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Department of Surgery (C.W., B.K.L.-Y., L.M.R.) and of Cell Biology and Neuroanatomy (W.C.E.), University of Minnesota, Minneapolis, Minnesota 55455; Department of Medicine, Endocrine Section (C.E.G.-S.), Harry S. Truman Memorial Veterans Hospital, University of Missouri, Columbia, Missouri 65201
Address all correspondence and requests for reprints to: W. C. Engeland, Ph.D., Box 120 UMHC, 516 Delaware Street S.E., Minneapolis, Minnesota 55455.
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Introduction |
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There are two predominant theories that could be invoked to explain the growth and differentiation that occur within the fetal adrenal cortex, eventually leading to adult zonation. The zonation theory hypothesizes that each zone develops independently of the other (5). The cell migration theory hypothesizes that cells proliferate in the zona glomerulosa, then migrate and differentiate centripetally until they reach the zona reticularis where they ultimately die (6). A similar theory has also been used to explain the regeneration of the adrenal cortex after enucleation or transplantation (7), implying that regeneration recapitulates the ontogeny of the gland. Although previous investigations have attempted to describe the timing of fetal adrenal zonation, the lack of specific cell markers to define cortical phenotype has left questions unanswered.
Past studies have relied on cellular ultrastructure, such as the type of mitochondrial cristae or number of lipid droplets, to characterize cortical phenotype throughout development (8, 9, 10, 11). Although this method may be accurate for the identification of mature cortical cells, it is not clear whether ultrastructural criteria can be used to define a developing cell and its functional capacity. In contrast, monitoring the expression of the genes that encode zone-specific steroidogenic P450c11 enzymes could be an effective method for defining the appearance of functional phenotypes during adrenal organogenesis. In the adult gland, production of 11ß-hydroxylase (P45011ß) and aldosterone synthase (P450aldo) is specific for the zona fasciculata/reticularis and the zona glomerulosa, respectively. Mellon et al. (12) examined the expression of P45011ß and P450aldo messenger RNA (mRNA) in RNA prepared from whole rat fetuses, offering an approximation of when adrenal cells express zone-specific steroidogenic enzymes. However, no attempt was made to examine the distribution of different cellular phenotypes and thus, this study could not delineate the pattern or timing of zonal development. Recently, Mitani (13) examined the expression of P450aldo and P45011ß protein in the developing rat adrenal at the cellular level by immunohistochemistry; however, studies were presented only for rats from embryonic day 18 (E18) and older and did not asses the ability of the identified cells to produce steroids.
The present study defines the timing and pattern of development of the zona glomerulosa and the zona fasciculata/reticularis in the fetal rat adrenal using in situ hybridization histochemistry and immunohistochemistry. P450aldo and P45011ß mRNA and protein were monitored to identify areas within developing adrenals expressing the glomerulosa or fasciculata/reticularis cell phenotype. RIAs for aldosterone and corticosterone in adrenal homogenates were used to confirm the functional capacity of the cells expressing these enzymes.
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Materials and Methods |
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Animals
Male and day 14 timed-pregnant Sprague-Dawley rats (Harlan
Sprague-Dawley, Inc., Indianapolis, IN) were housed under a 12-h light,
12-h dark cycle (lights on 0600–1800 h) with food and water available
ad libitum. Animals were maintained and cared for in
accordance with the NIH Guide for the Care and Use of Laboratory
Animals. Experimental procedures were approved by the University of
Minnesota Animal Care Committee.
Protocols
Tissue preparation. Pregnant and male adult rats were killed
by CO2 asphyxiation; fetuses were immediately excised and
decapitated; day 1 (D1) newborn rats were decapitated within 1–2 h
after separation from their mothers. Eight to sixteen adrenals from day
16 (E16), 17 (E17), 18 (E18), 18.5 (E18.5), 19 (E19), and 20 (E20)
fetuses, D1 newborns and adult male rats were collected for
histological analysis. Adrenal glands were removed, cleaned of
surrounding fat, then mounted in O.C.T. compound and frozen in
isopentane cooled in liquid nitrogen. Tissue sections (14
µM) were cut in a cryostat, thaw-mounted on ProbeOn
slides, and stored desiccated at -20 C.
Analytical methods
Immunohistofluorescence. The P450aldo polyclonal antibody
was generated by immunizing New Zealand White rabbits with the peptide
MAP-KVRQNARGSLTMDVQQ, corresponding to amino acids 175–192 of the
P450aldo enzyme. The P45011ß monoclonal antibody was generated in
Swiss-Webster mice immunized with the peptide KNVYRELAEGRQQSC,
corresponding to amino acids 272–285 of the P45011ß enzyme, and
conjugated to chicken serum albumin. The spleens corresponding to the
mice with the highest titer were fused to a SP-2 myeloma cell line, and
the clones were selected for their ability to bind immobilized
sonicated zona glomerulosa mitochondria from rats on a low sodium diet.
Immunohistofluorescent staining for P450aldo and P45011ß was
simultaneously performed on tissue sections that were fixed with
Zamboni’s fixative (0.2% picric acid, 2% paraformaldehyde in 0.16
M phosphate buffer) for 10 min at room temperature then
washed with PBS (3 x 5 min). Sections were blocked with NDS
[1:10 in PBS with 0.1% Triton X-100 (PBST)] for 30 min at
room temperature and incubated with primary antisera (rabbit
anti-P450aldo, 1:100 in PBST and mouse anti-P45011ß, 1:125 in PBST)
for 16–18 h at 4 C. Sections were then rinsed in PBST (3 x 10
min) and incubated with secondary antibody (Cy3-labeled DAR and
FITC-labeled DAM, 1:200 and 1:100, respectively, in PBST) for 1 h
at room temperature. To reduce nonspecific labeling, secondary
antibodies were incubated before use with 10% normal rat serum and 5%
NDS for 1 h at room temperature and then centrifuged at 1200
x g at 4 C for 30 min; the supernatant served as the
secondary antibody. Sections were then rinsed in PBST (3 x 10
min) and then coverslipped with SlowFade. Control slides consisted of
preabsorbing the anti-P450aldo antibody with a truncated version of the
immunizing peptide, using a nonimmune rabbit serum in place of
anti-P45011ß and omitting the primary antibodies from the staining
process. Control slides were negative for staining for all
developmental ages studied (data not shown).
In situ hybridization. The in situ hybridization technique was performed as outlined previously (14). Slides were incubated at 42 C overnight with 5–10 ng of 35S-labeled probe per ml of hybridization solution. Sections were washed 5 x 15 min in 1 x SCC at 54 C, dehydrated, air-dried, and exposed to Biomax film. Some sections were dipped in NTB-2 emulsion and exposed for 6–10 days. Dipped slides were developed, counterstained with bisbenzimide and dehydrated.
Oligonucleotide probes, synthesized by Keystone Labs (Menlo Park, CA), were designed to detect P450aldo [nucleotides (nt) 918–953] (15) and P45011ß (nt 814–849) (16) mRNA. The specificity of probes used to detect steroidogenic enzyme mRNAs was confirmed by three methods. First, it was demonstrated that the hybridization signal could be blocked by addition of excess (100x) unlabeled probe to the hybridization solution; second, for each antisense probe, hybridization was not affected by addition of unlabeled probe antisense to another steroidogenic enzyme mRNA; and third, use of labeled sense probes for each of the steroidogenic enzyme mRNA resulted in no hybridization signal (data not shown). For the E18 adrenal sections, in situ hybridization was performed by a modification of the method above (Levay-Young and Engeland, submitted). Briefly, the probe sequences listed above were resynthesized (Life Technologies, Bethesda, MD) with a 3' extension (-5'-A10GGGCCCCGGG-3') designed to hybridize to a second "labeling" oligonucleotide (5'-T30CCCGGGGCCC-3'). Probe and labeling oligonucleotides were hybridized and then extended with [33P]-dATP (Amersham, Chicago, IL) and exo-Klenow DNA polymerase (Promega, Madison, WI). The partially double stranded probes generated in this way had approximately 10-fold higher specific activity than probes used above, but the hybridization specificity of the modified probes was unchanged. In addition, the [33P]-labeled probes produce the same pattern of hybridization for P450aldo and P45011ß mRNA in fetal and adult adrenals as that observed using the [35S]-labeled probes (data not shown).
Photomicroscopy. Optical images were collected using a monochrome CCD camera (Cohu, San Diego, CA), captured with a Scion LG-3 frame grabber and processed on a Macintosh IIcx computer using the public domain NIH Image 1.6 program (by W. Rasband (NIH) and available from the Internet by anonymous ftp from zippy.nimh.nih.gov). Images were then pseudocolored and overlapped with Adobe Photoshop 3.0 or 4.0 software.
RIA. Adrenals were homogenized with 20% ethanol in PBS, then centrifuged at 1200 x g at 4 C for 5 min. Supernatants were collected for steroid analysis, and pellets were resuspended in 1 N NaOH for measurement of protein content. Adrenal corticosterone and aldosterone content were measured by RIA kits. The intraassay and the interassay CV for corticosterone was 7.6% and 13.3%, respectively; the intraassay and the interassay CV for aldosterone was 15.9% and 21.8%, respectively. Cross-reactivity for the corticosterone RIA, as defined by the manufacturer, was 0.34% for deoxycorticosterone and 0.03% for aldosterone. Cross-reactivity for the aldosterone kit, as defined by the manufacturer and verified in our laboratory, was 0.033% for 18-OH-corticosterone and 0.002% for corticosterone. Steroid content was expressed relative to adrenal protein measured using the BCA protein assay kit.
Statistical analysis. Values are expressed as the mean ± SEM; the range is given where n = 2. Before analysis, adrenal protein, aldosterone and corticosterone content data were subjected to logarithmic transformation to reduce statistical variance. Differences between groups were assessed by ANOVA. When ANOVA showed a significant difference within an experiment, the Fisher’s LSD test was used to identify differences between groups. For all statistical analyses, differences were considered significant when the test yielded a P < 0.05.
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Results |
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). Nonstaining areas
correspond to medullary cells (defined as SA-1 positive (17)/P45011ß
negative cells; data not shown) and blood vessels (11). The staining
pattern for P45011ß remained relatively constant from E16 to D1 (Fig. 1, A–E) as SA-1 positive/P45011ß negative medullary cells merged in
the center of the gland. Positive staining for P450aldo was also
detected at E16; however, staining was observed as single cells, or
small clusters of cells, dispersed throughout the adrenal (Fig. 1A).
This spatial distribution was markedly different from that found in the
adult gland, in which P450aldo staining was limited to a 4- to
5-cell-wide zone under the capsule (Fig. 1F). At E17, there appeared to
be a decrease in the number of P450aldo positive cells compared with
the E16 gland (Fig. 1B), but the distribution was unchanged. By E18 and
E18.5 an increased number of P450aldo positive cells was observed, and
the distribution of these cells had changed from E16, becoming more
localized under the capsule and somewhat separate from the region of
P45011ß staining (Fig. 1C). This is more clearly seen in E19 glands
that demonstrated a marked increase in P450aldo staining, occurring as
a discontinuous ring of P450aldo positive cells adjacent to the capsule
with some residual staining within the region of P45011ß staining
(Fig. 1D). At E19, a clear distinction between an outer and inner
cortex was apparent; in addition, a region of cells located between
P450aldo and P45011ß positive cells did not stain, corresponding in
enzymatic expression to the zona intermedia of the adult gland. Unlike
the scattered P45011ß negative medullary cells seen at days 16–18.5,
these zona intermedia-like P45011ß negative cells were also SA-1
negative (data not shown). The postnatal day 1 (D1) adrenals had a
pattern of P450aldo staining that was similar to that of the adult
gland. (Fig. 1E). As in the adult, there were no double labeled cells
(i.e. cells positive for both P45011ß and P450aldo)
detected in any fetal rat adrenal examined.
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, C, E, and G) and adult glands
examined and interspersed with P45011ß negative patches of
presumptive medullary cells.
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). The absence of hybridization at these time points is likely
due to a lack of sensitivity of the in situ hybridization
technique used, combined with a low level, single cell distribution of
P450aldo expression, as was seen at the protein level. Expression of
P450aldo mRNA was first detected in E18 adrenals as small, localized
patches of specific hybridization under the capsule (Fig. 2D). At E19,
P450aldo mRNA hybridization was in the form of a ring, with
intermittent unhybridized gaps, underlying the capsule (Fig. 2F). This
ring of P450aldo mRNA hybridization was more continuous in D1 and adult
glands (Fig. 2H), and closely matched the distribution of P450aldo
protein discussed above.
Measurement of aldosterone and corticosterone
Table 1 shows corticosterone and
aldosterone content of adult, D1 and fetal adrenals. Corticosterone was
present in all fetal adrenal homogenates and increased significantly
between E17 and E18. Corticosterone content then decreased at E19 to
markedly low levels at D1. Although the corticosterone content of adult
adrenals was also lower than that measured in all except E16 fetal
glands, it was significantly higher than the corticosterone content of
D1 adrenals. Aldosterone was also present in all fetal adrenal
homogenates measured and increased between E17 and E18, and again
between E19 and D1. Unlike corticosterone content, there was no
difference in aldosterone content between D1 and adult glands.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence and distribution of fasciculata/reticularis cells throughout the adrenal at E16, gradually changing to produce an adult-like cortex surrounding a medulla at D1 is not surprising. Corticosterone content of the developing adrenal increases between E17 and E18, and then drops significantly at birth, which is in agreement with previous studies (18, 19, 20). Adrenal corticosterone content at D1 was reduced relative to that measured in adult glands that had been harvested after stress; this observation suggests that reduced corticosterone content at D1 may reflect the onset of the stress hyporesponsive period (21). The most novel finding of this study is that the ontogeny of the zona glomerulosa appears to proceed through two developmental stages. The first stage occurs as early as E16 and is characterized by the presence of single or small clusters of glomerulosa cells distributed throughout the adrenal. During an interval between E16 and E17, expression of the glomerulosa phenotype decreases in the inner gland, with only a few aldosterone producing cells remaining in the inner cortex. The second stage is characterized by the appearance of functional glomerulosa cells underlying the capsule between E18 and E19 and a subsequent increase in cell number to produce an adult-like zona glomerulosa by D1.
It is possible that the transition between the two stages of zona glomerulosa development occurs via migration of glomerulosa cells from the inner to outer cortex. However, both the rapid change in glomerulosa cell distribution, and the virtual disappearance and subsequent reappearance of glomerulosa cells over a 2-day period, suggests that migration is an unlikely mechanism. It seems more likely that glomerulosa cells present in the inner cortex at E16 either differentiate into fasciculata/reticularis cells (22) or die (23) between E16 and E17. Subsequently, cells adjacent to the capsule would differentiate to become glomerulosa cells and then proliferate (24) to form the adult-like zonation. Based on work in adult adrenal regeneration and plasticity, potential progenitors of the glomerulosa phenotype include capsular (7, 25, 26) and intermediate (3) cells. The early expression of P45011ß denoting the zona fasciculata/reticularis, followed by the appearance of P450aldo in cells underlying the capsule in late gestation, supports the independent establishment of each zone during fetal development as proposed by the zonation theory (5).
The mechanisms that mediate the two stages of zona glomerulosa
development are unknown; we favor the hypothesis that each stage is
regulated independently of the other. The disappearance of P450aldo
positive cells between E16 and E17 following a presumed differentiation
from an undifferentiated precursor cell is reminiscent of transient
17
-hydroxylase mRNA expression in a subpopulation of mouse adrenal
cells between E12.5 and birth (27), and of P45011B3 gene expression
that is elevated only in neonatal adrenals (28, 29). The subsequent
abrupt appearance of P450aldo protein and mRNA in the adult-like
subcapsular zona glomerulosa toward the end of gestation suggests that
classic regulators of glomerulosa function initiate this event,
independent of the earlier events.
One potential regulator of this second stage of zona glomerulosa development is the renin-angiotensin system. It has been shown that midgestational fetal bovine cells respond to AII through the AII type 1 receptor (AT1) by increasing P450c18 activity (the aldosterone synthase P450 enzyme found in bovine adrenals) and aldosterone production in vitro (30). In the rat, transcripts for both AT1 receptor subtypes (AT1A and AT1B) are found in the zona glomerulosa beginning at E17, and AT1A mRNA expression peaks at E19 (31). The AII type 2 receptor (AT2) has also been identified in the periphery of the developing adrenal by E15 (32). Because the AT1 receptor is implicated as the primary receptor through which AII regulates aldosterone production, and the AT2 receptor may also regulate growth and differentiation in developing tissues, it follows logically that AII may upregulate expression of P450aldo in the outer cortex during late gestation. Of course, this raises the question of what regulates AII receptor subtype expression in the zona glomerulosa.
Whereas the relevance of glucocorticoids in the normal development of the fetus is well documented (33), a similar role for mineralocorticoids is still unknown. None the less, it is likely that fetal aldosterone production has physiological significance during development. Recently, Brown et al. (34) investigated the ontogeny of mineralocorticoid receptor (MR) mRNA and 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) mRNA expression in the mouse and found co-expression of these mRNAs in the kidney and colon between E17.5 and birth. Because 11ß-HSD2 is believed to inactivate glucocorticoids, which have a higher affinity for the MR than aldosterone, these data support the idea that aldosterone produced late in gestation can bind to MR in fetal tissues to affect growth or function. Although it is not clear what function glomerulosa cells present in the gland before E19 have in fetal physiology, it will be interesting to determine what controls the initial appearance and final fate of these early P450aldo expressing cells. These issues will be clarified by a careful examination of the differentiation of adrenal steroidogenic cells during development.
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Acknowledgments |
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Received March 4, 1998.
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