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Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143-0556
Address all correspondence and requests for reprints to: Robert B. Jaffe, M.D., Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143-0556.
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Abstract |
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The enzymes CYP21A2 (P450 21 hydroxylase, or P450c21), CYP11B1 (11ß hydroxylase or P450c11) and CYP11B2 (aldosterone synthase) are necessary for glucocorticoid and mineralocorticoid synthesis but have not been localized previously in an ontogenic manner in the primate fetal adrenal gland. Therefore, we used immunocytochemistry (ICC) to assess specific zonal localization and developmental regulation of CYP21A2 and CYP11B1/CYP11B2 in the human (13–24 weeks’ gestation) and rhesus monkey (109d-term) fetal adrenal gland. In the fetal rhesus, ICC was performed with and without metyrapone administration to the fetus to assess the effects of endogenously increased fetal ACTH.
In the human fetal adrenal, CYP21A2 immunoreactivity (IR) was present in only a few isolated cells in the DZ but was detectable in almost all cells in the TZ and FZ. In the fetal rhesus, CYP21A2-IR was present in cells throughout the DZ and TZ and, to a lesser degree, in the FZ. Staining intensity increased with advancing gestational age and was up-regulated in the DZ and TZ, but not the FZ, of the metyrapone-treated fetuses.
In the human fetal adrenal gland, CYP11B1/CYP11B2-IR was absent in the DZ but present in the TZ and FZ. In the fetal rhesus monkey adrenal, CYP11B1/CYP11B2-IR was present in all cells of the TZ and FZ but was absent from the DZ until near term. After metyrapone, CYP11B1/CYP11B2 -IR was induced in the DZ and was up-regulated in the TZ and FZ.
Taken together, these data indicate that in the primate fetal adrenal gland, the FZ has the capacity to synthesize DHEA and DHEAS beginning early in development, the TZ has the capacity to synthesize cortisol after midgestation, and the DZ has the capacity to synthesize mineralocorticoids, but not until near term. The spatial localization of steroid metabolizing enzymes and steroid products in the human and rhesus monkey fetal adrenal suggests analogies of the three functional zones of the fetus (DZ, TZ, and FZ) to their adult counterparts (zona glomerulosa, zona fasciculata, and zona reticularis) and their steroid products (mineralocorticoids, glucocorticoids and androgens, respectively), although the reason for the presence of CYP11B1/CYP11B2 - and CYP21A2-IR in the FZ remains to be elucidated.
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Introduction |
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Our previous studies of the human and subhuman primate fetal adrenal
gland (1, 2, 3) have led to the conclusion that the gland has three
functional zones, at least during the last trimester of human
pregnancy: 1) the outer, definitive zone, comprised of cells
that have a proliferative morphologic appearance, appears to function
as a reservoir of precursor stem cells and does not acquire a
steroidogenic phenotype enabling it to secrete mineralocorticoids until
close to term (1, 2, 3, 4); 2) the transitional zone, located at
the interface between the inner, fetal zone and the definitive zone,
which begins to function steroidogenically by about 24–28 weeks of
human pregnancy (1, 2, 5) and possesses enzymes necessary to produce
cortisol de novo (cytochrome P450 side-chain cleaving
enzyme, CYP11A1 or P450scc; cytochrome P450 17
hydroxylase/17, 20
lyase, CYP17 or P450c17); and 3ß-hydroxysteroid
dehydrogenase-
4–5-isomerase (3ßHSDII or 3ßHSD),
although the two other enzymes involved in cortisol synthesis,
cytochrome P450 21 hydroxylase (CYP21A2 or P450c21) and cytochrome P450
11ß-hydroxylase (CYP11B1 or P450c11), previously have not been
localized specifically in an ontogenic manner in either the human or
subhuman primate fetal adrenal gland. In incubations of fetal rhesus
monkey adrenals, 11ß hydroxylation and 21 hydroxylation were higher
in the inner (likely transitional) zone than outer (likely definitive)
zone at 135 days of gestation (6); and 3) the large inner fetal
zone, which begins functioning early in pregnancy and possesses
the enzymes necessary for the synthesis of the androgen
dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) (CYP11A1, CYP17;
and sulfokinase).
Previous studies (7) indicated that glucocorticoid synthesis by the human fetal adrenal is possible early in intrauterine life, and study of congenital adrenal hyperplasia, a group of genetic abnormalities resulting in a deficiency of enzymes involved in corticosteroid synthesis, indicates that, under normal circumstances, the ACTH-cortisol axis is intact in utero (8). A possible explanation for the fetal synthesis of cortisol early in gestation, at a time when 3ßHSD is not yet expressed in the fetal adrenal gland, is that the gland uses progesterone secreted by the placenta as substrate for cortisol biosynthesis, and only during the latter part of pregnancy does it acquire the capacity to synthesize cortisol de novo. Indeed, perfusion of radiolabeled progestin to the previable human fetus (9) and incubation of fetal adrenal tissue with radiolabeled progesterone (10) indicate that the human fetus can effect the biosynthesis of cortisol from progestins both in vivo and in vitro.
The biosynthesis of the mineralocorticoid, aldosterone, requires the presence of CYP11A1, 3ßHSDII, aldosterone synthase encoded by the CYP11B2 gene, and CYP21A2, but not CYP17. Our previous studies in the human and rhesus monkey fetal adrenal gland demonstrated that the outer definitive zone expresses CYP11A1 and 3ßHSDII, but not CYP17 (1, 2). However, 3ßHSD was not expressed until near term, and CYP21A2, CYP11B1 and CYP11B2 had not been studied extensively, particularly near term.
Therefore, in the present study we determined the localization and temporal expression of CYP21A2 and CYP11B1/CYP11B2 in the human and subhuman primate fetal adrenal gland using immunocytochemical techniques. We also determined whether the time in gestation at which these enzymes are detected could be advanced by increasing endogenous ACTH production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thirty-one pregnant rhesus monkeys (Macaca mulatta) were used in this study. The animals were obtained from the timed breeding colony at the California Primate Research Center, Davis, CA, and maintained in accordance with the NIH Guide for the Care and Use of Animals. The protocols were approved by the Committee on Animal Care, UCSF. In 21 pregnant rhesus monkeys between 109–125 days (n = 7), 134–156 days (n = 7), and 159–172 days (n = 7), the fetuses were delivered by hysterotomy for collection of fetal adrenal tissue.
In 10 pregnant rhesus monkeys, surgery was performed between 126 and 140 days under halothane anesthesia using aseptic techniques (1–2% halothane in N2O/O2) for implantation of a fetal peritoneal catheter as described previously (11). Metyrapone was dissolved in vehicle (8% ethanol in 0.9% saline) and infused (16 mg/day) via the fetal peritoneal catheter for 3 days (n = 2), 5 days (n = 1) or 7 days (n = 1) using a miniinfusion pump (Pulsamat, Ferring, Suffern, NY) as described previously (11). Control animals (130–147 days) received vehicle (8% ethanol in 0.9% saline) for 7 d (n = 2). In the other four fetuses, the catheters were nonfunctional because they were either detached from the fetus (n = 2) or were occluded (n = 2); these animals were included in the control group.
Adrenals were bisected and fixed by immersion in 0.1 M phosphate-buffered paraformaldehyde (4%) and embedded in paraffin. Sections (6 µm) were prepared and mounted on pretreated slides (Superfrost Plus, Fisher Scientific International, Inc., Pittsburgh, PA) to localize CYP21A2 and CYP11B1/CYP11B2 using immunocytochemical techniques.
Adrenal immunohistochemistry
To localize CYP21A2 and CYP11B1/CYP11B2 protein in paraffin
sections of adrenal glands, we used the avidin-biotin-peroxidase
technique (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA) with an Immunopure metal-enhanced
diaminobenzidine (DAB) substrate (Pierce, Rockford, IL) as
the chromogen. All steps were performed at room temperature unless
otherwise noted. The sections were deparaffinized using Histoclear
(National Diagnostics, Manville, NJ), rehydrated through graded
alcohols, and rinsed in 0.1 M Tris-buffered saline (TBS),
pH 7.4. To permeabilize the tissue and inhibit endogenous peroxidases,
the sections were incubated for 20 min in TBS containing 0.1% saponin,
0.02 M glycine, and 3% H202 and
then washed in TBS (3 x 5 min). Nonspecific binding was blocked
by incubation for 15 min in TBS containing 3% normal goat serum and
1% BSA (3%NGS/1%BSA). The polyclonal primary antibody was diluted in
3%NGS/1%BSA in TBS (CYP21A2 1:1000, CYP11B1/CYP11B2 1:7500) and the
sections incubated overnight at 4 C in a humidified chamber. The
sections were washed in TBS before incubation in the blocking solution
(3% NGS/1% BSA in TBS; 20 min). The sections were incubated with a
biotinylated goat-antirabbit secondary antibody, which was diluted in
3% NGS/1% BSA in TBS (1:500; 60 min). The sections were washed in TBS
before incubation with the ABC reagent (1:200 dilution in TBS; 60 min)
and washed again in TBS. The signal was detected using the DAB
substrate, and the brown reaction product was observed under the light
microscope. The sections were dehydrated in graded alcohols, cleared in
Histoclear, and mounted.
The CYP21A2 and CYP11B1/CYP11B2 polyclonal antibodies, which were generously provided by Professor Pieter Swart (Department of Biochemistry, University of Stellenboch, South Africa), were raised in rabbits against immunopurified ovine CYP11B1/CYP11B2 and CYP21A2 enzymes and have been characterized previously (12). The CYP11B1/CYP11B2 antibody does not distinguish between 11ß-hydroxylase and aldosterone synthase, which are encoded by separate but highly similar genes, CYP11B1 and CYP11B2, respectively (13, 14). Therefore, this two-enzyme complex is indicated as CYP11B1/ CYP11B2.
To further assess the specificity of the antibodies, 100 µg of human adrenal protein were compared with 100 µg of maternal sheep adrenal protein on 5–15% gradient SDS-PAGE. By Western blotting with the CYP21A2 antibody, a strong band at 55K was detected, and with the CYP11B1/CYP11B2 antibody, a unique band at 50K was observed. These molecular weights confirm original published data for CYP11B1/CYP11B2 and with data from P. Swart on the original Western blot using the CYP21A2 antibody.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A–C,
Table 1). In contrast, CYP21A2-IR was
detectable in all cells in the transitional (TZ) and fetal zones (FZ);
however, some adrenal cortical cells were stained more intensely than
others (Fig. 1, A–C, and Table 1). At this early stage of gestation,
the adrenal medulla has not formed a discrete structure in the center
of the gland (15), and islands of cells are found throughout the
adrenal cortex (Fig. 1A). These islands of adrenomedullary cells did
not stain for CYP21A2-IR; they are counterstained blue with Mayer’s
hematoxylin and identified by the arrow (Fig. 1A and Table 1).
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, D–F, and Table 1). In all fetal monkey adrenals examined,
CYP21A2-IR was absent in the adrenomedullary cells. The staining
intensity for CYP21A2-IR increased with advancing gestational age
between the age groups of 109–125 days, 130–156 days, and 159–172
days (Table 1). After metyrapone treatment, CYP21A2-IR was up-regulated
in the DZ and TZ compared with age-matched controls (Fig. 1, E and
G, and Table 1), but the intensity of CYP21A2-IR was not changed in the
FZ (Fig. 1H). At the border of the FZ and medulla, the FZ cells were
not stained. In the metyrapone-treated fetuses, CYP21A2-IR was also
absent from the adrenomedullary cells (Fig. 1H).
In the adult human adrenal gland, intense CYP21A2-IR was present in the
zona glomerulosa and zona fasciculata (Fig. 1I), whereas staining was
almost undetectable in the zona reticularis (Fig. 1J). The
adrenomedullary cells did not stain for CYP21A2-IR (Fig. 1J). The
intensity of CYP21A2-IR in the adult adrenal gland was greater than
seen at any stage of fetal development (Fig. 1J and Table 2). Therefore, a reduced concentration of
antibody was used for immunostaining the adult gland.
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, A and B,
and Table 1). The islands of adrenomedullary cells that were found
throughout the adrenal cortex did not stain for CYP11B1/CYP11B2-IR
(Fig. 2B).
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, C
and D, and Table 2); however, staining was absent from the DZ until
near term (Fig. 2E and Table 2). After metyrapone treatment,
CYP11B1/CYP11B2-IR was induced in the DZ and was up-regulated in the TZ
and FZ, compared with age-matched controls (Fig. 3, A–D, and Table 2). In all fetal
monkey adrenals examined from both metyrapone-treated and untreated
monkeys, CYP11B1/CYP11B2-IR was absent in the adrenomedullary cells
(Fig. 3, B and D). In the adult human adrenal gland, CYP11B1/CYP11B2-IR
was present in the zonae glomerulosa, fasciculata and reticularis (Fig. 2, G and H). Staining was more intense in the adult adrenal cortex than
in the fetal adrenal cortex at any stage of fetal development and a
lower concentration of antiserum was used for visualization. The adult
adrenomedullary cells did not have CYP11B1/CYP11B2-IR (Fig. 2H).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Elucidation of the localization and ontogenesis of CYP21A2, CYP11B1, and CYP11B2 has led to an integrated concept of the zonal production and temporal sequence of steroid hormone biosynthesis in the human and rhesus monkey fetal adrenal glands.
Definitive zone. Early in gestation, the absence of expression of genes encoding steroid biosynthetic enzymes in the DZ (1) and the preponderance of PCNA, which reflects cells that are actively proliferating (4), point to the DZ cells (which lack morphologic characteristics of steroid-producing cells at this stage of gestation) as progenitor cells that move centripetally to populate the rest of the gland. Late in gestation, when expression of the genes and production of the proteins for CYP11A1, CYP17, 3ßHSDII (1, 2), CYP21A2, CYP11B1, and CYP11B2 (18 and present study) are found, and the cells have adopted a steroidogenic phenotype, mineralocorticoid (aldosterone) production is initiated. Thus, the definitive zone acquires the nascent steroidogenic capacity of the adult zona glomerulosa.
Transitional zone. The presence of the steroid biosynthetic
enzymes necessary for cortisol biosynthesis (CYP11A1, CYP17, 3ßHSDII,
CYP21A2, and CYP11B1) during the latter third of gestation (1, 2, 5,
18, present study), the lack of 3ßHSDII in the fetal zone at all
stages of gestation, and the lack of CYP17 in the definitive zone
throughout gestation, point to the transitional zone as the sole site
of de novo cortisol synthesis in the latter part of
pregnancy. The acceleration of induction of these enzymes by
metyrapone-induced ACTH secretion in the rhesus monkey in
vivo (2, present study) indicates that this biosynthetic process
is under ACTH regulation, either directly or via locally produced
growth factors, of which insulin-like growth factor-II (IGF-II) (11, 19), basic fibroblast growth factor (bFGF) (20), activin (21), and
transforming growth factors-alpha (TGF) (22) and TGFß (21), may
play significant roles. These data indicate that the transitional zone
has steroidogenic activity qualitatively analogous to the zona
fasciculata of the adult. The data also are compatible with the
utilization of placental progesterone as substrate for cortisol
synthesis by the fetal adrenal gland in early pregnancy, thus obviating
the need for 3ßHSDII at that time.
Fetal zone. Several observations indicate that the fetal zone is the site of synthesis of the principal androgen of the fetal adrenal (DHEAS), as the zona reticularis is in the adult: 1) the presence of CYP11A1 and CYP17 early in gestation and the absence of 3ßHSDII throughout pregnancy in the fetal zone (1, 2); 2) the in vitro demonstration of basal production and ACTH stimulation of DHEAS by separated fetal zone tissue in vitro (7) and of the rhesus fetal adrenal gland in vivo (23); and 3) the stimulation of the enzymes required for DHEA formation and their regulation by metyrapone-induced ACTH in the rhesus monkey in utero (2). DHEAS serves as a precursor for placental estrogens, which may play a role in the initiation of primate parturition (24, 25).
The reason for the expression of CYP21A2 and CYP11B1/CYP11B2 in the fetal zone, albeit more weakly than in the transitional and definitive zones, is not immediately apparent. This may, however, explain in part the capacity of the fetal zone cells in culture to produce glucocorticoids, although the cells would still have to develop 3ßHSDII activity. Another explanation for this phenomenon is that some transitional zone cells were admixed with fetal zone cells and, in the presence of ACTH, produced cortisol. A similar pattern is observed in the corresponding zona reticularis of the adult (26, present study).
In summary, the spatial location of steroid metabolizing enzymes and steroid products in the human and rhesus monkey fetal adrenal gland suggests analogies of the three functional zones of the fetus (definitive, transitional, fetal) to their adult counterparts (zonae glomerulosa, fasciculata and reticularis, respectively) and their steroid products (mineralocorticoids, glucocorticoids and androgens, respectively). Whether these three fetal zones are the actual precursors of their adult counterparts, or whether the adult zones are formed anew after the fetal adrenal remodeling that occurs in the peripartal period, remains to be elucidated.
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Footnotes |
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2 Present address: Baker Medical Research Institute, Prahran,
Victoria, Australia 3181. C. J. Martin Fellow of the National
Health and Medical Research Council of Australia.
Received December 29, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase/17,20-lyase, and 3ß-hydroxysteroid
dehydrogenase isomerase steroidogenic enzymes in human and rhesus
monkey fetal adrenal glands: reappraisal of functional zonation. J
Clin Endocrinol Metab 77:1184–1189[Abstract]
,21-trihydroxypregn-5-en-20-one by the intact human foetus at
midpregnancy. Biochim Biophys Acta 152:648–650[Medline]
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