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Department of Biochemistry, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan
Address all correspondence and requests for reprints to: Dr. Fumiko Mitani, Department of Biochemistry, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan 160-8582. E-mail: fmitani{at}med.keio.ac.jp
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Abstract |
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Introduction |
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We previously demonstrated immunohistochemically that aldosterone synthase cytochrome P450 (P450aldo) and steroid 11ß-hydroxylase (P45011ß) are specifically localized in zG and zF, respectively (4, 5, 6), defining the biochemical basis for the functional zonation of the rat adrenal cortex. Using these enzymes as markers for the respective cortical zones, we further found a new zone between zG and zF, where cells stained with neither anti-P450aldo nor anti-P45011ß antibodies; in other words, they did not express both P450aldo and P45011ß (6). We also obtained evidence that DNA-synthesizing cells were present in and around this zone, and that cells labeled with 5-bromo-2'-deoxyuridine (BrdU) migrated inward from around the new zone in a time-dependent manner. Therefore we have proposed that this zone is an undifferentiated zone and contains stem cells for the adrenal cortex.
Development of the adrenal gland has been studied from histological and biochemical points of view (2, 7, 8, 9). According to Roos (7), the adrenal gland on gestational day 18 seems to be histologically mature, with a distinct capsule, a basophilic zG, and palisaded zF and zR. In fact, it was reported that the considerable amount of glucocorticoid was produced at this stage of gestation (10, 11). However, the amount of aldosterone produced was very low (10) compared with that after birth. Therefore, the discrepancy between the morphological features and the steroidogenic capacity of fetal adrenal glands should be clarified at the molecular level. Employing the adrenocortical zone-specific markers, i.e. P450aldo and P45011ß, together with BrdU and tyrosine hydroxylase for labeling DNA-synthesizing cells and medullary cells, respectively, we investigated the maturation of the adrenal gland, focusing on whether or not the undifferentiated zone described above is involved.
The results indicate that the basic structure of the adrenal gland, i.e. a distinct cortex and medulla, and functional zonation in the cortex including the undifferentiated zone (6), was almost established at the time of birth. In addition, after birth, DNA-synthesizing cells were found around the undifferentiated zone and then they migrated with time from that area inwards. This contrasts with the distribution seen during the fetal period, where DNA-synthesizing cells were scattered throughout the adrenal gland and then proliferated without significant migration. A preliminary report of this work has appeared elsewhere (12).
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Materials and Methods |
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Immunohistochemical studies
Immunohistochemical localization of steroidogenic enzymes was
performed on 6-µm sections of a fresh-frozen adrenal gland as
previously described (5, 6). The antibodies used were raised, in
rabbits, against rat P450aldo (5), rat P45011ß (5), and bovine
P450scc (cholesterol side-chain cleavage enzyme) (13). Since an
oligopeptide used to raise antibody against P45011ß (5) was the same
as the corresponding amino acid sequence of P450c11B3 (14), the
antibody used to identify P45011ß in this study probably
cross-reacted with P450c11B3 as well. Antiguinea pig P450c21 (steroid
21-hydroxylase) antibody (15) and antibovine Ad4-binding protein
(Ad4BP) antibody (16) were generous gifts from Drs. S. Takemori and S.
Kominami of Hiroshima University (Hiroshima), and Dr. K. Morohashi of
the National Institute for Basic Biology (Okazaki), respectively.
Commercially available antibodies for rat tyrosine hydroxylase, bovine
phenylethanolamine-N-methyl transferase, Fos family protein
(17), and rat resident macrophages (18) were products of Sigma Chemical Co. (St. Louis, MO), PROTOS Biotech Co. (New York, NY),
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and BMA
Biochemicals Ltd. (Augst, Switzerland), respectively. Double staining,
i.e. staining with two different antigens of a single
specimen, was carried out by essentially the same method as that of
Nakane (6, 19).
To detect DNA-synthesizing cells, pregnant rats were injected ip with 50 mg 5-bromo-2'-deoxyuridine (BrdU) (Sigma Chemical Co.)/kg body wt 1 h before they were killed (20, 21), and then the fetuses were collected from the uterine horns. BrdU was also injected ip into postnatal rats 1 h before killed. Immunohistochemical staining with BrdU of fresh-frozen or paraffin adrenal sections was performed using a mouse monoclonal anti-BrdU antibody (Becton Dickinson and Co., Mountain View, CA) as described previously (22). Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) (23) was performed on alcohol-fixed paraffin sections (4-µm) of the adrenal gland using the monoclonal antibody PC-10 (DAKO Japan Co., Ltd., Kyoto Japan) (24, 25).
Enzymatic histochemical localization of alkaline phosphatase and
dipeptidyl aminopeptidase IV
To visualize capillary networks of the adrenal gland, fresh
frozen-adrenal sections (6-µm) were stained histochemically for the
activities of alkaline phosphatase (ALP) and dipeptidyl aminopeptidase
IV (DAP IV) (26, 27) according to the method of Batra et al.
(27). Double staining with ALP and DAP IV of the same section has been
used to demonstrate the capillary bed in many organs (26); the reaction
for ALP reveals the arterial part of capillaries, whereas DAP IV
activity reveals the endothelium in the venous part of the capillary
bed. The function of DAP IV in the endothelium, however, has not been
clarified, and it is only suggested that DAP IV takes a part in the
degradation of some vasoactive peptides (26).
In situ hybridization analysis
In situ hybridization analyses were performed on
fresh-frozen adrenal sections with digoxigenin (DIG)-labeled cRNA
probes (28) for P450aldo, P45011ß, P450scc, and angiotensin 1B
(AT1B) receptor messenger RNAs (mRNAs) by the slightly
modified method of Holtke and Kessler (29). cDNA fragments encoding
P450aldo [nucleotide positions 784–948 (30)], P45011ß (784–945
(31)], P450scc (32) (1038–1381 (33)], and AT1B receptor
(583–2009) (34) were cloned into pGEM-4Z (Promega Biotech, Madison, WI). DIG-labeled sense and antisense RNA
probes were prepared with T7 and SP6 RNA polymerase, respectively, in
the presence of DIG-labeled UTP (Roche Molecular Biochemicals, Mannheim, Germany). RNA probes were then partially
degraded to an average size of 100–150 nucleotides by alkaline
hydrolysis. Rat AT1B receptor cDNA was a generous gift from
Dr. K. Sandberg of NIH (Bethesda, MD).
Fresh-frozen sections on 3-aminopropyltriethoxysilane (APS)-coated glass slides were stood at room temperature and then at 37 C for 30 min each. The slides were then incubated at room temperature sequentially in 4% PFA/PBS for 40 min, acetone for 5 min, and 0.1% Triton X-100/PBS for 5 min. In between the treatments, the slides were washed with PBS. They were then successively treated at room temperature with proteinase K (10 µg/ml) (Sigma Chemical Co.) for 30 min (for adult adrenal glands) or 15 min (for fetal and early postnatal adrenal glands), 4% PFA/PBS for 5 min, and 0.2% glycine/PBS for 10 min. The slides were immersed in a prehybridization medium comprising 2 x SSC (1 x SSC: 0.15 M NaCl and 0.015 M trisodium citrate) and 50% formamide for 1 h at 45 C. DIG-labeled RNA probes were diluted to 0.5 µg/ml with a hybridization medium (20 mM Tris/HCl, pH 8.0, containing 2.5 mM EDTA, 0.3 M NaCl, 50x Denhardt’s solution [Sigma Chemical Co., 0.02% Ficol/0.02% BSA/0.02% polyvinylpyrolidine], 10% dextran sulfate, 1 mg/ml yeast transfer RNA[Sigma Chemical Co.], and 50% deionized formamide). The hybridization was carried out under a coverslide in a moist chamber at 45 C. After about 16 h, the coverslides were gently removed and the glass slides were washed in 2 x SSC containing 50% formamide for 60 min at 45 C, followed by a brief wash with 10 mM Tris/HCl-0.5 M NaCl, pH 8.0, for 5 min twice. They were then treated with ribonuclease A (Sigma Chemical Co.; 20 µg/ml in 10 mM Tris/HCl-0.5 M NaCl, pH 8.0) for 30 min at 37 C, and washed with 2 x SSC containing 50% formamide at 45 C for 1 h twice and at room temperature for 1 h once. The slides were next washed in buffer 1 (100 mM Tris/HCl, pH 7.5, containing 150 mM NaCl), and then incubated with 0.5% blocking reagent (Roche Molecular Biochemicals) in buffer 1 at room temperature for 30 min. After a brief wash in buffer 1, the slides were treated with 5% normal sheep serum in buffer 1 for 30 min. The slides were then incubated at room temperature for 1 h with ALP-conjugated sheep anti-DIG antibody (Roche Molecular Biochemicals) in buffer I containing 5% normal sheep serum. The unbound antibodies were removed by washing with buffer 1 three times for 10 min each. The slides were then equilibrated with buffer 2 (0.1M Tris/HCl, pH 9.5, containing 0.1 M NaCl and 0.05 M MgCl2), and incubated with an ALP substrate solution (0.4 mM 4-nitrobluetetrazolium chloride, 0.42 mM 5-bromo-4-chrolo-3-indolyl phosphate [Roche Molecular Biochemicals], and 0.27 mg levamisole/ml in buffer 2). The color reaction (dark purple-blue) was stopped by immersing the slides for 10 min in buffer 3 (10 mM Tris/HCl, pH 8.0, containing 1 mM EDTA). For control experiments to confirm the specificity of hybridization signals, DIG-labeled sense cRNA probes were used at the same concentrations as the corresponding antisense probes. However, no specific signal was observed.
Detection of fragmented DNA
Fragmented DNA in adrenal glands was detected in situ
by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick
end labeling (TUNEL) method of Gavrieli et al. (35). Adrenal
glands were fixed overnight at 4 C in 4% paraformaldehyde/PBS and then
embedded in paraffin. Four-micrometer-thick sections were
deparaffinized by heating at 50 C for 30 min and then washing three
times in xylol for a total of 10 min.
The rehydrated adrenal sections were subjected to in situ end labeling of fragmented DNA according to the instructions for an In situ Cell Death Detection Kit (Roche Molecular Biochemicals). A section as a positive control was incubated with DNase I (Roche Molecular Biochemicals) at 10 U/µl in 0.1 M Na-acetate, pH 5.5, containing 1 mM CaCl2 and 5 mM MgCl2 for 10 min at room temperature before incubation with the mixture comprising terminal deoxynucleotidyl transferase and biotinylated dUTP in the In situ Cell Death Detection Kit of Roche Molecular Biochemicals. All nuclei in the glands were stained, showing that they were completely fragmented.
Immunoblot analysis
Homogenates of adrenal glands from pre- and postnatal rats were
subjected to SDS-PAGE (7.5% polyacrylamide gel) (36), and then blotted
onto polyvinylidene difluoride membranes (4). Enhanced
chemiluminescence Western blot reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK) were employed for identification
of P45011ß after incubation of the membranes with anti-P45011ß
antibody. The intensity of signals accumulated on a phosphor screen was
measured after 30 min exposure at room temperature with a Molecular
Imager GS-525 (Nippon Bio-Rad Co., Tokyo, Japan) within
the range of linear responses.
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Results |
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shows the localization of
P450aldo, P45011ß, and tyrosine-hydroxylase (TH) together with that
of the microvasculature in rat adrenal glands during late gestational
development. At G-16, P450aldo-positive cells were undetectable in the
adrenal gland (Fig. 1, G-16:P450aldo), whereas P45011ß-positive cells
were present throughout the gland (Fig. 1, G-16:P45011ß), although
the intensity of the staining was somewhat faint in the outermost
portion of the gland. Thus, the bulk of cortical cells appeared to have
the properties of zF cells. Cells that did not stain with the
anti-P45011ß antibody were observed inside the gland, most of which
turned out to stain positive for tyrosine hydroxylase (TH) (Fig. 1, G-16:TH), which is a rate-limiting enzyme in catecholamine biosynthesis
and is known as a sensitive index of sympatho-adrenal precursors (1, 37). The TH-positive cells did not concentrate in the center of the
gland as seen in the adult medulla, but were scattered, indicating that
a medullary structure had not been formed at this stage of gestation.
Almost no ALP and DAP IV activities were detected in a G-16 fetal
adrenal gland (Fig. 1, G-16:ALP & DAP IV). Thus, the microvasculature
of the adrenal gland at G-16 was immature.
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, G-19:P450aldo), although a narrow layer consisting of relatively
small and tightly-packed cells corresponding to zG cells was observed
in the outermost portion of the gland. Within the gland, most of the
cells that did not stain for P45011ß were TH-positive ones as in the
adrenal gland of a G-16 fetus (Fig. 1, G-19:TH). Some of the
TH-positive cells also stained with antibody against
phenylethanolamine-N-methyl transferase, an
epinephrine-synthesizing enzyme specifically present in the medulla
(38) (data not shown). At this time, capillaries became histochemically
detectable in the adrenal gland (Fig. 1, G-19:ALP & DAP IV).
At G-20, P450aldo-positive cells were now seen in some areas of the
outermost portion of the cortex (Fig. 1, G-20:P450aldo), and a
P45011ß-positive cell layer was seen inside of the P450aldo-positive
cell layer (Fig. 1, G-20:P45011ß), indicating the beginning of the
functional zonation of the adrenal cortex. At around this stage,
TH-positive cells increased greatly in number, although they remained
intermingled with cortical cells (Fig. 1, G-20:TH).
Expression of P450aldo, P45011ß, tyrosine hydroxylase, and
formation of the microvasculature in rat adrenal glands during the
early postnatal period
The localization of P450aldo, P45011ß and tyrosine hydroxylase
(TH) together with that of the microvasculature in adrenal glands
during the early postnatal period is shown in Fig. 2. One day after birth (P-1), a
P450aldo-positive layer was clearly recognized in the outer part of the
gland, showing that the number of functionally active zG cells had
increased (Fig. 2, P-1:P450aldo). The intensity of P450aldo staining
increased afterwards with time examined up to P-25 (P-25:P450aldo). At
later stages, however, the intensity decreased (Fig. 2, adult:P450aldo).
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also shows the double staining of adrenal sections with
anti-P450aldo and anti-P45011ß antibodies. On postnatal day 1, a
narrow P450aldo-positive cell layer was present just beneath the
capsule, as described above, and then a wide P45011ß-positive cell
layer was observed (Fig. 2, P-1:P450aldo & P45011ß). Furthermore, in
between, a cell layer without P450aldo and P45011ß (6) was clearly
recognized for the first time, and this zone was thereafter constantly
observed in the gland (Fig. 2, P-25 and adult:P450aldo & P45011ß).
Meanwhile, TH-positive cells were still intermingled with
P45011ß-positive cells at P-1, although they were rather gathered in
the center of the gland (Fig. 2, P-1:TH). By the third day of life,
TH-positive cells, which were increasing in number, were coalesced into
a well-defined medulla (data not shown) like in adrenal glands at P-25
(Fig. 2, P-25:TH) and in adult rats (Fig. 2, adult:TH).
In the adrenal gland at P-25, a cortical area near the medulla still
strongly stained with anti-P45011ß antibody (Fig. 2, P-25:P450aldo &
P45011ß). Because such intensity of P45011ß staining was stronger
than that in zR of adult adrenal glands (Fig. 2, adult:P450aldo &
P45011ß, and refer to Ref. 6), it is likely that zR cells had not
appeared yet in P-25 rats. The observation that anastomosing sinusoidal
capillaries, which is one of typical features of zR, were not apparent
at the innermost edge of the cortex (P-25:ALP & DAP IV) may also
support this statement. zR cells at P-25, if any, might be very
immature and low in number. Within the following several days, zR
became clearly discernible morphologically in the innermost part of the
cortex (data not shown), showing the establishment of the cortical
zonation.
As described in Materials and Methods, the antibody raised against rat P45011ß and used in this study probably cross-reacted with P450c11B3 (14). The expression of P450c11B3 mRNA has been demonstrated in the zFR of rat adrenal cortex from several days to 1 month after birth (14). Therefore, immunohistochemical staining of early postnatal adrenal sections with the anti-P45011ß antibody may reveal also the presence of P450c11B3. However, no further examination was performed to discriminate between P45011ß and P450c11B3, because P450c11B3 has capacity of 11ß- and 18-hydroxylation of deoxycorticosterone, but no ability to produce aldosterone.
Localization of mRNAs for P450aldo, P45011ß, and P450scc in
adrenal glands during the late gestational and early postnatal
stages
The developmental expression of zone-specific enzymes described
above was further examined at the mRNA level by in situ
hybridization together with that of P450scc, which is the rate-limiting
enzyme functioning at the early common step in adrenocortical
steroidogenesis. As shown in Fig. 3, P450aldo mRNA was undetectable at G-18 (G-18:P450aldo mRNA) as the
P450aldo protein was not observed until G-20 (refer to the results in
Fig. 2), whereas P45011ß and P450scc mRNAs were detected already
throughout the adrenal gland at G-16, except for in TH-positive cells
and the outermost region of the gland. In the outermost region, signals
of P45011ß mRNA and P450scc mRNA were almost absent and weak,
respectively (G-18:P45011ß mRNA, P450scc mRNA).
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), whereas P450scc mRNA was expressed in P450aldo
mRNA-expressing zG cells and P45011ß mRNA-expressing zF cells, and
also cells in the undifferentiated zone at different intensities
(P-1:P450scc mRNA).
The signal intensity of P45011ß mRNA like that of P450scc mRNA right
after birth (Fig. 3, P-1), however, was weaker than those before birth
(Fig. 3, G-18). In the adrenal gland at P-25, the signals were
intensified again (Fig. 3, P-25). According to the results of
immunoblot analyses shown in Fig. 4, the
amounts of the P45011ß protein in homogenates of rat adrenal glands
at G-18, G-20, P-1 and P-10 were not significantly different from each
other. The analytical immunoblot data thus agreed well with the
immunohistochemical results (refer to Figs. 1 and 2), which showed
almost no difference in the intensity of P45011ß staining in adrenal
glands at G-20 and P-1. The significance of the decrease in the
P45011ß mRNA level right after birth will be discussed in the
following section.
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).
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, G-18,
G-20), which also stained with the antibody against P45011ß. From P-1
(Fig. 5, P-1) through 1 week after birth, however, the staining
intensity of the Fos-family protein was weak. Such a phenomenon
correlated well with the weak signal of P45011ß mRNA during the
neonatal period as described above (Fig. 3, P-1:P45011ß mRNA),
although it remains to be determined why the the down-regulation of
the P45011ß mRNA level did not lead to a decrease in the amount
of the P45011ß protein (refer to Fig. 4). The strong intensity of the
Fos-family protein staining was seen again in the nuclei of cortical
cells around 2 weeks after birth (Fig. 5, P-16). Nuclei in zG cells did
not stain with the anti-Fos-family protein antibody (see zG at P-16 and
in adults in Fig. 5).
Expression of angiotensin receptor mRNA in adrenal glands during
development
We have previously shown that the expression of P450aldo is
stimulated by angiotensin II (41, 42), which acts via the angiotensin
receptor (AT). Therefore, whether or not the expression of P450aldo
during adrenal development is related with expression of the
angiotensin receptor was investigated by in situ
hybridization. Among the angiotensin receptor subtypes so far reported,
AT1B receptor was chosen for this purpose because it is
known to be expressed predominantly in zG of the adult adrenal cortex
and is thought to be involved mainly in water and salt homeostasis
(34). As shown in Fig. 6, the adrenal
gland at G-16 already showed a signal of AT1B receptor mRNA
in its outer part (Fig. 6, G-16). The signal was always detected
afterwards in morphologically recognized-zG cells. The signal was
undetectable in zFR and the medulla.
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and Discussion).
Proliferating cells in adrenal glands during late gestational and
early postnatal development
To study the relationship between the differentiation of the
adrenal gland so far observed and replicating cells in the gland, the
localization of DNA-synthesizing cells and their behavior were
investigated during development.
In fetal adrenal glands at G-16 and G-18, cells containing BrdU-labeled
nuclei (BrdU-labeled cells), i.e. 19 ± 2% of total
adrenal cells, were found scattered throughout the gland (Fig. 7, G-16 and G-18:BrdU). From G-19 to the
neonatal period, BrdU-labeled cells, i.e. 6 ± 2% of
total adrenal cells, were found in two regions, one was the outermost
portion and the other was around the central region of the gland (Fig. 7, P-1:BrdU). One week after birth, these cells, i.e. 4
± 1% of total adrenal cells, were found in a subcapsular portion
including the undifferentiated zone (6) (Fig. 7, P-7:BrdU). Thirty days
after birth, most BrdU-labeled cells, i.e. 0.9 ± 0.2%
of total adrenal cells, were found around the undifferentiated zone and
the uppermost portion of zF (Fig. 7, P-30:BrdU), with some at the
border between the cortex and medulla like in adult adrenal glands (6).
Figure 7 also includes the localization of proliferating cell nuclear
antigen (PCNA), which is one of the endogenous markers for the
replicating cells (23). As shown in Fig. 7:PCNA, the localization of
PCNA-containing cells in fetal and early postnatal adrenal glands was
almost the same as that of BrdU-labeled cells, but their numbers were
greater than those of BrdU-labeled cells. This may be due to the fact
that BrdU is incorporated into the nuclei of cells in the S-phase (20, 21), whereas PCNA is present in the nuclei of cells in both the S- and
late G1-phases (23).
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). Three developmental stages were
chosen as times for BrdU-administration: G-16, the stage at which the
adrenal gland first appeared as an encapsulated ovoid mass; G-18, the
stage just before establishment of the cortical zonation; and P-10, the
stage of establishment of the cortical zonation and the medullary
structure.
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, A-1), as shown already
in Fig. 7. Two days later, i.e. at G-18, cells labeled with
BrdU at G-16 were still observed scattered over the gland (Fig. 8, A-2), although their number had increased to almost twice as many as
that at G-16. Similarly, cells incorporating BrdU at G-18 were found
throughout the whole adrenal gland as described (Fig. 7), and 2 days
later (i.e. G-20) about twice as many positive cells as at
G-18 were still present uniformly in the gland (data not shown). Ten
days (i.e. P-7) to 16 days (i.e. P-13) after
injection, BrdU-labeled cells were found to have moved with time closer
to the medulla except for some BrdU-labeled cells in the outermost
region of the gland. On the 23rd day (i.e. P-20) after
injection, only several BrdU-positive cells with very weak intensity
were detectable in the innermost portion of the cortex. These results
show that cells labeled with BrdU at G-18 proliferated without movement
in one direction during the fetal period, but after birth they migrated
centripetally and finally underwent degeneration at the border between
the innermost edge of the cortex and the medulla. When BrdU was
injected again into P-20 rats, which had received the first BrdU
injection at G-18, BrdU was incorporated into the nuclei of cells
around the undifferentiated zone, indicating again that cells in and
around the undifferentiated zone have replicating ability, and function
as stem cells for the adrenal cortex.
The results of the pulse-chase study after injection of BrdU into rats
at P-10 are shown in Fig. 8-B. BrdU-labeled cortical cells were present
abundantly in the outer portion, including the undifferentiated zone,
(Fig. 8, B-1) and then migrated toward the inside of the gland with
time (2 days after injection: Fig. 8-B-2, and 7 days after injection:
Fig. 8-B-3), leaving some BrdU-labeled cells in the peripheral region
of the gland behind. Furthermore, the numbers of BrdU-labeled cells in
both the cortex and medulla increased about twice in the first 2 days
(Fig. 8, B-2) and furthermore about 3-fold in 7 days (Fig. 8, B-3). In
early postnatal stages as well as in adulthood (6), the migration of
proliferating cortical cells from the undifferentiated zone may be
important for the growth and differentiation of the adrenal cortex.
Sites for physiological cell death in the adrenal gland during
development
It has been considered that the development of the adrenal gland
together with cortical zonation requires a dynamic balance among
growth, functional differentiation and physiological cell death, like
in many other tissues (43, 44, 45, 46). Therefore, the sites for physiological
cell death, which is called apoptosis or programmed cell death (44, 47), were investigated in the adrenal gland by detecting fragmented-DNA
in nuclei. The nuclear DNA-fragmentation has been reported to be one of
the reliable diagnostic criteria for the occurrence of apoptosis (35).
During the late gestational and neonatal stages, signals of
fragmented-DNA were found scattered throughout the adrenal gland (as a
typical example: Fig. 9, G-20
fragmented-DNA), but after birth they gradually became confined to the
border between the innermost edge of the cortex and the medulla (as a
typical example: Fig. 9, P-10 fragmented-DNA, and also refer to Ref.
48).
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, G-20 resident macrophage), while around the
time when the cortical zonation was established, they were found
abundantly in the center of the gland, i.e. the innermost
portion of the cortex and the medulla (Fig. 9, P-10 resident
macrophage). Thus Ki-M2R-positive cells were present around the area
where cells with fragmented DNA-containing nuclei existed (Fig. 9, fragmented DNA). Ki-M2R-positive cells seem to be involved in the
disposal of dying cells in the adrenal gland. These phenomena, taken together, imply a mechanism for cell renewal in the adrenal cortex where zonation is established and macrophages is localized in the center of the cortex; cortical cells present in and around the undifferentiated zone (6) have replicating ability, migrate centripetally, and finally undergo degeneration within resident macrophages present in the innermost part of the cortex.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although P45011ß (Fig. 1, G-16:P45011ß) as well as P450scc and
P450c21 (data not shown) were expressed already in adrenal glands at
G-16, P450aldo was detected for the first time in the outermost portion
of the adrenal gland at around G-20, just 1 day before birth (Fig. 1, G-20:P450aldo), showing the appearance of functionally active zG cells.
In fact, the amount of aldosterone in the fetal adrenal gland has been
reported to increase from around G-20, with a peak at around the time
of birth (10). In addition, renin (54), angiotensin converting enzyme
(55), and angiotensinogen (56), which are necessary to produce
angiotensin II, have been found to increase at around the time of
birth. The appearance of P450aldo at around G-20 observed in this study
(Fig. 1) is well coincident with these events. These reported phenomena
may further explain why there is no expression of P450aldo until G-20
even if AT1B receptor mRNA is already expressed in G-16
fetal adrenal glands (Fig. 6).
In addition, the onset of expression of P450aldo may also be related to the concentration of ACTH in the plasma of fetuses. According to the results of our recent studies involving adult rats, suppression or stimulation of ACTH secretion resulted in an increase or a decrease in the number of P450aldo-positive cells in zG, respectively (57). The ACTH concentration in rat fetal plasma has been reported to increase until G-19 and to decrease thereafter (58). Therefore, the low concentration of ACTH in plasma after G-19 (10, 11) may result in the expression of P450aldo. In any event, our results demonstrate that functional zonation of the adrenal cortex, including the undifferentiated zone (6), is established around the time of birth. According to the results of Nakahara et al., a lipid free-transitional zone (59), which resembles the undifferentiated zone (6), was morphologically recognizable at around the time of birth (9).
The cytokinetics of adrenocortical cells have been studied to
investigate the mechanism(s) underlying the process of the cortical
zonation by using [3H]-thymidine or BrdU as a marker for
replicating cells (22, 60, 61, 62, 63). Most studies have shown that
adrenocortical cells present in the subcapsular portion of the gland
have replicating ability, and those cells migrate centripetally from
that area toward the center, suggesting that stem cells of the cortex
are present in the outermost portion and differentiate to zF cells and
zR cells during migration. These results support one of currently
prevailing hypotheses for adrenocortical cytogenesis, i.e.
migration theory, originally expressed by Gottschau (64). The existence
of stem cells in adult adrenal glands has been inferred also from
observations, such as regeneration of the adrenal cortex after
enucleation (22, 65, 66) or the growth and differentiation of some
adrenocortical cells in cultures under certain conditions (67, 68). On
the other hand, we have recently proposed that stem cells of the
adrenal cortex of adult rats exist in and around the undifferentiated
zone between zG and zT, based on the functional and morphological
observations (6). Compared with numerous reports on the cell renewal
system in the adult adrenal gland, those concerning the cytogenic
system in the fetal adenal gland are very few. The earliest adrenal
glands so far examined were from rats at G-20, i.e. 1 day
before birth, where cortical cells in the outer portion of the gland
predominantly incorporate [3H]-thymidine and then they
migrate centripetally (61) like in the adult adrenal gland. According
to our present results, in the adrenal gland at G-20 the cortical
zonation begins, and most DNA-synthesizing cells are present in the
outer portion of the gland as in the adult adrenal gland. In this
study, the adrenal glands of fetuses at earlier stages than G-20,
i.e. G-16 and G-18, were employed for the first time to
investigate the localization and behavior of DNA-synthesizing cells in
fetal glands. Adrenal glands at these stages of gestation were immature
both structurally and functionally, i.e. the cortical cells
and the medullary cells were intermingled with each other, and the
cortical zonation was not yet recognizable (Fig. 1). In such adrenal
glands, DNA-synthesizing cells were found scattered throughout the
adrenal gland (Fig. 7). In addition, they proliferated rapidly without
significant migration (Fig. 8). These results are consistent with the
view of Morley et al. (69) in that the centripetal migration
of replicating cells observed in the adult adrenal cortex is not a
major mechanism for growth of the fetal adrenal cortex. They observed a
variegated and radial pattern of ß-galactosidase reporter staining in
the adrenal cortex of adult transgenic mice carrying a steroid
21-hydroxylase promoter/ß-galactosidase reporter gene, but a
variegated island pattern of ß-galactosidase reporter staining in the
glands of fetal transgenic mice (69). Taken together, these results
suggest that there exists a distinct cellular mechanism(s) contributing
to adrenal development at gestational stage, which is different from
that in mature adrenal glands.
Finally, whether or not cortical cells of the undifferentiated zone,
i.e. stem cells of cortical cells, observed in adult adrenal
gland (6) exist in fetal adrenal glands should be mentioned. As
described, in fetal adrenal glands before G-20, cortical zonation and
even separation between the cortex and medulla are not observed, and
there exist abundant DNA-synthesizing cells throughout. Therefore, in
preliminary experiments, anti-Ad4BP/SF-1 (70, 71) antibody was employed
to label all cortical cells in fetal adrenal glands. Ad4BP/SF-1 has
been reported to be a steroidogenic tissue-specific nuclear receptor
(70, 71), and in the adrenal gland it is expressed in the nuclei of
cortical cells including cells of the undifferentiated zone (16). As
shown in Fig. 10 (A and the magnified
view in B), most cells with Ad4BP-positive nuclei (black
dots) in G-16 fetal adrenal glands were found to be
P45011ß-positive (gray color), but some Ad4BP-positive
cells in the outermost portion seemed not to stain with the
anti-P45011ß antibody, showing they might be undifferentiated cells.
The faint or almost no signal of P45011ß mRNA in the outermost
portion of the G-18 fetal gland (Fig. 3) may also support this
possibility. Another possibility that such undifferentiated cells exist
in a small number inside the fetal adrenal gland cannot be ruled out.
It seems, however, technically difficult to detect such
undifferentiated cortical cells in fetal adrenal glands, because no
specific markers are known for these cells. It is thus obviously
important to identify specific markers to detect undifferentiated cells
in the adrenal cortex, and such factors are now under investigation in
our laboratory.
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Acknowledgments |
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Footnotes |
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Received October 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,lyase and
P450c21) and steroidogenic activities in guinea-pig adrenal cortex. J
Endocrinol 119:191–200
. Nature 326:515–517[CrossRef][Medline]
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