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Original Studies |
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, San Francisco, California 94143-0556. E-mail: robert_jaffe{at}quickmail.ucsf.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the second and third trimesters, the human fetal adrenal cortex grows rapidly due mainly to the striking enlargement of the FZ (5, 6). The cellular mechanism (i.e. hypertrophy and/or hyperplasia) underlying FZ growth is not clearly understood. As FZ cells do not have a mitotic phenotype, we have proposed that FZ growth initially occurs primarily by cellular hypertrophy (5). However, direct biochemical evidence supporting this hypothesis is lacking. Interestingly, projections of cell cords extending from the DZ into the FZ indicate centripetal migration of DZ cells. These observations support the migration theory of adrenal cortical development (for review, see Ref. 7). Thus, it is possible that the cellular dynamics by which the human fetal adrenal cortex grows involve hyperplasia of DZ cells, centripetal migration of these cells, and their eventual differentiation into FZ cells, which then undergo hypertrophy and perhaps subsequent hyperplasia.
The FZ is a transient structure; soon after birth (in the first 2–3 weeks) it atrophies and/or is remodeled. As a consequence, the total weight of the glands decreases by approximately 50% (6, 8). During this postnatal transition, the remaining cortical cells (mainly DZ cells) remodel to form the adult pattern of cortical zonation. The mechanism underlying the postnatal demise of the FZ remains uncertain. Early studies attributed it to hemorrhage and necrosis (8); however, these were later found to be inconsistent features (9). As the regression of the FZ is a developmentally controlled process, we hypothesized that it is specifically regulated and involves apoptosis (programmed cell death) of FZ cells. Apoptosis is clearly involved in the postnatal remodeling of the rat adrenal cortex (10, 11). The hormonal regulation of cellular apoptosis is likely to involve specific cytokines that stimulate the biochemical cascade leading to the target cell’s death. Several factors have been identified that are antimitogenic for FZ cells. Activin A and transforming growth factor-ß (TGFß) inhibit the proliferation of FZ cells in culture (12, 13). However, it is not known whether they inhibit growth by stimulating apoptosis.
In the present study, we examined the cellular dynamics underlying growth of the human fetal adrenal cortex and its postnatal regression by assessing the zonal localization of cellular proliferation and apoptosis in fetal and postnatal human adrenal glands. To identify possible regulators of FZ apoptosis, we determined whether activin A and TGFß modulate apoptosis of cultured FZ cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Midgestation (11–23 weeks gestation; assessed by fetal foot length; n = 12) human fetal adrenals were obtained immediately after laminaria-primed dilatation and evacuation. Third trimester (24–36 weeks; n = 6) and postnatal (3 days to 1 month postnatal; n = 7) adrenal glands were obtained from archival material obtained at the time of necropsy by the Department of Pathology, University of California, San Francisco (UCSF). All third trimester adrenals were obtained from infants born prematurely and treated with betamethasone. Adult adrenals were obtained at the time of recovery of kidneys from organ donors (30–58 yr of age) for potential renal transplantation. Segments of adrenals were fixed in 4% paraformaldehyde in phosphate-buffered saline or placed in ice-cold medium for subsequent cell culture. Use of human material was approved by the Human and Environmental Protection Committee, UCSF.
Cell culture
Human fetal adrenal glands were decapsulated to remove both the fibrous capsule and the adherent underlying DZ as described previously (14). Cells were plated on 3.5-cm dishes (Falcon Plastics, Los Angeles, CA) at a density of 500,000 cells/plate. Culture medium consisted of a 1:1 (vol/vol) mixture of DMEM H16 and Ham’s F-12 with 10% FCS, 2 mmol/L glutamine, and 50 mg/mL gentamicin. All media and additives were obtained from the Cell Culture Facility at UCSF. Cells were incubated at 37 C in a humidified environment consisting of 5% CO2 in air. After 48-h incubation to allow cell attachment, medium was changed, and ACTH (10 nmol/L; Cortrosyn, Organon, West Orange, NJ) and test substances were added. Recombinant human activin A, recombinant human inhibin A, TGFß, and follistatin were provided by Genentech, Inc. (South San Francisco, CA). For serum-free culture, cells were allowed to attach for 48 h in the presence of FCS, after which fresh culture medium without FCS was added.
In situ detection of cell proliferation
Fixed adrenal specimens were embedded in paraffin, sectioned (5 µm), mounted on gelatin-alum-coated glass slides, deparaffinized, and then rehydrated in graded ethanols. To localize proliferating cells, we used a modification of the peroxidase-antiperoxidase immunocytochemical staining technique (15) to detect proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase that is specifically expressed in cells during S phase (16). Immediately before staining, the sections were hydrated in 0.05 mol/L Tris-buffered saline (TBS), pH 7.4, pretreated with methanol containing 5% H2O2, for 30 min at room temperature and then washed in three changes of TBS. To unmask antigens, the specimens were treated with Antigen Retrieval Citra Solution (Bio Genex, San Ramon, CA) for 10 min at 100 C. The sections were then incubated with mouse antihuman PCNA (Boehringer Mannheim, Indianapolis, IN), diluted 1:500 in 2% normal horse serum and 0.3% Triton-X 100 in TBS. Normal mouse serum (1:200 and 1:500) and buffer controls were included to determine nonspecific staining. After 2 h at room temperature, slides were washed in TBS and incubated with horse antimouse Ig (1:200) for 45 min at room temperature. Slides were then washed in TBS, incubated with rabbit peroxidase/antiperoxidase (1:100) for 30 min at room temperature, and washed in TBS and then in Tris buffer alone. The sections were treated with Tris buffer containing 0.05% 3,5-diaminobenzidine tetrahydrochloride and 0.01% H2O2, and the appearance of brown reaction product was observed under the light microscope. The sections were stained with propidium iodide (5 µg/mL in H2O) for 10 min at room temperature and then mounted.
In situ detection of cell apoptosis
Detection of apoptotic cells was performed by in situ analysis of DNA fragmentation in histological sections using a nonradioactive in situ end labeling (ISEL) method (ApopTag, Oncor, Gaithersburg, MD). In brief, slides were pretreated with proteinase K (20 mg/mL; Sigma Chemical Co., St. Louis, MO) and washed, after which endogenous peroxidase was quenched with 0.5% H2O2 for 20 min. Digoxigenin-linked deoxy-UTP residues were incorporated onto 3'-hydroxyl ends of double or single stranded DNA using terminal deoxynucleotidyl transferase. Antidigoxigenin antibody fragment conjugated with the reporter enzyme peroxidase was then added, and staining was performed with diaminobenzidine as the chromogen. The apoptotic index in different adrenal cortical zones was assessed by counting at least 100 nuclei and determining the proportion (expressed as a percentage) that was ISEL positive.
Assessment of apoptosis in cultured cells
Apoptosis was examined in cultured FZ cells by assessing the extent of DNA internucleosomal cleavage (i.e. DNA laddering). Cells were harvested from plates after treatment with trypsin and were pelleted by centrifugation at 300 x g at 4 C. Cell pellets were snap-frozen in liquid nitrogen and then stored at -70 C until analysis. DNA was isolated from cells (17) and quantified by spectrophotometry at 260 nm. Aliquots of DNA (500 ng) from each sample were labeled at the 3'-ends with [32P]dideoxy-ATP (3000 Ci/mmol; Amersham, Arlington Heights, IL) using terminal transferase (25 U/sample; Boehringer Mannheim, Indianapolis, IN), as previously described by Tilly and Hsueh (17). Labeled samples (150 ng) were fractionated through 2% agarose gels. After electrophoresis, gels were dried for 2 h in a slab gel dryer without heat and exposed to Kodak X-Omat AR films (Eastman Kodak Co., Rochester NY) at -70 C.
Statistical analysis
All experiments were repeated a minimum of three times. Data were analyzed by ANOVA followed by Student-Newman-Keuls test to identify significantly different (P < 0.05) data groups.
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Results |
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In first and second trimester human fetal adrenal specimens, PCNA
staining was detected in FZ and DZ cells(Fig. 1
). Between 10–14 weeks gestation, the
mitotic indexes (percentage of PCNA-positive cells) in the DZ
(26.46 ± 2.95%) and in the FZ (21.26 ± 2.57%) were not
significantly different. Between 15–20 weeks gestation, the mitotic
index increased significantly (P < 0.05) in both
groups (FZ, 33.84 ± 5.21%; DZ, 67.45 ± 7.58%) relative to
levels before 15 weeks. This increase persisted between 21–24 weeks
gestation (FZ, 39.5 ± 4.22%; DZ, 58.63 ± 6.83%).
Interestingly, after 14 weeks, the mitotic index of the DZ was
significantly greater (P < 0.05) than that of the FZ
(Fig. 2). In adrenal specimens obtained
from infants born prematurely and treated in utero with
glucocorticoid, the mitotic indexes in the FZ and DZ were significantly
decreased (Fig. 2). No staining was detected in the control sections
reacted with buffer in place of the primary antisera (data not
shown).
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In all human fetal and neonatal adrenal specimens, ISEL-positive
nuclei were scant. In general, ISEL-positive nuclei were detected
mainly in the central portions of the FZ (Fig. 3), although in some of the early
gestation (<18 weeks) specimens the very scant ISEL-positive nuclei
were scattered in both zones. The number of ISEL-positive nuclei
remained low in the DZ throughout gestation. In contrast, the number of
ISEL-positive nuclei in the central portions of the FZ increased with
increasing gestational age and peaked during the perinatal period (Fig. 3). Specimens from the immediate postnatal period (3 days to 1 month)
showed the greatest number of apoptotic cells, which were found mainly
in the inner FZ. In contrast, adult adrenal cortical cells contained
only a few apoptotic nuclei, and these were scattered throughout the
cortex. Only faint, uniform background staining was observed in the
control specimens in which terminal transferase was omitted from the
ISEL reaction.
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To provide biochemical confirmation of apoptosis, DNA was
extracted from FZ cells under various culture conditions. As serum
deprivation induces apoptotic cell death in vitro (18),
serum-free culture was used as a positive control for apoptosis.
Apoptotic DNA appears in a ladder pattern of 180-bp multimers,
signifying internucleosomal cleavage by activated endonucleases (19).
As expected, serum-free culture increased the proportion of low mol wt
DNA relative to cells exposed to 10% FCS. Addition of activin A (10
ng/mL) or TGFß (1 ng/mL) for 24 h in the presence of 10% FCS
increased the proportion of low mol wt DNA relative to 10% FCS alone
(Fig. 4A). Concomitant addition of the
activin-binding protein, follistatin (100 ng/mL), prevented induction
of apoptosis by activin A (Fig. 4B). The effects of activin A and
TGFß on induction of apoptotic DNA cleavage after 24 h in
culture did not differ in the presence or absence of ACTH (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our present findings are consistent with a high degree of cellular proliferation in the DZ, particularly after the 14th week of gestation when approximately 60% of DZ cells stain positively for PCNA. Thus, our data provide biochemical evidence that hyperplasia is the predominant feature of cell growth in the DZ. However, the idea that hyperplasia is minimal in the FZ is not supported by our data. PCNA-positive FZ cells were detected in specimens from all gestational ages, suggesting that hyperplasia may contribute to the growth of the FZ. This is not surprising, as hypertrophy alone would not be sufficient to account for the rapid rate of FZ growth during midgestation, particularly in the adrenals of fetuses with congenital adrenal hyperplasia.
Although we have no data concerning the role of hypertrophy in human FZ growth, we have shown in the fetal rhesus monkey that growth of the FZ in response to increased endogenous ACTH secretion occurs initially by hypertrophy (23). In rodents, similar studies have shown that ACTH initially increases the protein/DNA ratio (indicative of hypertrophy), but after several days the ratio is restored to pretreatment values despite a marked increase in cortical size (24). Clearly, cellular hypertrophy appears to be an acute mechanism for adrenal cortical growth in response to ACTH, whereas the chronic response to ACTH includes hyperplasia. In the present study, all adrenal specimens derived from infants delivered prematurely and treated with glucocorticoids exhibited a markedly reduced mitotic index in both cortical zones. As glucocorticoids inhibit endogenous ACTH secretion, these findings are consistent with ACTH-stimulated hyperplasia of human fetal adrenal cortical cells.
Numerous studies of adrenal cortical development (for review, see Ref. 7) have resulted in two general models to explain the cytogenesis of the adrenal cortical zones: 1) the zonal model and 2) the cell migration model. In the zonal model, cell proliferation occurs within each cortical zone, which grows and functions independently of the other. In the migration model, each zone is derived from a common pool of progenitor cells located in the periphery of the cortex, which then migrate inward and differentiate to populate the inner cortical zones. This model implies that all cortical cells have a common origin and that their phenotype changes according to their location within the cortex. Studies in the rodent adrenal (25) and ultrastructural analyses of human fetal adrenals (21) tend to support this concept. It is likely, therefore, that cells proliferate in the periphery of the cortex and subsequently migrate centripetally (by active migration or passive mitotic pressure). During their inward migration, they may differentiate to form the specific cortical zones only to undergo senescence when they reach the center of the gland.
Evidence of centripetal migration of lipid-containing cells in the human fetal adrenal cortex has been reported (20). Jirasek (21) described the daughter cells resulting from mitoses in the DZ forming cords that invade the outer layers of the FZ. The disparate levels of proliferation between the DZ and FZ coupled with evidence of centripetal migration suggest that the migration model of adrenal cortical cytogenesis best describes the dynamics of human fetal adrenal growth. The adrenal cortical zones, therefore, could be interdependent and derived from a common pool of cells in the periphery. Thus, it is likely that growth of the FZ not only involves limited proliferation of existing FZ cells, but also differentiation and hypertrophy of inwardly migrating cells from the DZ.
Apoptosis appears to be a critical component of adrenal cortical development. In their studies of postnatal adrenal cortical remodeling in the rat, Wyllie and co-workers (10, 11) introduced the concept of apoptosis as a basic process in tissue remodeling and showed that in the adrenal it occurs mainly in the inner portions of the cortex. Similarly, by morphological criteria, Jirasek (21) detected cellular apoptosis in the human fetal adrenal, primarily in the central portions of the FZ. In the present study, we have confirmed and extended these findings using a technique that identifies apoptotic cells in situ based on the detection of increased accumulation of cleaved DNA. In general, apoptotic nuclei were greater in the central areas of the FZ than in the DZ. The number of apoptotic nuclei was very low in midgestation fetal adrenals, but increased markedly during the postnatal period. Thus, we propose that cell growth of the human fetal adrenal cortex is a dynamic process in which cells proliferate in the periphery, migrate centripetally, differentiate to form the specialized cortical compartments (and possibly continue to proliferate within these compartments), and then undergo senescence when they reach the center of the cortex. The sizes of the human fetal adrenal cortex and its constituent zones represent the net effect of forces that modulate these dynamic parameters of growth. Interestingly, recent studies by Wolkersdorfer et al. (26) showed that in the adult human adrenal cortex, cellular apoptosis was greatest in the external zona glomerulosa and decreased in the internal zonae fasciculata and reticularis. Although the reason for this reversal of the apoptotic pattern is unknown, it does suggest that the role of apoptosis in adrenal cortical growth and remodeling may be different during fetal and adult life. Recently, Wolkersdorfer and Bornstein suggested that apoptosis plays a critical role in adrenal cortical zonation and that is may be differentially regulated by interactions with cytokines, such as major histocompatibility complex II (7).
Our data suggest that regression of the FZ and its consequent involution postnatally involve apoptosis. In the immediate postnatal period, apoptotic cells were identified mainly in the inner portion of the regressing FZ. In early gestation specimens, apoptotic cells were detected, but were much fewer in number. As expected, a higher ratio of proliferating to dying cells was found at midgestation, when the adrenal cortex is growing rapidly. Our data from adult adrenals demonstrate apoptotic cells predominantly in the inner cortex, the zona reticularis, whereas cells were seen mainly in the outer cortex, the zona fasciculata (data not shown).
In the anencephalic human fetus, the adrenal is small, and the FZ often is reduced in size, particularly after 14 weeks (27). ACTH treatment of an anencephalic fetus in utero restored adrenal size and the FZ (8), suggesting that fetal pituitary ACTH is required for normal cortical growth. However, FZ regression and the postnatal involution of the gland do not coincide with an apparent decrease in ACTH exposure (28). Thus, other factors are probably involved in triggering the loss of cortical volume and the regression of the FZ postnatally. In considering possible modulators of FZ apoptosis, we studied activin A and TGFß, two peptides known to inhibit FZ cell proliferation in vitro (12, 13). Activin A is involved in the regulation of growth and differentiation in a variety of tissues and induces apoptosis in several cell types, including macrophages (29) and liver cells in vivo and in vitro (30). As we previously found that the activin A subunit messenger ribonucleic acid and intact immunoreactive protein are expressed in the human adrenal cortex (12), we postulated that it may play a regulatory role in human fetal adrenal remodeling by inducing apoptosis of FZ cells. The immunocytochemical and biochemical data presented here are consistent with this hypothesis. As expected, concomitant addition of follistatin, an activin-binding protein, prevented the induction of apoptosis by activin. We also previously showed that the inhibitory action of activin A on mitogenesis is specific to the FZ, and not the DZ or adult adrenal cells (12). Thus, we propose that activin A and possibly the related peptide TGFß are candidates for mediators of FZ remodeling by apoptosis during the postnatal period.
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Acknowledgments |
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Footnotes |
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2 Present address: Mothers and Babies Research Centre, John Hunter
Hospital, Locked Bag 1, Newcastle, New South Wales 2310,
Australia.
Received June 25, 1998.
Revised September 30, 1998.
Revised November 13, 1998.
Accepted November 18, 1998.
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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 the human and rhesus
fetal adrenal gland: reappraisal of functional zonation. J Clin
Endocrinol Metab. 77:1184–1189.
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