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Renin immunohistochemistry in the adrenal gland of the mouse fetus and neonate.

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THE ANATOMICAL RECORD 227:124-131 (1990)
Renin lmmunohistochemistry in the Adrenal
Gland of the Mouse Fetus and Neonate
Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Hokkaido University,
Kita-ku Kita 18-jou Nishi 9-choume Sapporo 060 ( Y . K., Y . H., M . S.); Department of
Veterinary Anatomy, Faculty of Agriculture, Tottori University, Tottori 680 (H. K.); and
Department of Applied Biochemistry, University of Tsukuba, Zbaraki 305 ( K . M.), Japan
The development of renin-containing cells in fetal and neonatal
adrenal glands of the mouse was studied using immunohistochemistry. On days
13-14 of gestation, immunoreactivity for renin was first observed in a few cortical
cells of the gland, appearing a s small patchy or granular reaction products in the
perikaryon. The mitotic configurations of the cells demonstrating immunoreactivity were noted. On day 16 of gestation, a number of intensively immunoreactive
cells were distributed in the aortal side of the cortical zone. On day 18 of gestation,
and day 1 postparturition, a small number of potent immunoreactive cells were
still found in the cortical area. Immunoreactivity of the cytoplasm was observed in
the cells, some showing a n intensive reaction and others possessing numerous tiny
granules just below the cell membrane. On days 3, 5, and 7 after birth, no renincontaining cells were found in the adrenal gland. The ratio of the numbers of
renin-positive cells in certain areas to the numbers in the entire cortical area was
significantly increased on day 16 of gestation, but there was no sexual difference
in the ratios. The ratios were decreased subsequently until day 1 after birth. The
possible significance of renin synthesis in specific adrenal cells in fetal life is
discussed with respect to a n important involvement of angiotensin I1 in the morphogenesis of the adrenal gland of the mouse.
Renin, which is synthesized mainly by the juxtaglomerular cells of the kidney, has a triggering role in
the control of circulating blood pressure by cleaving
angiotensinogen to form angiotensin I (Davis and Freeman, 1976). Recently, renin (or renin-like) activities or
renin-containing tissues in the extrarenal regions have
been reported in several organs, namely, in the submandibular gland (Cohen e t al., 1972; Tanaka et al.,
1980), brain (pineal, anterior, and intermediate pituitary) (Deschepper et al., 1985; Haulica et al., 1975;
McKenzie et al., 1985),adrenal gland (Naruse and Inagami, 1982; Naruse et al., 1983; Ryan, 1972), testis
(Parmentier et al., 1983)) uterus (Eskildsen, 19721,
ovary (Fernandez et al., 1985a,b),placenta (Poisner et
al., 1981), and vascular wall (Gould e t al., 1964; Swales
et al., 1983). It is generally accepted that these extrarenal renins may function as local, independent reninangiotensin systems in individual organs rather than
as a circulating one such as that originating from the
kidney (Deschepper and Ganong, 1988). However,
there have only been a few morphological identifications of the cell types in individual organs.
We have studied the development of the intrarenal
and extrarenal renin-angiotensin system ontogenetically using immunohistochemical techniques for enzyme renin (Kon et al., 1984, 1989). Thus, in the
present study, we examined the renin-containing cells
in fetal and neonatal adrenal glands of mouse from the
viewpoint of the development of the renin-angiotensin0 1990 WILEY-LISS, INC
aldosterone system, and we discuss here the functional
meaning of the regional occurrence of renin in developmental adrenals.
Mouse (Balbk strain) fetuses from 11 to 18 days of
gestation and neonates, aged 1 to 7 days, were used in
this examination. For demonstration of gestational
stages, adult male and female mice were placed together overnight, and day 0 of the gestational period
was determined with the appearance of a vaginal plug.
All animals examined in this study were anesthetized with ether. Whole fetal specimens or dissected
adrenal glands were fixed in Bouin’s solution overnight. Tissues were dehydrated by graded ethanol series, embedded in paraffin, and sectioned transversely
o r sagittally into 5-km-thick slices through the adrenal
glands. The sections were deparaffinized, rehydrated,
and processed by routine staining with hematoxylineosin, periodic acid Schiff-hematoxylin, azan-trichrome, or Grimerius’ impregnation, and by a n immunohistochemical method using specific antimouse
submandibular gland renin rabbit-serum (Kon et al.,
1989).Grimelius’ impregnation was carried out for the
detection of the argyrophilic cells, especially the adre-
Received March 13, 1989; accepted August 14, 1989.
Fig. 1. Sagittal section of the adrenal gland on day 12 of gestation.
The gland is composed of a cluster of undifferentiated cells (arrow).
Hematoxylin-eosin, x 80
Fig. 2. Transverse section of the adrenal gland on day 13 of gestation. a: Argyrophilic cells are scattered within the adrenal parenchyme. Adrenal glands are surrounded by arrows. Grimelius’ impregnation. x 80. b: Note a few renin-immunoreactive cells containing
small patchy or granular reaction products (arrows). Immunohistochemical method. x 160. c: Renin immunoreactivity in the wall of an
intra-adrenal vessel (arrow). Immunohistochemical method. x 160.
nal medulla cells. Briefly, this method consisted of the
following successive incubation in: 1)0.003% silver nitrate solution for 3 hours a t 60" C; 2) reducing solution
containing 1 % hydroquinone and 5% sodium sulfite for
1 minute a t 45" C; and 3) 5% sodium thiosulfate for 2
minutes a t room temperature. The immunostaining sequence for renin comprised the following steps: 1)
treatment with 0.1% hydrogen peroxide and with absolute methanol for 30 minutes, respectively; 2) preincubation in 1% normal goat serum for 1 hour a t room
temperature; 3) incubation with rabbit antimouse renin antiserum 1:2,000-3,000 for 48 hours a t 4" C; 4)
incubation with goat antirabbit immunoglobulin G serum 1 : l O O for 1 hour a t room temperature; 5) incubation with soluble rabbit PAP serum 1:lOO for 1hour at
room temperature; and 6) exposure to 3,3'-diaminobenzidine-HC1. Controls for the immunohistochemical
staining consisted of normal rabbit serum o r phosphate-buffered saline substituted for the serum of the
specific antibody. The sections were counter-stained
slightly with hematoxylin.
Because renin-antiserum was raised against male
mouse submandibular gland renin, morphometrical
analysis was employed to determine if the development
of immunoreactivity for renin in the male adrenal
gland was different from those in female. For morphometrical analysis, the transverse serial sections of adrenal glands obtained from timed animals, namely, on
days 14, 16, and 18 of gestation and on day 1 after
birth, were used, and the sex of each fetus was determined with observation of the primordial genital organs. In sections impregnated a t each stage with
Grimelius' method, the areas of the whole parenchyme
or the medulla were divided and, in the neighboring
sections immunostained for renin, the renin-positive
areas were shown as numerical values. The data of
these measurements, obtained by using a computenized planimeter system (Cosmozone-1 system, software by Nikon, Inc.), were represented finally a s a percentage of the renin-positive area against the entire
cortical area.
Days 11 and 12 of Gestation
Although there were conglomerations of undifferentiated adrenal cells a t the caudoventral region of the
primordial metanephros, no capsule surrounding the
adrenal gland was present. This allowed no obvious
discrimination of the cortex and the medulla at this
stage (Fig. 1). No renin immunoreactivity was demonstrated within the adrenal tissue in this gestational
A bbreuiations
adrenal gland
Ao aorta
Cap capsule
MT metanephros
Day 13 of Gestation
The capsule, the cortex, and the medulla were still
immature; however, a few argyrophilic cells were
found to be sparsely scattered, and some were conglomerated within the parenchyma (Fig. 2a). Immunoreactivity for renin was demonstrated in the parenchymal
cells of the adrenal gland in only one fetus of the five
examined in this gestational period (Fig. 2b). The reaction products in the cytoplasm of the positive cells
appeared a s small patchy or granular forms. The wall
of the arteriole flowing into the adrenal tissue also
contained a renin immunoreactive cell (Fig. 2c).
Day 14 of Gestation
A thin, fibrous capsule partly constructed with the
mesothelial tissue was found to surround the gland.
Angiogenesis was advanced, and many blood capillaries were found to occur irregularly among the parenchymal cells. Because the aggregation of argyrophilic
cells started to form numerous small islets centripetally, i t was noted, in varying degrees, that the area of
separation of the gland split into a n inner medullary
and a n outer cortical zone.
In this period, the renin-containing cells were scattered within the gland and distributed numerously in
the medial region of the cortical zone near the aorta
(Fig. 3a), whereas cortical cells in the lateral regions
contained weaker or no immunoreactions. The renincontaining cells occurring in the medullary zone were
found neighbouring the small islets of medulla cells.
The immunoreactive cells were represented a s large,
round-to-oval shapes, where the cytoplasm possessed
small dots or rod-shaped immunoreactive inclusions
near the nucleus (Fig. 3b). In addition, there were some
mitotic cells showing slightly demonstrable immunoreactivity.
Day 16 of Gestation
The results obtained from serial sections exposed to
antirenin serum or impregnated with Grimelius'
method showed that renin-containing cells were localized only in the cortical zone (Fig. 4a, b). During this
gestational period, a number of intensive immunoreactive cells were distributed in the cortical zone near the
aorta, whereas the opposite region contained numerous
feebly reacting cells as well a s those found on day 14 of
gestation. The outer zone of the cortical tissue just below the capsule was not generally composed of immunoreactive cells. The continuous appearance of immunoreactive cells from the deep cortical zone to this outer
zone was rarely encountered, especially in the ventral
region of the gland (Fig. 412).
Immunoreactivity in the cytoplasm of positive cells
was observed to have various morphologies, namely,
from showing intense activity thoughout the cytoplasm
to demonstrating a number of small-sized granular appearances. Most granular configurations of the reaction products were generally detected in their peripheral cytoplasm. In the deep or intermediate zone of the
cortex, the intensely reactive cells were sparsely distributed rather than making cellular aggregations.
Furthermore, mitotic cells showing immunoreactivity
for renin were still observed throughout the cortical
zone (Fig. 4d).
Fig. 3. Renin-containing cells of the adrenal gland on day 14 of
gestation. a: Many renin-immunoreactive cells are observed in the
cortical zone near the aorta. Immunohistochemical method. x 80. b:
The cytoplasm of the positive cells contains small dots or rod-shaped
immunoreactive inclusions (arrows). Mitotic cell expressing immunoreactivity is observed (arrowhead). Immunohistochemical method.
x 310.
Fig. 4. The adrenal gland on day 16 of gestation. The serial sections
stained with immunohistochemistry for renin (a)and with Grimelius’
impregnation (b). x 80. c: Immunoreactivity located just below the
adrenal capsule (arrow). Immunohistochemical method. x 310. d: A
mitotic configuration containing renin reaction (arrow).Immunohistochemical method. x 500.
Day 18 of Gestation
The parenchymal cells had grown into a more tightly
packed arrangement, and the capsule decreased in
thickness. In the adrenal cortex stained with hematoxylin-eosin, one or more layers of cells just below the
capsule showed a basophilic appearance; however, the
cells of deeper zones were demonstrated as larger acidophilic ones, most of which showed a diffuse, weak reaction for renin (Fig. 5a).
Ten or more potent immunoreactive cells were found
in solitude or in aggregations in the medial region of
the adrenal cortex near the aorta per a 5-pm-section.
Although immunoreactive cells were also found in the
opposite side of the cortex, most were distributed solitarily. Immunoreactive cells decreased their cytoplasmic reactivity in this period; however, numerous granules with marked reactivity were located just below the
cell membrane (Fig. 5b), and a few mitotic immunoreactive cells were still observed in the middle cortical
zone as well a s those found on day 16 of gestation.
Day 1 After Birth
Immunoreactive cells still markedly decreased in
number and were scattered in the middle cortical region facing the aorta (Fig. 6a). The immunoreactivity
of their cytoplasm ranged from intensive reactions to
possessing a few tiny granules below the cell membrane and vacuolized figure (Fig. 6b).
Days 3, 5, and 7 After Birth
No renin-containing cells were found in any areas of
the adrenal gland (Fig. 7a, b). The ratios of the reninpositive area to the whole area of the cortex, determined during day 14 of gestation and day 1 after birth
in the male and female, respectively, are shown in Figure 8. The positive area in the day-16 of gestation fetus
showed a significant increase. The averages were
44.9% in the male and 36.9% in the female, but there
was no sexual difference between the two groups. The
ratios decreased subsequently until day 1 after birth.
Our data demonstrate the presence of renin-immunoreactivity in cortical cells in the adrenal gland of the
mouse, a t least during fetal and neonatal life. Although it has been reported that the adrenal gland is
possessed of the enzyme renin in vivo and in vitro, the
results for the cells or regions containing renin do not
always correspond to each other because of differences
in cell distributions among animal species: examples
include the adrenal glomerulosa region in the rat (Deschepper et al., 1986; Doi et al., 1984; Nakamaru e t al.,
19851, the innercortex of the adrenal gland in the
mouse (Naruse e t al., 1984), and the medullary tissue
in the bovine (Ryan, 1972).
In any case, the fact that many renin-containing
cells, some even showing mitotic figures, were demonstrated on day 16 of gestation corresponds with the
results in the kidney, where renin-positive cells were
also detected numerously in the same prenatal life
(Kon et al., 1989). It is suggested that this gestational
period may be a n important period in the ontogeny of
the system regulating blood pressure.
In the present study, immunoreactivity in the cyto-
plasm on day 14 was detected a s dots or rod-shaped
figures near the nucleus, whereas that on days 16 to 18
showed small granular contours just below the cell
membrane. Such movement of immunocytochemical
production suggests that the renin observed in adrenal
cortical cells is not taken from circulating blood, but it
is synthesized in the cytoplasm. This renin, when
moved to peripheral positions in the cytoplasm, may be
released to the intercellular spaces. Further studies on
the ultrastructural detection of renin should be carried
The present results-showing that the number of renin-containing cells in the glands decreased significantly during perinatal development and that no cells
were demonstrable using immunohistochemistry 3
days after birth-may lead to the following hypotheses.
First, the adrenal gland may not be the main source
of circulating renin, but it does possess a preparatory
role in the maintenance of blood pressure. Such a hypothesis has been accepted concerning the significant
elevation of adrenal renin activity in experimantal bilateral nephrectomy in mouse and rat (Naruse and Inagami, 1982; Naruse et al., 1984).
Second, a n amount of renin too small to be demonstrable with immunohistochemical procedures may
perform a n important physiological function in the release of aldosterone (Doi et al., 1983; Vecsei et al.,
1978). In the present study, the enzyme renin may be
derived from the cortical cells located in fascicular and
reticular zones. The idea that renin originally existing
in the adrenal gland is involved in the release of aldosterone, generally known to be produced by zona glomerulosa cells via angiotensin series (Vecsei et al., 1978),
suggests a possible role in the paracrine function of
renin-containing cells. Such a possibility has been suggested previously by some investigators (Deschepper et
al., 1986; Naruse et al., 1984).
Finally, it is possible that variable masking of antigenic determinants, interference against combinations
of antigens and antibodies with unknown proteins, or
some antigenic alterations of extrarenal renin occur in
the adrenal tissue during the later development of the
mouse. Further studies are required to clarify these
The other components functioning a s a reninangiotensin system without renin examined in this paper have been reported to exist in the adrenal gland.
Fig. 5. Adrenal gland stained with immunohistochemistry on day 18
of gestation. a: A few cells immunoreactive for renin are observed in
the middle cortical zone (arrows). Immunohistochemical method.
x 120. b: Numerous granules with marked reactivity are located a t
the cell membrane (arrow).Immunohistochemical method. x 310.
Fig. 6. The adrenal gland on day 1 after birth. a: A few renincontaining cells with intense or feeble reactions are distributed individually in the middle cortical region (arrows). Immunohistochemical
method. x 80. b: Higher magnification showing some granules below
the cell membrane and a vacuolated figure in the cytoplasm (arrows).
Immunohistochemical method. x 310.
Fig. 7. Adrenal glands on days 3 (a) and 7 (b) after birth. Immunoreactivity for renin is not found in the gland. Numerous immunoreactive cells are found in blood vessels of the metanephros. Immunohistochemical method. x 40.
Figs. 5-7
1 4 t h day
1 6 t h day
18th day
1st day after birth
Gestational a n d neonatal periods
Fig. 8. The ontogenetical changes of the ratio of the renin positive area against the whole cortical area
from day 14 of gestation to day 1 of birth in the male and female, respectively. There is no sexual
difference between the two groups.
For example, angiotensin I, angiotensin 11, angiotensin
111, and angiotensin-converting enzyme have been
measured in the rat adrenal gland (Mendelsohn, 1982;
Nakamaru et al., 1985), and mRNAs of renin and angiotensinogen have been detected with the Northern
blot technique (Campbell and Habener, 1986). These
investigations suggest that the extrarenal adrenal renin-angiotensin system provides a local independent
regulating function.
Our present results may be of some significance in
elucidating the ontogenetical development of the
extrarenal renin-angiotensin system in the mouse.
Because the initial appearance of renin immunoreactivity was observed in adrenal cortical cells accompanied by cell divisions at days 13-14 of gestation, there
is a n assumption that the angiotensin series also
appear in the gland a t or after the fetal period, as in
the present observations (Celio et al., 1985). Angiotensin I1 is known to have various functions in tissues; for
instance, in the bovine adrenal gland i t is a potent
promoting factor for the mitosis of cortical cells (Gill et
al., 1977). Furthermore, angiotensin I1 has also been
shown to be a significant stimulating factor for
angiogenesis and prostaglandin synthesis in several
organs, and the prostaglandin in the adrenal gland
has also been reported to stimulate steroidogenesis
(Fernandez et al., 1985; Flack et al., 1969). These
results suggest that there is a n important involvement
of angiotensin I1 produced by renin synthesis in the
morphogenesis of the adrenal gland in the fetal stage
of the mouse.
This work was supported by a Grant-in-Aid for Scientific Research (no. 63760233) from the Ministry of
Education, Science and Culture, Japan.
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