developing FZ (Fig. 1Go). However, after immunostaining for P450c17, and particularly for b5, it was clear that within the FZ there were populations of cells that exhibited different expression characteristics, once again from the earliest stages examined. At 50 d gestation, P450c17 expression was strongest in FZ cells immediately underlying the DZ (Fig. 1Go, A and C). In contrast, cytochrome b5 expression was most prominent in clusters of cells located toward the center of the developing cortex (Fig. 1Go, B and D). In fact, the expression of b5 at 80 d gestation (second trimester) was restricted entirely to cells surrounding the developing medulla or occasionally in cells embedded within it (Fig. 2AGo). Similarly, b5 was expressed in perimedullary cells at 150 d (Fig. 2BGo), but was also seen in a few larger cells scattered through the FZ (Fig. 2BGo and inset). By 160 d (term), b5 expression involved an additional population of cells in the outer FZ, on occasion forming a solid band of cells beneath the TZ (Fig. 2CGo).



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  		Figure 1. Expression of P450c17 (A and C) and b5 (B and D) in the developing adrenal cortex of the fetal macaque at 50 d gestation. A, Expression of P450c17. Note that P450c17 expression (red staining) is highest in those cells of the developing FZ that lie immediately beneath the outer DZ. B, Expression of b5 in an adjacent section from the same specimen. Note that cytochrome b5 expression in the FZ is strongest in cells located in a deeper layer than those expressing P450c17. C, View of 50 d fetal adrenal at low magnification showing the distribution of P450c17 expression in cells of the outer cortex underlying the DZ. D, Low magnification view showing b5 expression in the 50 d fetal adrenal in cells with a more medullary location to those expressing P450c17. Bar, 25 µm.

 


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  		Figure 2. Expression of b5 in various cells in different locations of the fetal macaque adrenal gland detected by immunocytochemistry at different gestational days. A, At 80 d gestation (second trimester), b5 expression (arrows) is evident in the developing adrenal cortex involving cells surrounding the medulla (M). B, Similar perimedullary expression of b5 at 150 d gestation, but also in the larger cells of the FZ, as shown in the inset. C, Cytochrome b5 expression in the outer cortex at term (160 d), showing expression in cells in the region underlying the DZ that show a smaller cytoplasmic/nuclear ratio than positive cells seen in the FZ. The lower magnification view seen in the inset illustrates the outer margins of expression (arrows) near the DZ and the diminished expression throughout the FZ. All bars, 10 µm.

 
Gestational age not only affected the location of cells expressing b5, but, more dramatically, their number and levels of expression. The restriction of b5 to the perimedullary region at 80 d was a reflection of a general decline in expression that extended through the third trimester. Specifically, cytochrome b5 was not detected in the specimens collected at 100 and 115 d gestation, the transition from the second to the third trimester (Fig. 3AGo), but was observed again in cells at the corticomedullary junction at 130 d, the early third trimester (Fig. 3BGo), and more generally in cells scattered throughout the FZ at ages examined thereafter (Fig. 3CGo). Indeed, by 160 d (term), it was obvious that this represented a marked increase in the number and intensity of cytochrome b5 staining in cells throughout the cortex over those seen at earlier gestational ages. Occasional groups of cells appeared to form long tracts (Fig. 3CGo) extending toward and even reaching the medulla in some instances. Additionally, cytochrome b5 expression became detectable in cells in the outer cortex that were generally smaller in size than those of the FZ, with a correspondingly greater nuclear/cytoplasmic ratio, not unlike cells of the TZ (Figs. 2CGo and 4AGo). Little or no cytochrome b5 expression was observed in the cells of the DZ at any fetal age (Fig. 3Go, A and C, and Fig. 4AGo). In all cases staining, when detected, was clearly cytoplasmic in location, and background staining was minimal. The lack of staining of sections incubated without the primary antiserum (negative controls) was similar to results obtained from adrenals lacking evidence of cytochrome b5 expression at 100 and 115 d gestation (Fig. 3AGo).



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  		Figure 3. Development of b5 expression in the macaque fetal adrenal cortex at various gestational ages. A, At 115 d gestation (early third trimester) there is a general lack of cytochrome b5 expression in the fetal adrenal cortex. In particular, note the lack of staining in the DZ and, with the exception of a very few scattered cells, little evidence of b5 expression in the FZ. Bar, 20 µm. B, At 130 d gestation (midthird trimester), expression of b5 is evident in cortical cells surrounding the developing adrenal medulla (M). Bar, 20 µm. C, At 160 d (term), the induction of b5 in the TZ is evident, and there is the occasional appearance of streaming of positive cells extending into the FZ. Note the lack of expression in the DZ and FZ, but the appearance of expression in cells at the corticomedullary junction (inset). Bar, 50 µm.

 


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  		Figure 4. Zonal expression of b5, P450c17, and 3ßHSD in fetal macaque adrenal gland at term (160 d). A, b5 expression is shown clearly in the FZ and inner cells of the TZ, with little or no expression detectable in the DZ. B, P450c17 expression shown throughout the TZ, at lower levels in the FZ, and absent from the DZ. C, 3ßHSD expression clearly restricted to the DZ and at low levels or absent in the TZ and FZ. Bar, 50 µm.

 
Developmental changes were also noted in the appearance and apparent levels of P450c17 expression, which were immediately apparent in the cortex of adrenal glands at 50 d gestation in cells immediately beneath the developing DZ (Fig. 1AGo), as described above. Similar to b5, levels of P450c17 expression appeared to have decreased overall from 80–130 d and was restricted to just a few cells of the TZ when detectable (data not shown). However, by 150 and 160 d gestation, P450c17 staining was more intense, forming an almost complete band of cells under the DZ and also involving cells within the FZ (Fig. 4BGo). Thus, positive cells became more numerous and stained more intensely in both the TZ and FZ at these later stages of fetal development. As was seen with cytochrome b5 expression (Fig. 4AGo), P450c17-positive cells were less numerous in regions near the medulla, creating a mosaic pattern of expression mostly in the outer half of the FZ. Immunostaining in the late gestation fetal adrenal cortex also emphasized the complexity of cortical development and zonal maturation based on the differences in the location and levels of enzymes and accessory proteins in serial sections. It appeared that those cells exhibiting the highest expression of cytochrome b5 in late gestation lay in the outer FZ and inner layer of the TZ (Fig. 4AGo). In contrast, TZ cells exhibiting the highest level of P450c17 (Fig. 4BGo) lay just outside of the cells that were staining most intensely for cytochrome b5. Staining for 3ßHSD was light or undetectable in specimens collected at gestational ages earlier than 150 d, but was strong and clearly defined in the DZ (Fig. 4CGo) in specimens from 150 and 160 d (term). Staining for 3ßHSD was detectable in the TZ, although at notably lower levels than observed in the DZ. Regardless of stage of gestation, 3ßHSD expression was light or more often undetectable in the FZ (Fig. 4CGo).

The specificity of the cytochrome b5 and P450c17 antisera were demonstrated by immunoblot analysis of porcine testicular tissue homogenate. A single immunoreactive band was observed after incubation with the antihuman b5 antiserum. The apparent size, 16 kDa, was as expected and consistent with the length of the deduced sequence (Fig. 5AGo, first lane). Subsequent incubation of the same membrane with antihuman P450c17 demonstrated a second band, at 50 kDa, again the correct, expected size (Fig. 5AGo, second lane). The sensitivity of the antihuman P450c17 antiserum was tested directly against purified recombinant human and bovine P450c17 proteins. The antiserum detected as little as 0.1 pmol human P450c17, but bovine P450c17 was detected only down to 1 pmol (Fig. 5BGo). At the exposures necessary to reach this level of sensitivity, 10 pmol recombinant human P450c17 appeared as a smear.



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  		Figure 5. Immunoblot analysis of porcine testicular protein; recombinant human and bovine P450c17 protein were subjected to electrophoresis by SDS-PAGE and transferred to membranes. A, b5 expression was detected as a single clear band (16 kDa) in porcine testicular protein (50 µg) after incubation of the membrane with antihuman b5 and detection as described in Materials and Methods. The same membrane was incubated subsequently with antihuman P450c17 and developed a second time (b5+c17) to detect P450c17 expression (50 kDa) as a second, clear, additional band. B, The sensitivity and specificity of the antihuman P450c17 antibody was tested further by detection of 0.1, 1.0, and 10 pmol recombinant human (H) and bovine (B) P450c17. Note that the antisera detected 0.1 pmol human, but not bovine, P450c17.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The expression of cytochrome b5 (22) and adrenal androgen secretion (7, 8, 10, 27) are properties associated with cells of the zona reticularis. Cytochrome b5 also appears to be a useful marker of cellular differentiation and the potential for androgen synthesis by human adrenal tumors (28, 29, 30). Thus, the appearance of this protein may mark the onset of differentiation of this cortical zone, coincident with adrenal cortical activation. At the same time, the relative lack of cytochrome b5 expression in the fetal adrenal cortex for most of pregnancy emphasizes a fundamental phenotypic difference between the FZ cells and the cells of the zona reticularis of mature animals. This contrasts with the expression of cytochrome b5 in the FZ of the human fetal adrenal gland (31) and the uniform pattern of expression of cytochrome b5 in the zona reticularis of mature rhesus adrenal (22). There is a clear parallel between the FZ and the mature zona reticularis of the human and nonhuman primate adrenal relative to the lack of 3ßHSD expression (5, 32, 33, 34), yet our data leave doubt as to the similarity in differentiated phenotype, and perhaps steroidogenic function, based on cytochrome b5 expression. Although expression was not prominent in the DZ at any stage of development, the induction of cytochrome b5 at gestation d 150 and 160 occurred only sparsely in cells scattered throughout the FZ, if more consistently in a thin band of cells in the TZ. Therefore, whether the FZ differentiates into the reticularis postnatally or is otherwise replaced by it, our data provide evidence of a clear, previously unrecognized or undescribed, morphological distinction between these compartments of the nonhuman primate adrenal cortex.

These data document the ontogeny of cytochrome b5 expression in the fetal adrenal cortex of a nonhuman primate from d 50 of gestation (late first trimester) to term, the first developmental study of this protein in a nonhuman primate. In addition, this report extends observations on the expression of P450c17 in the rhesus fetal adrenal gland to include development from d 50–100 of gestation. Cytochrome b5 and P450c17 expression, both apparently required for efficient androgen synthesis (17, 20, 35, 36, 37), were obvious in the developing adrenal cortex at 50 d gestation. However, further adrenal development, encompassing 80–145 d, appeared to be associated with a general decline in b5 and P450c17 expression until late in gestation, when there was a preparturient increase in expression of b5, P450c17, and 3ßHSD. This late gestational induction of cytochrome b5 and P450c17 expression was observed to involve cells of the TZ and FZ, consistent with the process of adrenal maturation before birth (5, 31). A similar temporal pattern of adrenal steroidogenesis, characterized by a midgestational depression of P450c17 expression and corticoid synthesis, has also been observed during development in bovine (38, 39), porcine (40), and ovine fetuses (41). There have been few comparable studies in primates, but Resko et al. (42) observed a decline in the level of circulating androstenedione in both male and female rhesus fetuses between d 40 or 50 of gestation and d 90–100 and a subsequent increase through late gestation. Challis et al. (43) showed a dramatic increase in amniotic concentrations of androstenedione over the last 2 wk of gestation that was suppressed by glucocorticoid treatment. More importantly, there is a late gestational rise in fetal serum DHEAS concentrations (14, 15). Pepe and Albrecht (44), directly assessing fetal adrenals at different stages of gestation in the baboon, demonstrated an increase of over 4-fold in P450c17 activity between midgestation and term. All of these data are consistent with the results reported here by immunocytochemistry indicating that there is a decline in adrenocortical function between the early and late stages of fetal adrenocortical development in the rhesus monkey and perhaps other nonhuman primates.

The results of the present study also provide evidence consistent with a role of b5 in the control of androgen synthesis by P450c17 in the human and nonhuman primate adrenal cortex. Specifically, the apparent induction of cytochrome b5 expression at 150 and 160 d gestation, both the increase in intensity of staining of individual cells and the increased number of cortical cells expressing the protein, correlates with the increase in DHEAS secretion and prepartum adrenal growth, as noted previously (14, 15). It is still not entirely clear whether b5 and P450c17 are expressed in the same cells of the fetal adrenal cortex as they appear to be in the mature zona reticularis (22). Coexpression of these proteins within the same microsomal environment is requisite for their molecular interaction, and therefore for efficient 17,20-lyase activity, based on our current understanding of human P450c17 function (18, 19). Although there are obvious differences in the regional expression patterns for b5 and P450c17, there appears to be at least some overlap between these regions at 50 d gestation, and again at term, when both appear to be induced. In fact, the apparent overlap of expression seen at 150 and 160 d gestation occurs at the interface between the TZ and the FZ, which may represent the origins of the reticularis. Regardless, our data are consistent with a cellular pattern of adrenal maturation represented by the expression of cytochrome b5. Further studies are necessary to clarify the events involved in reticularis development from the FZ in the neonatal period, but it appears that cytochrome b5 expression will be a valuable tool in such evaluations.


   Acknowledgments
 
The authors thank the animal technical staff at the California Regional Primate Research Center for assistance with animals, and Jo Corbin for help with the preparation of antisera.


   Footnotes
 
This work was supported in part by USDA Grant 98-35203-6439; Grant HD-36913 (to A.J.C.); Grants DK-49317, HL-55175, and RR-00169 (to A.F.T.); and Office of Naval Research Grant N00014-96-1-0255 (to C.R.P.).

Abbreviations: b5, Cytochrome b5; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; DZ, definitive zone; FZ, fetal zone; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; P450c17, 17{alpha}-hydroxylase/17,20-lyase cytochrome P450; TZ, transitional zone.

Received September 11, 2001.

Accepted for publication December 7, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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