J. Biomedical Science and Engineering, 2011, 4, 219-234 JBiSE
doi:10.4236/jbise.2011.43031 Published Online March 2011 (http://www.SciRP.org/journal/jbise/).
Published Online March 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Electron microscopic radioautographic study on
mitochondrial RNA synthesis in adrenal cortical and
medullary cells of aging mice
Tetsuji Nagata
Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto, 390-8621 Japan and Department of
Anatomy, Shinshu Institute of Alternative Medicine and Welfare, Nagano, 380-0816 Japan.
Received 22 March 2010; revised 20 July 2010; accepted 23 July 2010.
ABSTRACT
In order to study the aging changes of intramito-
chondrial RNA synthesis of mouse adrenal cells, 10
groups of developing and aging mice, each consisting
of 3 individuals, total 30, from fetal day 19 to postna-
tal newborn at day 1, 3, 9, 14, adult at month 1, 2, 6
and senescent animals at month 12 (year 1) and 24
(year 2) were injected with 3H-uridine, an RNA pre-
cursor, sacrificed 1 hr later and the adrenal tissues
were fixed and processed for electron microscopic
radioautography. On electron microscopic radio-
autograms obtained from each animal, the number of
mitochondria per cell, the number of labeled mito-
chondria with 3H-uridine showing RNA synthesis per
cell and the mitochondrial labeling index in each
adreno-cortical cells, in 3 zones, as well as in each
adreno-medullary cells, 2 types of cells in the medulla,
the adrenalin cells and the noradrenalin cells, were
calculated and the results in respective aging groups
were compared with each others. The results demon-
strated that the number of mitochondria in adreno-
cortical cells in 3 zones, the zona glomerulosa, fas-
ciculata and reticularis of respective mice at various
ages increased from fetal day 19 to postnatal month 1
reaching the plateau from month 1 to 24 due to de-
velopment and aging of animals, respectively, while
the number of labeled mitochondria per cell and the
labeling index of intramitochondrial RNA synthesis
incorporating 3H-uridine increased from fetal day 19
to postnatal month 2, reaching the maxima and de-
creased slightly from month 6 to month 24. On the
other hand, the number of mitochondria per cell in
the medulla increased from fetal day 19 to postnatal
month 1 reaching the plateau from month 1 to 24,
while the number of labeled mitochondria per cell
and the labeling index of intramitochondrial RNA
synthesis increased from fetal day 19 to postnatal day
14, reaching the maxima and decreased from month
1 to 24. From the results, it was demonstrated that
the activity of intramitochnodrial RNA synthesis in
both the cortical and medullary cells in developing
and aging mice adrenals changed due to aging of in-
dividual animals.
Keywords: Mouse; Mitochondria; Adrena l Cortex and
Medulla; EM Radioautography; RNA Synthesis
1. INTRODUCTION
Intramitochondrial nucleic acid syntheses, both DNA
and RNA, in mammalian and avian cells were first
demonstrated morphologically by the present author by
means of electron microscopic radioautography in pri-
mary cultured cells of the livers and kidn eys of mice and
chickens in vitro [1,2] and then in some other established
cell lines such as HeLa cells [3-6] or mitochondrial frac-
tions prepared from in vivo cells [7-9]. It was later
commonly found in various cells and tissues not only in
vitro obtained from various organs in vivo [10-14], but
also in vivo cells of various organs such as the salivary
glands [15], the liver [16-29], the pancreas [30,31], the
trachea [32], the lung [33], the kidneys [34], the testis
[35,36], the uterus [37,38], the adrenals [39-42], the
brains [43], and the retina [44-48] of mice, rats and
chickens. The relationship between the intramitochon-
drial DNA synthesis and cell cycle was formerly studied
and it was clarified that the intramitochondrial DNA
synthesis was performed without nuclear involvement
[3]. However, the relationship between the aging of in-
dividual animals and the DNA synthesis in respective
cell types in these organs has not yet been clear. Recently,
the relationship of both the DNA and RNA synthesis to
the aging of animals was first clarified in the hepatocytes

T. Nagata / J. Biomedical Science an d Engineering 4 (2011) 219-234
220
of mice [19-21]. Later, the relationship of the DNA syn-
thesis to the aging of animals in the adrenocortical cells
was also clarified [41,42]. However, the relationship of
the RNA synthesis to the aging of animals in the adrenal
medullary cells has not yet been clarified. This paper
deals with the relationship between the RNA synthesis
and the aging in the adreno-medullary cells of mice in
vivo at various developmental stages from fetal day 19
to postnatal month 2 and further to adult and senescent
stages up to month 24 (year 2) during aging by means of
electron microscopic radioautography as a part of serial
studies on special cytochemistry [49] and radioautogra-
phology [5 0].
2. MATERIALS AND METHODS
2.1. The Animals
The adrenal tissues were obtained from 10 groups of
developing and aging normal ddY strain mice, from fetal
day 19 to postnatal newborn at day 1, 3, 9, 14, adult at
month 1, 2, 6, 12 and 24, each consisting of 3 litter
mates of both sexes, total 30. The embryonic age was
based on observation of the vaginal plug of the female
mice (vaginal plug = day 0). All the animals were housed
under convention al conditions and bred with normal diet
(mouse chow Clea EC2, Clea Co., Tokyo, Japan) with
access to water ad libitum in our laboratory. They were
administered with 3H-uridine, an RNA precursor, and the
adrenal tissues were fixed and processed for electron
microscopic radioautography. All the procedures used in
this study concerning the animal experiments were in
accordance with the guidelines of the animal research
committee of Shinshu University School of Medicine,
Matsumoto, Japan, where this experiment was carried
out, as well as the principles of laboratory animal care in
NIH publication No. 86-23 (revised 1985).
2.2. Electron Microscopic Radioautography
All the animals were injected intraperitoneally with
3H-uridine (Amersham, England, specific activity 877
GBq/mM) in saline, at 9 a.m., one hour before sacrifices.
The dosage of injections was 370 KBq/gm body weight.
The animals were perfused at 10 a.m., one hour after the
injection, via the left ventricles of the hearts with 0.1 M
cacodylate-buffered 2.5% glutaraldehyde under Nembu-
tal (Abbott Laboratories, Chicago, ILL, USA) anesthesia.
The right adrenal glands were taken out, excised and 3
small pieces of the adrenal tissues (1mm × 1mm × 1mm)
were immersed in the same fixative at 4˚C for 1 hr., fol-
lowed by postfixation in 1% osmium tetroxide in the
same buffer at 4˚C for 1 hr., dehydrated in graded series
of ethanol and acetone, and embedded in epoxy resin
Epok 812 (Oken, Tokyo, Japan).
For electron microscopic radioautography, semithin
sections at 0.2 µm thickness, thicker than conventional
ultrathin sections containing more radiolabeled com-
pound than ultrathin sections in order to shorten the ex-
posure time, were cut in sequence on a Porter-Blum
MT-2B ultramicrotome (Dupont-Sorvall, Newtown, MA,
USA) using glass knives. The sections were collected on
collodion coated copper grid meshes (VECO, Eerbeek,
Netherlands), coated with Konica NR-H2 radioauto-
graphic emulsion (Konica, Tokyo, Japan) by a wire-loop
method [30,49-52]. They were stored in dark boxes con-
taining silica gel (desiccant) at 4˚C for exposure. After
the exposure for 10 months, the specimens were proc-
essed for development in freshly prepared gold latensi-
fication solution for 30 sec at 16˚C and then in fresh
phenidon developer for 1 min at 16˚C in a water bath,
rinsed in distilled water and dried in an oven at 37˚C
overnight, stained with lead citrate solution for 3 min,
coated with carbon for electron microscopy. The electron
microscopic (EM) radioautograms were examined in a
JEOL JEM-4000EX high voltage electron microscope
(JEOL, Tokyo, Japan) at an accelerating voltage of
400kV for observing thick specimens [50].
2.3. Quantitative Analysis of Electron
Micrographs
For quantitative analysis of electron micrographs, twenty
EM radioautograms showing cross sections of either
adreno-cortical cells or adreno-medullary cells sectioned
through the centers of their nuclei and cell bodies se-
lected at random from each group, based on the electron
microscopic photographs taken after observation on at
least 100 adrenocortical cells in the cortex from respec-
tive animals, and at least 10 cells from respective zones,
i.e. zona glomerulosa, zona fasciculata and zone reticu-
laris, were analyzed to calculate the total number of mi-
tochondria in each adrenocortical cell in respective
zones, and the number of labeled mitochondria covered
with silver grains by visual grain counting. In the me-
dulla, likewise, at least 100 adreno-medullary cells fro m
respective animals, and at least 10 cells from the 2 types
of cells, adrenalin and noradrenalin cells in the medulla,
were analyzed to calculate the total number of mito-
chondria in each cell and the number of labeled mito-
chondria covered with silver grains by visual grain
counting.
On the other hand, the number of silver grains in the
same area size as a mitochondrion outside cells was also
calculated in respective specimens as background fog,
which resulted in less than 1 silver grain (0.03/mi- to-
chondrial area) almost zero. Therefore, the grain count in
each specimen was not corrected with the background
fog. From all the data thus obtained the averages and

T. Nagata / J. Biomedical Science and Engineering 4 (2011) 219-234
221
standard deviations in respective aging groups were
computed with a personal computer (Macintosh type
8100/100, Apple Computer, Tokyo, Japan). The data were
stochastically analyzed using variance and Student’s
t-test. The differences were considered to be significant
at P value < 0.01.
3. RESULTS
3.1. Morphological Observations
The adrenal tissues obtained from ddY strain mice at
various ages from embryo day 19 to postnatal year 2
(month 24), consisted of both adreno-cortical cells in 3
layers, zona glomerulosa, zona fasciculate, zona reticu-
laris, and the adreno-medullary cells in the medulla.
The adreno-cortical tissues obtained from ddY strain
mice at various ages from embryo day 19 to postnatal
month 24, consisted of 3 layers, zona glomerulosa, zona
fasciculata and zona reticularis, showed gradual devel-
opment. At embryonic day 19 and postnatal day 1, the
adreno-cortical cells were composed mainly of polygo-
nal cells, while the specific orientation of the 3 layers,
zona glomerulosa, zona fasciculata and zona reticularis,
was not yet well established. At postnatal day 3, orienta-
tion of 3 layers, zona glomerulosa, zona fasciculata and
zona reticularis, became evident. At postnatal day 9 and
14, the specific structure of 3 layers was completely
formed and the arrangements of the cells in respective
layer became typical especially at day 14 (Figures 1-3).
Observing the ultrastructure of the adrenocortical cells,
cell organelles including mitochondria were not so well
developed at perinatal and early postnatal stages from
embryonic day 19 to postnatal day 3. However, these
cell organelles, mitochondria, endoplasmic reticulum,
Golgi apparatus, appeared well developed from the ju-
venile stage at postnatal day 14 (Figures 1-3) to the
adult stages at postnatal month 1, month 2 (Figures 4-6),
month 6 and further to the senescent stage at month 12
(Figures 7-9) and 24.
The zona glomerulosa (Figures 1, 4 and 7) of mouse
adrenal cortex is the thinnest layer found at the outer
zone, covered by the capsule, consisted of closely
packed groups of columnar or pyramidal cells forming
arcades of cell columns. The cells contained many
spherical mitochondria and well developed smooth sur-
faced endoplasmic reticulum but a compact Golgi appa-
ratus in day 14 (Figure 1) to month 1, 2 (Figure 4), 6,
and to month 12 (Figure 7) and 24 animals. The zona
fasciculata was the thickest layer, consisted of polygonal
cells which were larger than the glomerulosa cells, ar-
ranged in long cords disposed radially to the medulla
containing many lipid droplets (Figures 2, 5 and 8). At
postnatal month 1, 2 and 6, the specific structure of 3
layers was completely developed and the arrangements
of the cells in respective layer became typical as adult
tissues (Figures 7-9). By high power magnification at
this stage, the cytoplasmic matrix can be observed full of
numerous mitochondria and considerable number of
lipid droplets. Observing the ultrastructure of the adre-
nocortical cells at the juvenile and adult stages, cell or-
ganelles including mitochondria were well developed
from postnatal day 14 to month 1, month 2, month 6,
month 12 and month 24. The mitochondria in the zona
fasciclulata (Figures 2, 5 and 8) were less numerous and
were more variable in size and shape than those of the
glomeruloza cells, while the smooth surfaced endoplas-
mic reticulum were more developed and the Golgi appa-
ratus was larger than the glomerulosa. In the zona re-
ticularis (Figures 3, 6 and 9), the parallel arrangement of
cell cords were anastomosed showing networks contin-
ued to the medullar cells. The mitochondria were less
numerous and were more variable in size and shape than
those of the glomeruloza cells like the fasciculata cells,
as well as the smooth surfaced endoplasmic reticulum
was developed and the Golgi apparatus was large like
the fasciculata cells. Thus, the structure of the
adreno-cortical cells showed changes due to develop-
ment and aging at respective developmental stages.
Figure 1. Electron microscopic radioautogram of the
zona glomerulosa of a juvenile mouse at postnatal day
14, labeled with 3H-uridine showing RNA synthesis
(several silver grains) in the nucleus as well as in a
few mitochondria. ×3000.

T. Nagata / J. Biomedical Science an d Engineering 4 (2011) 219-234
222
Figure 2. Electron microscopic radioautogram of the
zona fasciculata of a juvenile mouse at postnatal day
14, labeled with 3 H-uridine showing RNA synthesis
(several silver grains) in the nucleus as well as in a
few mitochondria. ×3000.
Figure 3. Electron microscopic radioautogram of the
zona reticularis of a juvenile mouse at postnatal day
14, labeled with 3H-uridine showing RNA synthesis
(several silver grains) in the nucleus as well as in a
few mitochondria. ×3000.
Figure 4. Electron microscopic radioautogram of the
zona glomerulosa of a mature adult mouse aged at
postnatal month 2, labeled with 3 H-uridine showing
RNA synthesis (several silver grains) in the nucleus as
well as in several mitochondria. ×3000.
Figure 5. Electron microscopic radioautogram of the
zona fasciculata of a mature adult mouse aged at
postnatal month 2, labeled with 3
H-uridine showing
RNA synthesis (several silver grains) in the nucleus as
well as in a few mitochondria. ×3000.

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223
Figure 6. Electron microscopic radioautogram of the
zona reticularis of a mature adult mouse aged at post-
natal month 2, labeled with 3H-uridine showing RNA
synthesis in the nucleus as well as in a few mitochon-
dria. ×3000.
Figure 7. Electron microscopic radioautogram of the
zona glomerulosa of an old adult mouse aged at post-
natal month 12, labeled with 3H-uridine showing RNA
synthesis (few silver grains) in the nucleus as well as
in a few mitochondria. ×3000.
Figure 8. Electron microscopic radioautogram of the
zona fasciculata of an old adult mouse aged at post-
natal month 12, labeled with 3H-uridine showing RNA
synthesis (few silver grains) in the nucleus and in a
few mitochondria. ×3000.
Figure 9. Electron microscopic radioautogram of the
zona reticularis of an old adult mouse aged at postna-
tal month 12, labeled with 3H-uridine showing RNA
synthesis in the nucleus and a few mitochondria.
×3000.

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224
The adreno-medullary tissues, on the other hand, con-
sisted of 2 types of cell, the adrenalin cells and noradrena-
lin cells. Some of the medullary cells possessed many
granules of medium electron density which were be-
lieved to correspond to the adrenalin granules, while some
other cells possessed many granules of very high elec-
tron density which were believed to correspond to the
noradrenalin granules [41,42,50]. However, the numbers
of mitochondria found in their cytoplasm were not so
many at prenatal day 19. At postnatal day 1, day 3 and
day 9, the 2 types of cells differentiated and the nu mbers
of granules, both adrenalin and noradrenalin granules,
increased respectively. Likewise, the numbers of mito-
chondria also increased from prenatal day to postnatal
days.
In the medulla, at postnatal day 14 to month 1, month
2 and month 6, the numbers of adrenalin and noradrena-
lin granules as well as mitochondria increased from
17-18/cell to 23-24/cell [50]. At postnatal month 1 and 2,
the ultrastructures of 2 cell types were completely de-
veloped and the arrangements of the cells in the medulla
became typical as adult tissues [50]. In the present study,
the ultrastructure of 2 cell types appeared almost the
same as in the previous study [49]. Thus, the ultrastruc-
ture of the adreno-medullary cells did not show any
changes due to aging at respective senescent stages at
postnatal month 12 and month 24.
3.2. Radioautographic Observations
Observing EM radioautograms, the silver grains were
found over the nuclei of some adreno-cortical cells la-
beled with 3H-urdine demonstrating RNA synthesis in all
aging stages from perinatal stages at embryonic day 19,
postnatal day 1 and day 3, day 9 and day 14 (Figures
1-3) and adults at month 1, month 2 (Figures 4-6),
month 6, month 12 and month 24 (Figures 7-9). Those
labeled cells were found in all the 3 layers, the zona
glomerulosa (Figures 1, 4 and 7), the zona fasciculata
(Figures 2, 5 an d 8) and the zona reticularis (Figures 3,
6 and 9), at respective aging stages. In the labeled
adreno-cortical cells in 3 layers the silver grains were
mainly localized over the euchromatin of the nuclei or a
few or several silver grains were found over cytoplasmic
organelles, especially over some of the mitochondria
showing RNA synthesis incorporating 3H-uridine. The
localizations of silver grains over the mitochond ria were
mainly on the mitoch ondrial matrices and some over the
mitochondrial me mbranes when observ ed by high power
magnification.
In the adreno-medullary cells, on the other hand, the
silver grains were also found over the nuclei of some
medullary cells labeled with 3H-uridine in all aging
stages from perinatal stages at embryonic day 19, post-
natal day 1 and day 3, day 9, 14 and adults at month 1,
month 2, month 6, month 12 and month 24. Those la-
beled cells were found in all the 2 types of cells, the
adrenalin cells and the noradrenalin cells, at various ag-
ing stages from embryo day 19, to postnatal day 1, 3, 9,
14, month 1, 2, 6, 12 and 24. In the labeled adre-
no-medullary cells the silver grains were mainly local-
ized over the euchromatin of the nuclei or a few or sev-
eral silver grains were found over cytoplasmic organ-
elles, especially over some of the mitochondria showing
RNA synthesis incorporating 3H-uridine. The localiza-
tions of silver grains ov er the mitochondria were mainly
on the mitochondrial matrices and some over the mito-
chondrial membranes when observed by high power
magnification.
3.3. Quantitative Analysis
3.3.1. Number of Mitochondria per Cell
Preliminary quantitative analysis on the number of mi-
tochondria in either 10 adreno-cortical cells or adreno-
medullary cells whose nuclei and cytoplasm were la-
beled with silver grains and other 10 cells whose nuclei
and cytoplasm were not labeled in each aging group re-
vealed that there was no significant difference between
the number of mitochondria and the lab eling ind ices (P <
0.01). Thus, the number of mitochondria and the label-
ing indices were calculated regardless whether their nu-
clei were labeled or not. The results obtained from the
number of mitochondria in adreno-cortical cells in the 3
layers of respective animals in 10 aging groups at peri-
natal stages, prenatal embryo day 19, postnatal day 1, 3,
9 and 14, showed an gradual increase from the prenatal
day 19 (glomerulosa 12.5, fasciculata 14.5, reticularis
15/2/cell) to postnatal day 14 (glomerulosa 35.1, fas-
ciculata 33.2, reticularis 35.3/cell), and to adu lt stages at
postnatal month 1 (glomerulosa 50.7, fasciculata 50.8,
reticularis 49.2/cell), then slightly decreased at month 2
(glomerulosa 42.4, fasciculata 37.6, reticularis 44.1/cell),
but kept plateau from month 6 (glomerulosa 49.8, fas-
ciculata 49.2, reticularis 50.6/cell), to month 12 (glome-
rulosa 54.7, fasciculata 53.8, reticularis 50.2/cell) and
month 24 (glomerulosa 49.5, fasciculata 52.1, reticularis
50.6/cell), as is shown in Figures 10-12. The increase
from embryo day 19 to postnatal month 1 was stochasti-
cally significant (P < 0.01).
The results ob tained from the n umber of mitochondr ia
in adreno-medullary cells of respective animals in 10
aging groups at perinatal stages, from prenatal embryo
day 19 to postnatal d ay 1, 3, 9, 14, and month 1, showed
an gradual increase from the prenatal day 19 (adrenalin
17.5, noradrenalin 18.5/cell) to postnatal day 1, day 3,
day 9, day 14 (adrenalin 24.4, noradrenalin 24.6/cell),
and to adult stages at postnatal month 1 (adrenalin 24.7,

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225
Figure 10. Histogram showing aging changes of the average numbers of mitochoindria per cell in each adre-
no-cortical cell in the 3 layers of respective animals in 10 aging groups.
Figure 11. Histogram showing aging changes of the average numbers of labeled mitochoindria with
3H-uridine showing RNA synthesis per cell in each adreno-cortical cell in the 3 layers of respective animals in
10 aging groups.

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226
Figure 12. Histogram showing aging changes of the average labeling index of mitochondria labeled with
3H-uridine showing RNA synthesis per cell in each adreno-cortical cell in the 3 layers of respective animals in
10 aging groups.
noradrenalin 22.9/cell), month 2 (adrenalin 24.3, nora-
drenalin 24.1/cell) and month 6 (adrenalin 24.8, nora-
drenalin 24.2/cell). However, the number of mitochon-
dria at senescent stage from month 12 (adrenalin 24.3,
noradrenalin 24.1/cell) to 24 (adrenalin 23.6, noradrena-
lin 23.2/cell) appeared to decrease slightly. The increase
of mitochondrial numbers in both adrenalin and nora-
drenalin cells from embryonic day 19 to postnatal month
2 and 6 was considered to be significant at P value <
0.01. However, the slight decrease from month 6 to
month 2 4 w as n ot significant.
3.3.2. Mitoch ondrial RN A Synthe sis
The results of visual grain counting on the number of
mitochondria labeled with silver grains obtain ed fro m 10
adreno-cortical cells in the 3 layers of each animal la-
beled with 3H-uridine demonstrating RNA synthesis in
10 aging groups at perin atal stages, prenatal embryo day
19, postnatal day 1, 3, 9 and 14, month 1, 3, 6, 12 and 24,
are plotted in Figures 11-12. The results demonstrated
that the numbers of labeled mitochondria with 3H-uridine
showing RNA synthesis per cell gradually increased
from prenatal embryo day 19 (glomerulosa 1.3, fascicu-
lata 1.7, reticularis 1.7/cell) to postnatal da y 1 (glomeru-
losa 2.8, fasciculata 3.1, reticularis 3.3/cell), day 3 (glo-
merulosa 4.1, fasciculata 4.9, reticularis 5.5/cell), day 9
(glomerulosa 4.6, fasciculata 5.1, reticularis 5.3/cell),
day 14 (glomerulosa 5.1, fasciculata 5.7, reticularis
4.7/cell), and month 1 (glomerulosa 5.8, fasciculata 5.6,
reticularis 5.4/cell) and month 2 (glomerulosa 6.3, fas-
ciculata 6.6, reticularis 6.1/cell), reaching the maximum,
then decreased to month 6 (glomerulosa 6.2, fasciculata
5.9, reticularis 6.4/cell), month 12 (glomerulosa 5.2,
fasciculata 6.5, reticularis 6.1/cell) and 24 (glomerulosa
5.1, fasciculata 5.6, reticularis 5.4/cell) as is shown in
the histogram (Figure 11).
The results of visual grain counting on the number of
mitochondria labeled with silver grains obtain ed fro m 10
adreno-medullary cells of each animal labeled with
3H-uridine demonstrating RNA synthesis in 10 aging
groups at perinatal stages, prenatal embryo day 19,
postnatal day 1, 3, 9 and 14, month 1, 3, 6, 12, and 24,
showed that the numbers of labeled mitochondria with
3H-uridine showing RNA synthesis per cell gradually
increased from prenatal embryo day 19 (adrenalin 0.5,
noradrenalin 0.5/cell), to postnatal day 1 (adrenalin 0.7,
noradrenalin 0.6/cell), day 3 (adrenalin 0.7, noradrenalin
0.8/cell), day 9 (adrenalin 0.8, noradrenalin 0.8/cell), day

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14 (adrenalin 0.8, noradrenalin 0.9/cell), reaching the
maximum, and decreased to month 1 (adrenalin 0.5, no-
radrenalin 0.5/cell), month 2 (adrenalin 0.45, noradrena-
lin 0.4/cell), month 6 (adrenalin 0.41, noradrenalin
0.36/cell), and month 12 (adrenalin 0.4, noradrenalin
0.32/cell) and 24 (adrenalin 0.38, noradrenalin 0.35/cell).
The data were stochastically analyzed using variance
and Student’s t-test. The increases of the numbers of
labeled mitochondria in both adrenalin and noradrenalin
cells from embryo day 19 to postnatal day 14, as well as
the decreases from day 14 to month 24 were stochasti-
cally significant (P < 0.01).
3.3.3. The Labeling Index
Finally, the labeling indices of adreno-cortical cells
showing RNA synthesis in respective aging stages were
calculated from the number of labeled mitochondria
(Figure 11) dividing by the number of total mitochon-
dria per cell (Figure 10), which were plotted in Figure
12, respe ctively.
The results showed that the labeling indices gradually
increased from prenatal day 19 (glomerulosa 10.4, fas-
ciculata 11.4, reticularis 11.1%) to postnatal newborn
stage at postnatal day 1 (glomerulosa 12.6, fasciculata
12.1, reticularis 13.1%) and day 3 (glomerulosa 14.5,
fasciculata 17.6, reticularis 19.6%), and to juvenile stage
at postnatal day 9 (glomerulosa 16.6, fasciculata 22.3,
reticularis 18.0%), reaching the maximum, and de-
creased to day 14 (glomerulosa 14.5, fasciculata 17.1,
reticularis 13.4%) and to the adult stage at month 1
(glomerulosa 11.4, fasciculata 11.0, reticularis 10.7%)
and month 2 (glomerulosa 10.0, fasciculata 11.4, reticu-
laris 10.7%), to month 6 (glomerulosa 12.4, fasciculata
12.0, reticularis 12.6%) to month 12 (glomerulosa 9.5,
fasciculata 11.7, reticularis 11.3%) and finally to senes-
cence at month 24 (glomerulosa 10.3, fasciculata 10.7,
reticularis 10.7%), as is shown in the histogram (Figure
12). Likewise, the labeling indices of adreno-medullary
cells showing RNA synthesis in respective aging stages
were calculated from the number of labeled mitochon-
dria, dividing by the number of total mitochondria per
cell. The results showed that the labeling indices gradu-
ally increased from prenatal day 19 (adren alin 2.8, nora-
drenalin 2.6%) to postnatal newborn day 1 (adrenalin 2.8,
noradrenalin 2.4%), day 3 (adrenalin 3.3, noradrenalin
2.9%), day 9 (adrenalin 3.4, noradrenalin 2.3%) to juve-
nile stage at day 14 (adrenalin 3.6, noradrenalin 3.8%),
reaching the maximum, and decreased to adult stages at
month 1 (adrenalin 2.1, noradrenalin 2.2%), month 2
(adrenalin 1.8, noradrenalin 1.6%), month 6 (adrenalin
1.7, noradrenalin 1.5%), month 12 (adrenalin 1.6, nora-
drenalin 1.4%) and 24 (adrenalin 1.6, noradrenalin 1.5%)
as shown in Figures 13-17. From the results, the in-
creases of the mitochondrial labeling indices in both
adrenalin and noradrenalin cells from embryo day 19 to
postnatal day 14, as well as the decreases from day 14 to
month 24 were stochastically significant (P < 0.01).
In the previous study, the labeling indices of mito-
chondrial DNA synthesis in 2 cell types of adrenal me-
dulla in respective aging stages were already calculated
from the number of labeled mitochondria and the number
of total mitochondria per cell. The results showed that
the labeling indices of DNA gradually increased from
prenatal day 19 (adrenalin 2.8, noradrenalin 2.6%) to
postnatal newborn day 1 (adrenalin 2.8, noradrenalin
2.4%), day 3 (adrenalin 3.3, noradrenalin 2.9%), day 9
(adrenalin 3.4, noradrenalin 2.3%) to juvenile stage at
day 14 (adrenalin 3.6, noradrenalin 3.8%), reaching the
maximum, and decreased to adult stages at month 1
(adrenalin 2.1, noradrenalin 2.2%), month 2 (adrenalin1.8,
Figure 13. Electron microscopic radioautogram of the me-
dulla of a juvenile mouse at postnatal day 14, labeled with
3H-uridine showing RNA synthesis (several silver grains) in
the nucleus as well as in a few mitochondria of both
adrenalin and noradrenalin cells. × 3 000.
Figure 14. Electron microscopic radioautogram of the me-
dulla of an old adult mouse aged at postnatal month 12,
labeled with 3H-uridine showing RNA synthesis in the nu-
cleus and a few mitochondria of both adrenalin and
noradrenalin cells. 3000 ×.

T. Nagata / J. Biomedical Science an d Engineering 4 (2011) 219-234
228
Figure 15. Histogram showing number of mitochondria per cell.
Figure 16. Histogram showing number of mitochondria labeled with 3H-ridine.

T. Nagata / J. Biomedical Science and Engineering 4 (2011) 219-234
229
Figure 17. Histogram showing the labeling index labeled with 3H-uridine.
noradrenal i n 1. 6%), month 6 (adrenalin 1.7, noradrenalin
1.5%), month 12 (adrenalin 1.6, noradrenalin 1.4%) and
24 (adrenalin 1.6, noradrenalin 1.5%) as discussed in the
previous study. From the results, the increases of the
mitochondrial labeling indices in both the adrenalin and
noradrenalin cells from embryo day 19 to postnatal day
14, as well as the decreases from day 14 to month 24
were stochastically significant (P < 0.01). The labeling
indices of RNA revealed in the present study, on the
other hand, gradually increased from prenatal day 19
(adrenalin 2.8, noradrenalin 2.6%) to postnatal newborn
day 1 (adrenalin 2.8, noradr enalin 2.4%), day 3 (adr ena-
lin 3.3, noradrenalin 2.9%), day 9 (adrenalin 3.4, nora-
drenalin 2.3%) to juvenile stage at day 14 (adrenalin 3.6,
noradrenalin 3.8%), reaching the maximum, and de-
creased to adult stages at month 1 (adrenalin 2.1, nora-
drenalin 2.2%), month 2 (adrenalin 1.8, noradrenalin
1.6%), month 6 (adrenalin 1.7, noradrenalin 1.5%),
month 12 (adrenalin 1.6, noradrenalin 1.4%) and 24
(adrenalin 1.6, noradrenalin 1.5%). From the results, the
increases of the mitochondrial labeling indices of RNA
synthesis in both adrenalin and noradrenalin cells from
embryo day 19 to postnatal day 14, as well as the de-
creases from day 14 to month 24 were stochastically
significant (P < 0.01).
4. DISCUSSION
From the results obtained at present, it was shown that
the labeling indices of the adreno-cortical cells in 3 lay-
ers of each animal labeled with 3H-uridine demonstrat-
ing RNA synthesis in 10 groups gradually increased
from prenatal embryo day 19 to postnatal day 1, 3, 9,
reaching the maxima, and decreased to day 14, month 1,
2, 6, 12 and 24, due to aging and senescence. On the
other hand, the adreno-medullary cells showing RNA
synthesis gradually increased from prenatal day 19 to
postnatal newborn day 1, 3, 9, 14, reaching the maxi-
mum, and decreased to adult stages at month 1, 6, 12,
and 24, slightly different from DNA synthesis.
As for the macromolecular synthesis in various cells
in various organs of experimental animals observed by
light and electron microscopic radioautography, it is well
known that the silver grains due to radiolabeled 3H-thy-
midine demonstrate DNA synthesis [1-3,48,49], while
the grains due to 3H-uridine demonstrate RNA synthesis
[22,25,26,28]. The previous results obtained from the
studies on the adreno-cortical cells of aging mice by
light microscopic radioautography revealed that silver
grains indicating DNA synthesis incorporating 3H-thy-
midine were observed over the nuclei of some adreno-
cortical cells at perinatal stages from postnatal day 1 to
14 [39,40]. However, they did not observe the intrami-
tochondrial DNA synthesis. In the previous study [41],
the numbers of silver grains showing nuclear DNA syn-
thesis, as expressed by grain counting, did not give any
significant difference between the cells in the 3 layers in

T. Nagata / J. Biomedical Science an d Engineering 4 (2011) 219-234
230
the same aging groups. These results indicated that the
amount of DNA synthesized in one nucleus was almost
the same as in any other cells independent upon whether
the nucleus belong ed to any layers of th e adrenal cortex.
However, these differences between the 3 layers at re-
spective aging groups were not stochastically significant
(P < 0.01). These results indicated that the DNA syn-
thetic activity in the nuclei of 3 layers of the adrenal
cortex did not show any difference. To the contrary, the
numbers of mitochondria labeled with 3H-thymidine per
cell as well as the labeling indices between the aging
groups increased gradually from prenatal embryo day 19,
to postnatal day 1, 3, 9 and 14, to postnatal month 1 and
2, then decreased to month 6, 12 and 24. These de-
creases were stochastically significant (P < 0.01). These
results indicated that the mito chondria in adreno-cortical
cells proliferated from perinatal and postnatal newborn
stage to adult stage at postnatal month 1 and 2, reaching
the maxima, then lost their proliferating activities from
aged stage at month 6 to senescent stage up to month 24.
On the other hand, the radioautograms in the present
study showing incorporations of 3H-uridine into mito-
chondria indicating mitochondrial RNA synthesis re-
sulted in silver grain localization over the mitochondria
independently from the nuclei whether the nuclei were
labeled with silver grains or not in almost all the cells in
the 3 layers of the adreno-cortical cells from prenatal
embryo day 19 to postnatal day 1, 3, 9 and 14, to post-
natal month 1, 2, 6, 12 and 24, during the development
and aging. The numbers of labeled mitochondria show-
ing RNA synthesis increased from perinatal day to post-
natal adult stage at month 2, then kept plateau, while the
labeled mitochondria with 3H-uridine showing RNA
synthesis increased from perinatal stage to postnatal
adult stage at month 2, then decreased at month 24,
while the labeling indices increased from perinatal em-
bryonic day to postnatal newborn and juvenile stages at
day 9, then decreased from day 14 to senescence at
month 24, then decreased to the adult stages at month 1
and 2, to month 6, 12 and 24. These changes demon-
strate the respective aging changes. The results obtained
previously [41] indicated that mitochondria in the adre-
nocortical cells proliferated from newborn to adult stag-
es around month 1 and 2, showing mitochondrial DNA
synthesis, while the mitochondrial RNA synthesis in-
creased from newborn stage to postnatal day 9, then de-
creased from day 14, reaching the maxima, then de-
creased to month 24, but the RNA synthetic activity was
kept from day 14 to month 12 which decreased to se-
nescent at month 24 due to aging.
With regard to the DNA in mitochondria in animal
cells or plastids in plant cells, many studies have been
reported in various cells of various plants and animals
since 1960s [53-56]. Most of these authors observed
DNA fibrils in mitochondria which were histochemically
extracted by DN’ase. Electron microscopic observation
of the DNA molecules isolated from the mitochondria
revealed that they were circular in shape, with a circum-
ference of 5-6 µm [57]. It was calculated that such a
single molecule had a molecular weight of about 107
daltons [58]. Mitochondria of various cells also con-
tained DNA polymerase, which was supposed to func-
tion in the replication of the mitochondrial DNA [59].
On the other hand, the incorporations of 3H-thymidine
into mitochondria demonstrating DNA synthesis were
observed by means of electron microscopic radioauto-
graphy in lower organism such as slime mold [60,61],
tetrahymena [62] or chicken fibroblasts in tissue culture
under abnormal conditions [63]. However, these authors
used old-fashioned developers consisting of methol and
hydroquinone (MQ-developer), which produced coarse
spiral silver grains resulting in inaccurate localization
over cell organelles when observed by electron micros-
copy. All of these authors showed photographs of elec-
tron radioautographs with large spiral-formed silver
grains (2-3 µm in diameter) localizing not only over the
mitochondria but also outside the mitochondria. I n order
to obtain smaller silver grains, we first used elon-ascorbic
acid developer after gold latensification [1-6], which
produced comma-shaped smaller silver grains (0.4-0.8
µm in diameter), then later we used phenidon developer
after gold latensification, producing dot-like smaller
silver grains (0.2-0.4 µm in diameter) localizing only
inside the mitochondria showing ultrahigh resolution of
radioautograms [30,49-52]. These papers were the first
that demonstrated intramitochondrial DNA synthesis
incorporating 3H-thymidine with accurate intramito-
chondrial localization in avian and mammalian cells.
With regard to the resolution of electron microscopic
radioautography, on the other hand, many authors dis-
cussed the size of silver grains under various conditions
and calculated various values of resolutions [4,5,64-66].
Those authors who used the M-Q developers maintained
the resolution to be 100-160 nm [64,65], while those
authors who used the elon-ascorbic acid developer
[4,5,66] calculated it to be 25-50 nm. When we used
phenidon developer at 16˚C for 1 min after gold latensi-
fication, we could produce very fine dot-shaped silver
grains and obtained the resolution around 25 nm
[30,49-51,66,67]. For the analysis of electron radioauto-
graphs, Salpeter et al. [64] proposed to use the
half-distance and very complicated calculations through
which respective coarse spiral-shaped silver grains were
judged to be attributable to the radioactive source in a
certain territory within a resolution boundary circle.
However, since we used phenidon developer after gold

T. Nagata / J. Biomedical Science and Engineering 4 (2011) 219-234
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latensification to produce very fine dot-shaped silver
grains, we judged only the silver grains which were lo-
cated in the mitochondria which were dot-shaped very
fine ones to be attributable to the mitochondria without
any problem as was formerly discussed [3-5,49-52].
Then we also demonstrated intramitochondrial DNA
synthesis incorporating 3H-thymidine in some other es-
tablished cell lines originated fro m human being such as
HeLa cells [3-6] or mitochondrial fractions prepared
from in vivo mammalian cells such as rat and mouse
[7-9]. It was later commonly found in various cells and
tissues not only in vitro obtained from various organs in
vivo such as the cultured human HeLa cells [13,68],
cultured rat sarcoma cells [12], mouse liver and pancreas
cells in vitro [11,14,31], but also in vivo cells obtained
from various organs such as the salivary glands [15], the
liver [16-29], the pancreas [30,31], the trachea [32], the
lung [33], the kidneys [34], the testis [35,36], the uterus
[37,38], the adrenal glands [39-42], the brains [43], and
the retina [44-48] of mice, rats and chickens. Thus, it is
clear that all the cells in various organs of various ani-
mals synthesize DNA not only in their nuclei but also in
their mitochondria.
The relationship between the intramitochondrial DNA
synthesis and cell cycle was formerly studied in syn-
chronized cells and it was clarified that the intramito-
chondrial DNA synthesis was performed without nuclear
involvement [4]. However, the relationship between the
DNA synthesis and the aging of individual animals and
men has not yet been clarified except a few papers re-
cently published by Korr and associates on mouse brain
[69-72]. They reported both nuclear DNA repair, meas-
ured as nuclear unscheduled DNA synthesis, and cyto-
plasmic DNA synthesis labeled with 3H-thymidine in
several types of cells in brains such as pyramidal cells,
Purkinje cells, granular cells, glial cells, endothelial cells,
ependymal cells, epithelial cells as observed by light
microscopic radioautography using paraffin sections.
They observed silver grains over cytoplasm of these
cells by light microscopy and maintained that it was
reasonable to interpret these labeling as 3H-DNA outside
the nuclei, which theoretically belonged to mitochon-
drial DNA without observing the mitochondria by elec-
tron microscopy. From the results, they concluded that
distinct types of neuronal cells showed a decline of both
unscheduled DNA and mitochondrial DNA syntheses
with age in contrast that other cell types, glial an d endo-
thelial cells, did not show such age-related changes
without counting the number of mitochondria in respec-
tive cells nor counting the labeling indices at respective
aging stages. Thus, their results from the statistics ob-
tained from the cytoplasmic grain counting seems to be
not accurate without observing mitochondria directly. To
the contrary, we had studied DNA synthesis in the livers
of aging mice [16-29] and clearly demonstrated that the
number of mitochondria in each hepatocytes, especially
mononucleate hepatocytes, increased with the ages of
animals from the perinatal stages to adult and senescent
stages, while the number of labeled mitochondria and
the labeling indices increased from the perinatal stages,
reaching a maximum at postnatal day 14, then decreased.
Our previous studies [22,23] also clarified that the DNA
synthesis and cell proliferation by mitosis were the most
active in the nuclei of mononucleate hepatocytes at the
perinatal stages in contrast that binucleate cells were less
active at the perinatal stage but th e number of binucleate
hepatocytes increased at senescent stages and the results
suggest the possibility that the mitochondria in mononu-
cleate hepatocytes synthesized their DNA by themselves
which peaked at postnatal day 14 in accordance with the
proliferation of mononucleate hepatocytes while binu-
cleate hepatocytes increased after the perinatal stage and
did not divide but remained binucleate keeping many
mitochondria in their cytoplasm which were more in
number than mononucleate hepatocytes at the senescent
stage.
Thus, our previous papers [22-28] were the first that
dealt with the relationship between the DNA synthesis
and aging in hepatocytes of mice in vivo at v arious ages
by means of electron microscopic radioautography ob-
serving the small dot-like silver grains, due to incorpor a-
tions of 3H-thymidine, which exactly localized inside the
mitochondria. Our previous results [39-42] also r evealed
that an increase was observed by direct observation on
mitochondria at electron microscopic level and obtaining
accurate mitochondrial number and labeling indices in
adreno-cortical cells in 8 groups of developing mice.
There was a discrepancy between our results from the
hepatocytes [22,23] as well as the adrenocortical cells
[39-42] and the results from the several types of cells in
the brains by Korr et al. [69-72]. The reaso n for this dif-
ference might be due to the difference between the cell
types (hepatocytes or ad renal cells and the brain cells) or
the difference between the observation by electron mi-
croscopy, i.e., direct observation of mitochondria in our
results or light microscopy, i.e., indirect observation of
mitochondria without observing any mitochondria di-
rectly and misinterpretation by Korr et al. [69-72].
The results obtained from the adrenal glands of aging
mice at present should form a part of special cytochem-
istry [49], as well as a part of special radioautographol-
ogy [50], i.e., the application of radioautography to the
adrenal glands, as was recently reviewed by the present
author. We expect that such special radioautographology
and special cytochemistry should be further developed in
all the organs animals in the future.

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5. CONCLUSIONS
From the results obtained at present, it was concluded
that almost all the cells in the 3 layers of the adrenal
cortex of mice at various ages, from prenatal embryo day
19 to postnatal newborns, on day 1, 3, 9 and 14, and
adults to postnatal month 1, 2, 6, 12 and 24, were labeled
with silver grains showing RNA synthesis with
3H-uridine in their mitochondria. Quantitative analysis
on the number of mitochondria in adreno-cortical cells in
the 3 layers resulted in an increase from the prenatal day
to postnatal day 1, 3, 9, 14, and month 1 and 2, and 6,
reaching the maximum at postnatal month 12, then a
little decreased to month 24. To the contrary, the num-
bers of labeled mitochondria with 3H-uridine showing
RNA synthesis increased from perinatal stage to postna-
tal juvenile stage at day 9, and decreased to aging and
senescence, while the mitochondrial labeling index also
increased from prenatal day to postnatal day 1, 3 and 9,
reaching the maximum at postnatal day 9, then de-
creased to day 14, month 1, 2, 6, 12 and 24.
On the other hand, the number of mitochondria per
cell in the medulla increased from fetal day 19 to post-
natal month 1 reaching the plateau from month 1 to 24,
while the number of labeled mitochondria per cell and
the labeling index of intramitochondrial RNA synthesis
incorporating 3H-uridine increased from fetal day 19 to
postnatal day 14, reaching the maxima and decreased
from month 1 to 24.
These results demonstrate that the number of mito-
chondria in adrenal cells in both the cortex and medulla
increased by proliferating themselves synthesizing mi-
tochondrial DNA and RNA at perinatal stages to postna-
tal month 1-6 and decreased due to aging.
6. ACKNOWLEDGEMENTS
This study was supported in part by Grant-in-Aids for Scientific Re-
search from the Ministry of Education, Science and Culture of Japan
(No. 02454564) while the author worked at Shinshu University School
of Medicine as well as Grants for Promotion of Characteristic Research
and Education from the Japan Foundation for Promotion of Private
Schools (1997, 1998 1999, 2000) while the author worked at Nagano
Women’s Jr. College. The author is also grateful to Grant-in-Aids for
Scientific Research from the Japan Society for Promotion of Sciences
(No. 18924034, No. 19924204 and No. 20929003) while the author has
been working at Shinshu Institute of Alternative Medicine and Welfare
since 2005 up to the present time. The author thanks Dr. Kiyokazu
Kametani, Technical Official, Research Center for Instrumental Analy-
sis, Shinshu University, for his technical assistance in electron micros-
copy during the course of this study.
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