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Macromolecular Synthesis in Hepatocyte Mitochondria of
Aging Mice as Revealed by Electron Microscopic
Radioautography I: Nucleic Acid Synthesis
T. Nagata1,2*
1
Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto 3908621, Japan
2
Department of Anatomy, Shinshu Insititute of Alternative Medicine, Nagano 380-0816, Japan
We studied the aging changes of intramitochondrial nucleic acid synthesis, both DNA and RNA, in
hepatocytes of several groups of litter mate mice at various ages, from embryos to postnatal newborn,
juvenile, young adults and senescent year 1 and 2, by means of electron microscopic radioautography
after the injections with 3H-thymidine and 3H-uridine , respectively. From the results, it was found that
some of the mitochondria in the hepatocytes were labeled with silver grains showing DNA and RNA
syntheses. Quantitative analysis revealed that the intramitochondrial labeling indices with silver grains
increased from prenatal day to postnatal newborn, adult month 1, reaching the maxima, then decreased to
senile year 1 to 2, indicating the aging changes. Based upon our findings, available literatures on
macromolecular synthesis in mitochondria of various cells are reviewed.
Keywords: mitochondria; EM radioautography; DNA and RNA syntheses; mouse hepatocytes; aging
1. Introduction
Macromolecular synthesis such as nucleic acids syntheses in nuclei and cell bodies of various kinds of
cells in various organs of experimental animals has been extensively studied since many years by both
biochemical and morphological approaches [1-15]. Among of these studies, we first demonstrated the
intramitochondrial nucleic acid syntheses, both DNA and RNA, in mammalian and avian cells
morphologically by means of electron microscopic radioautography with accurate localization in
primary cultured cells of the livers and kidneys of mice and chickens in vitro [1,2] and then in some
other established cell lines such as HeLa cells [3-15] or mitochondrial fractions prepared from in vivo
cells [5]. These phenomena were later commonly found in various cells and tissues not only in vitro
obtained from various organs in vivo [16-21], but also in vivo cells of various organs such as the salivary
gland [22], the liver [23-40], the pancreas [41-43], the trachea [44], the kidney [45], the testis [46,47],
the uterus [48-51], the spleen [52,53], the adrenal gland [54,55], the brain [56] of mice and the eyes of
chickens [57-61] and mice [62,63] in our laboratory. The relationship between the intramitochondrial
DNA and RNA syntheses and cell cycle was formerly studied and it was clarified that the
intramitochondrial DNA and RNA syntheses were performed without any nuclear involvement [5].
This paper reviews the relationship between the nucleic acids, both DNA and RNA, syntheses in
hepatocyte mitochondria and aging of mice in vivo at various ages by means of electron microscopic
radioautography carried out in our laboratory [1-65] as well as available literatures. These studies supply
additional data to the serial studies on special cytochemistry [64] and radioautographology [65].
2. Radioautographic Procedures
2.1.Animals and RI Administration
*
Corresponding author : e-mail: [email protected], FAX: +81-263-46-2848
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Normal ddY strain mice of 20 groups, each consisting of 3 litter mates of both sexes, total 60, aged from
embryonic day 19 to postnatal day 1, 3, 9 and 14 and month 1, 2, 6, 12 and 24, were housed under
conventional 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 divided into 2 sub-groups and administered with
one of the RI-labeled macromolecular precursors, 3H-thymidine (DNA precursor, Amersham, England,
specific activity 877 GBq/mM) or 3H-uridine (RNA precursor, Amersham, England, specific activity
1.11 TBq/mM) at 9:00 a.m. simultaneously at a dosage of 370 KBq/gm body in saline. The animals
were perfused at 10 a.m., one hr. after the injection, via the left ventricles of the hearts with 0.1 M
cacodylate-buffered 2.5% glutaraldehyde under Nembutal anesthesia, then the liver tissues were taken
out 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 as well as the principles of laboratory animal care
in NIH publication No. 86-23 (revised 1985). For light microscopy, thick sections at 1 µm thickness
from respective specimens were cut in sequence and processed for radioautography with Konica NR-M2
radioautographic emulsion (Konica, Tokyo, Japan) by a dipping method [19,21,42,64,65]. For electron
microscopy, semithin sections at 0.2µm thickness were cut in sequence, collected on collodion coated
copper grid meshes and coated with Konica NR-H2 radioautographic emulsion (Konica, Tokyo, Japan)
by a wire-loop method [19, 20, 21, 42, 64,65]. The electron microscopic radioautograms were examined
in either a Hitachi H-700 electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 200
kV or a JEOL JEM-4000EX electron microscope (JEOL, Tokyo, Japan) at accelerating voltages of 300400 kV for observing thick specimens.
2.2. Quantitative Analysis of Electron Micrographs
For quantitative analysis of electron micrographs, twenty EM radioautograms showing cross sections of
mononucleate hepatocytes from each group, based on the electron microscopic photographs taken after
observation on at least 100 hepatocytes from respective animals, were analyzed to calculate the total
number of mitochondria in each hepatocyte and the number of labeled mitochondria covered with silver
grains by visual grain counting. On the other hand, the number of silver grains in 10 circles with the
same area size as a mitochondrion outside cells was also calculated in respective specimens as
background fog. The average number of silver grains per mitochondrial area was 0.01-0.03/area in the
respective groups, which resulted in less than 1 silver grain per area. Therefore, the grain count in each
specimen was not corrected with background fog. Thus, the mitochondrion that was labeled with more
than one silver grain was defined as labeled. The data were stochastically analyzed using variance and
Student's t-test. The differences were considered to be significant at P value <0.01 (n=20).
3. DNA Synthesis in Mouse Hepatocyte Mitochondria
3.1. Qualitative Observations on DNA Synthesis in Hepatocyte Mitochondria
Observing light microscopic and electron microscopic radioautograms labeled with 3H-thymidine, the
silver grains were found over the nuclei of some hepatocytes, demonstrating DNA synthesis mainly in
perinatal stages at embryonic day 19, postnatal day 1, day 3 (Fig. 1A) and day 9 (Fig. 1B). However,
those labeled hepatocytes were almost mononucleate cells (Figs. 1A-1B) and only a few binucleate cells
were found (Fig. 1C) among the mononucleate hepatocytes. In the labeled hepatocytes the silver grains
were mainly localized over the euchromatin of the nuclei and only a few or several silver grains were
found over the mitochondria of these mononucleate hepatocytes. To the contrary, most hepatocytes were
not labeled with any silver grains in their nuclei nor cytoplasm, showing no DNA synthesis even after
labeling with 3H-thymidine in aged adult and senescent animals at postnatal month 1, month 2, month 6,
month 12 and month 24. On the other hand, labeled binucleate hepatocytes over their nuclei were very
rarely found only at the perinatal stages from postnatal day 1, day 3, day 9 and day 14 but not after
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postnatal month 1 to senescent stages up to month 12 or 24. Among many unlabeled hepatocytes, most
mononucleate and binucleate hepatocytes were observed to be labeled with several silver grains over
their mitochondria due to the incorporations of 3H-thymidine especially at the perinatal stages from
embryonic day 19 to postnatal day 1, day 3, day 9 and 14 (Fig. 1D). The localizations of silver grains
over the mitochondria were mainly on the mitochondrial matrices but some grains over the
mitochondrial membranes and cristae when observed by high power magnification (Fig. 1D).
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Figs. 1 Electron microscopic radioautograms of hepatocytes of mice at various ages from newborn to adult mouse at
postnatal month 12, labeled with 3H-thymidine showing DNA synthesis in the nuclei as well as in some
mitochondria. Fig. 1A. Electron microscopic radioautogram of a mononucleate hepatocyte of a newborn mouse at
postnatal day 3, labeled with 3H-thymidine showing DNA synthesis (many silver grains) in the nucleus as well as in
a few mitochondria. Note that all the mitochondria at this stage are small and round and the number of mitochondria
are not so many. x 3,000. Fig. 1B. Electron microscopic radioautogram of three mononucleate hepatocytes of a
newborn mouse aged at postnatal day 9. Note that the number of mitochondria are more than Fig. 1A. x 3,000. Fig.
1C. Electron microscopic radioautogram of a binucleate hepatocyte of a young mouse aged at postnatal day 14. x
3,000. Fig. 1D. High power magnification electron microscopic radioautogram of a mononucleate hepatocyte of a
young mouse aged at postnatal day 14, labeled with 3H-thymidine showing DNA synthesis labeled with many silver
grains in the part of a nucleus (top) as well as in several mitochondria labeled with silver grains. Note that the silver
grains in the mitochondria are mainly localized over the mitochondrial matrices. x 15,000.
This figure was cited from Annals of Microscopy, Vol. 4: pp. 4-18, 2005.
3.2. Quantitative Analysis on DNA Synthesis in Hepatocyte Mitochondria
3.2.1.Number of Mitochondria per Cell
Preliminary quantitative analysis on the number of mitochondria in 10 mononucleate hepatocytes whose
nuclei were labeled with silver grains and other 10 mononucleate hepatocytes whose nuclei were not
labeled in each aging group injected with 3H-thymidine revealed that there was no significant difference
between the number of mitochondria and the labeling indices in both types of hepatocytes (P<0.01).
Thus, the numbers of mitochondria and the labeling indices were calculated in both types of hepatocytes
with labeled or unlabeled nuclei at respective aging stages. The results obtained from the number of
mitochondria in mononucleate hepatocytes showed increases from the prenatal day to postnatal day 14,
then to postnatal month 1-6, reaching the maximum, then decreased to year 1-2 as shown in Fig. 2 (top
left). As for the binucleate hepatocytes, on the other hand, because the appearances of binucleate
hepatocytes showing silver grains in their nuclei demonstrating DNA synthesis were not so many in the
adult and senescent stages from postnatal month 1 to 24, only binucleate cells at perinatal stages when
reasonable numbers of labeled hepatocytes were found in respective groups were analyzed. The number
of mitochondria in binucleate hepatocytes at postnatal day 1 to 14 kept around 80 which did not show
such remarkable changes (Fig. 2 top right) as in mononucleate cells.
3.2.2. Mitochondrial DNA Synthesis in Mouse Hepatocytes
The results of visual counting on the number of mitochondria labeled with silver grains obtained from 10
mononucleate hepatocytes of each animal labeled with 3H-thymidine demonstrating DNA synthesis in 7
aging groups at perinatal stages, prenatal embryo day 19, postnatal day 3, 9 and 14, month 1, 6 and 12,
are plotted in Fig. 2 (middle left). The labeling indices in respective aging stages were calculated from
the number of labeled mitochondria (Fig. 2 middle left) and the number of total mitochondria per cell
(Fig. 2 top left) which were plotted in Fig. 2 (bottom left), respectively. The results showed that the
numbers of labeled mitochondria with 3H-thymidine increased from prenatal embryo day 19 to postnatal
day 14, reaching the maximum, and then decreased to month 6 and again increased to year 1 (6.0/cell),
while the labeling indices increased from prenatal day 19 (11.8%) to postnatal day 14 (16.9%), reaching
the maximum, then decreased to month 6 (4.1%) and year 1 (6.4%) and year 2 (2.3%). The increase of
the total number of mitochondria in mononucleate hepatocytes was stochastically significant (P<0.01),
while the changes of number of labeled mitochondria and labeling index in mononucleate hepatocytes
were not significant (P<0.01).
4. RNA Synthesis in Mouse Hepatocyte Mitochondria
4.1. Qualitative Observations on RNA Synthesis in Hepatocyte Mitochondria
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Observing light microscopic radioautograms labeled with 3H-uridine, the silver grains were found over
both the karyoplasm and cytoplasm of almost all the cells not only at the perinatal stages from embryo
day 19 to postnatal day 1, 3, 9, 14, but also at the adult and senescent stages from postnatal month 1 to 2,
6, 12 and 24. By electron microscopic observation, silver grains in the hepatocytes were observed in
most hepatocytes at respective aging groups localizing not only over euchromatin and nucleoli in the
nuclei but also over many cell organelles such as endoplasmic reticulum, ribosomes, and mitochondria as
well as cytoplasmic matrix of both mononucleate (Figs. 3A, B, D) and binucleate (Fig. 3C) hepatocytes
from perinatal stage at embryonic day 19, postnatal day 1 (Fig. 3A), day 3 (Fig. 3B), day 9, day 14 (Fig.
3D), to adult and senescent stages at postnatal month 1, month 2, month 12 (Fig. 3C) and month 24. The
localizations of silver grains over the mitochondria were mainly on the mitochondrial matrices but a few
over the mitochondrial membrans and cristae when observed by high power magnification (Fig. 3D).
4.2. Quantitative Analysis on RNA Synthesis in Hepatocyte Mitochondria
Fig. 2.Transitional curves demonstrating aging changes of mitochondria in mononucleate (left) and binucleate
(right) hepatocytes labeled with 3H-thymidine. Mean ± standard deviations. Top: The averages of the total number
of mitochondria per cell in mononucleate and binucleate hepatocytes at respective aging groups from embryonic day
19 to postnatal year 2 were plotted. Middle: The averages of the total number of mitochondria labeled with 3Hthymidine showing DNA synthesis per cell in mononucleate and binucleate hepatocytes at respective aging groups
were plotted. Bottom: The averages of the labeling index of mitochondria labeled with 3H-thymidine showing DNA
synthesis (number of labeled mitochondria / number of total mitochondria) at respective aging groups were plotted.
This figure was cited from Annals of Microscopy, Vol. 4: pp. 4-18, 2005.
4.2.1.Number of Mitochondria per Cell
The numbers of mitochondria per cell were calculated in all the groups of animals. The results showed
that the numbers increased from the prenatal day to postnatal month 2-6, reaching the maximum, then
decreased to year 1-2 as shown in Fig. 4 (top left and right).
4.2.2. Mitochondrial RNA Synthesis in Mouse Hepatocytes
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The results of visual counting on the number of mitochondria labeled with silver grains obtained from 20
mononucleate hepatocytes of each animal labeled with 3H-uridine in 10 aging groups at perinatal stages,
prenatal embryo day 19, postnatal day 1, 3, 9 and 14, month 1, 6 and year 1 and 2, are plotted in Fig. 4
(middle left) which showed the maximum at month 3. The labeling indices in respective aging stages
were calculated from the number of labeled mitochondria (Fig. 4, middle left) and the number of total
mitochondria per cellular profile area (Fig. 4, top left) which were plotted in Fig. 4 (bottom left),
respectively. The results showed that the numbers of labeled mitochondria with 3H-uridine showing RNA
synthesis increased from prenatal embryo day 19 (3.3/cell) to postnatal month 1 (9.2/cell), reaching the
maximum, and then decreased to month 6 (3.5/cell) and again increased to year 1 (4.0/cell) and year 2
(4.3/cell), while the labeling indices increased from prenatal day 19 (12.4%) to postnatal month 1
(16.7%), reaching the maximum, then decreased to year 1 (4.8%) and year 2 (5.3%). On the other hand,
the number of mitochondria in binucleate cells reached the maximum at month 6, but the number of
labeled mitochondria and the labeling indices did not show significant changes (Fig. 4 right). It is well
known that considerable numbers of binucleate hepatocytes appear in adult and senescent stages [23,33].
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Figs. 3. Electron microscopic radioautograms of hepatocytes of mice at various ages from newborn to adult mouse
at postnatal month 12, labeled with 3H-uridine showing RNA synthesis in the nucleoli and the chromatin in the
nuclei as well as in some mitochondria in the cytoplasm. Fig. 3A. Electron microscopic radioautogram of a
mononucleate hepatocyte of a newborn mouse aged at postnatal day 1, labeled with 3H-uridine showing RNA
synthesis (many silver grains) in the nucleolus and the chromatin in the nucleus of the hepatocytes at center as well
as several silver grains in some mitochondria in the cytoplasm. Note that all the mitochondria at this stage are small
and round and the number of mitochondria are not so many. x 3,000. Fig. 3B. Electron microscopic radioautogram
of two mononucleate hepatocytes of a newborn mouse aged at postnatal day 3, labeled with 3H-uridine showing
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RNA synthesis (many silver grains) in the nucleolus and karyoplasm in the nucleus (center) as well as in another
nucleus at top right. Some of the mitochondria in both cells are labeled with silver grains. x 3,000. Fig. 3C.
Electron microscopic radioautogram of a binucleate hepatocyte of an adult mouse aged at postnatal month 12,
labeled with 3H-uridine showing RNA synthesis (several silver grains) in the two nuclei (a few silver grains) as well
as a few mitochondria (with a few silver grains). x 3,000. Fig. 3D. High power magnification electron microscopic
radioautogram of a mononucleate hepatocyte of a young mouse aged at postnatal day 14, labeled with 3H-uridine
showing RNA synthesis (several silver grains) in the nucleolus and karyoplasm in the nucleus at top left as well as in
a mitochondrion with 3 silver grains at lower left. Note that the silver grains in the mitochondrion are localized over
the mitochondrial matrix. x 15,000
Figs. 3A, 3B, 3C, 3D are reproductions of Figs. 1, 2, 8, 4, respectively, published in Microsc. Res.
Technique, Vol. 67, Issue 2, June 1, 2005, authored by T. Nagata and H. Ma, entitled "Electron
microscopic radioautographic study of RNA synthesis in hepatocyte mitochondria of aging mouse," Vol.
67: pp. 55-64, 2005.
Fig. 4. Transitional curves demonstrating aging changes of mitochondria in mononucleate (left) and binucleate
(right) hepatocytes labeled with 3H-uridine. Mean ± standard deviations. Top: The averages of the total number of
mitochondria per cellular profile area in mononucleate and binucleate hepatocytes at respective aging groups from
embryonic day 19 to postnatal year 2 were plotted. Middle: The averages of the total number of mitochondria
labeled with 3H-uridine showing RNA synthesis per cellular profile area in mononucleate and binucleate
hepatocytes at respective aging groups were plotted. Bottom: The averages of the labeling index of mitochondria
labeled with 3H-uridine showing RNA synthesis (number of labeled mitochondria / number of total mitochondria) at
respective aging groups were plotted.
Figs. 4 left and right are reproductions of Figs. 9, 10, respectively, published in Microsc. Res. Technique,
Vol. 67, Issue 2, June 1, 2005, authored by T. Nagata and H. Ma, entitled "Electron microscopic
radioautographic study of RNA synthesis in hepatocyte mitochondria of aging mouse," Vol. 67: pp. 5564, 2005.
We formerly observed that the quantity of DNA and RNA synthesis as expressed by grain counting in
karyoplasm and cytoplasm of both mononucleate and binucleate cells in mouse hepatocytes showed
increases and decreases reaching the maxima at postnatal month 2 in case of mononucleate cell and at
month 6 in case of binucleate cells [23,33-35]. It was noted that the differences of grain counts between
the mononucleate and binucleate cells in the same aging groups were stochastically significant. These
results indicated that the amount of DNA and RNA synthesized and distributed in karyoplasm and
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cytoplasm of each binucleate cell was much more than each mononucleate cell in respective aging
groups..
5. Nucleic Acid Synthesis in Mitochondria of Various Cells
Concerning to 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-thymidine demonstrate DNA synthesis [1-9,11-14,16-29,42,64,65], while 3H-uridine
demonstrates RNA synthesis [1,3-21, 24, 42,64,65]. The previous studies obtained from the livers of
aging mice revealed that silver grains incorporating either 3H-thymidine or 3H-uridine were observed not
only over the nuclei of some hepatocytes but also over the mitochondria showing intramitochondrial
DNA and RNA synthesis [2,3,5-10,42,64f,65].
5.1. Mitochondrial DNA Synthesis in Various Cells
With regards to the incorporations of 3H-thymidine into mitochondria demonstrating DNA synthesis,
many authors previously reported that it was observed by means of electron microscopic
radioautography in lower organism such as slime mold [66, 67], tetrahymena [68] or chicken fibroblasts
in tissue culture under abnormal conditions [69, 70] or liver and kidney cells of chicken and mouse
under normal conditions [1] as was formerly reviewed [71, 72]. Likewise, RNA synthesis in Agaricus
[73], Ochromonas [74], chicken liver [75,76], mouse liver [77], rat adrenal cortex [78] and human cells
[4-15] were also demonstrated. Most of these authors, however, used old-fashioned developers
consisting of methol and hydroquinone (MQ-developer) which produced coarse spiral silver grains
resulting in inaccurate localization over cell organelles, especially mitochondria, when observed by
electron microscopy. Thus, most of these authors, except Nagata [4-15], showed photographs of electron
radioautograms with large spiral-formed silver grains (2-3 µm in diameter) localizing not only over the
mitochondria but also outside the mitochondria. In order to obtain smaller silver grains, we first used
elon-ascorbic acid developer after gold latensification [4-15], which produced comma-shaped smaller
silver grains (0.3-0.8 µm in diameter) much better for localization over mitochondria than spiral silver
grains (2-3 µm in diameter), then later we used phenidon developer after gold latensification, producing
dot-like smaller silver grains (0.2-0.3 µm in diameter) localizing only inside the mitochondria showing
ultrahigh resolution of radioautograms [19-21,35,66,67] as shown in Figs. 1 and 3 in this study. Thus,
these papers from our laboratory were the first which demonstrated intramitochondrial DNA, RNA and
protein syntheses incorporating 3H-thymidine, 3H-uridine and 3H-leucine, respectively, with accurate
intramitochondrial localization in avian and mammalian cells in 1960s-1970s [1-14]. Concerning to the
resolution of electron microscopic radioautography, on the other hand, several authors discussed the
sizes of silver grains under various experimental conditions and calculated various values of resolutions
[4-7,79-81]. Those authors who used the M-Q developers maintained the resolution to be 100-160 nm
[82-83], while those authors who used the elon-ascorbic acid developer [5-7,82] calculated it to be 25-50
nm. When we used phenidon developer at 16 C for 1 min after gold latensification, we could produce
very fine dot-shaped silver grains and obtained the resolution around 25 nm [19-21,42,64,65,83]. For the
purpose of analyzing electron radioautographs, Salpeter et al. [80] suggested to use the half-distance and
proposed to use 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 latensification to produce very
fine dot-shaped silver grains, we judged only the silver grains which were located in the mitochondria
which were dot-shaped very fine ones to be attributable to the mitochondria without any problem with
the resolution around 25 nm as was formerly discussed [5-7].
On the other hand, the incorporations of 3H-thymidine into mitochondria demonstrating DNA
synthesis were formerly observed by means of electron microscopic radioautography not only in lower
organisms such as slime mold [66,67], tetrahymena [68,84] but also in higher animals such as chicken
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fibroblasts in tissue culture under abnormal conditions [69,70] or liver and kidney cells of chicken and
mouse under normal conditions [1,2,77]. Then we also demonstrated intramitochondrial DNA synthesis
incorporating 3H-thymidine in some other established cell lines originated from human being such as
HeLa cells [3-15] or mitochondrial fractions prepared from in vivo mammalian cells such as rat and
mouse [5]. It was later commonly found in various cells and tissues in vitro obtained from various organs
such as the mouse liver and pancreas cells in vitro [11,12], cultured rat sarcoma cells [13], cultured
human uterus cancer cells HeLa [14,15], but also in vivo cells obtained from various organs [22-65] of
chickens and mice. Thus, it is clear that all the cells in various organs of various animals synthesize
DNA and RNA not only in their nuclei but also in their mitochondria.
The relationship between the cell cycle and the intramitochondrial DNA as well as RNA syntheses
was formerly studied in synchronized cells and it was clarified that both the intramitochondrial DNA and
RNA syntheses were performed without any nuclear involvement [5]. However, the relationship
between the aging of individual animals and the nucleic acid syntheses has not yet been clarified except a
few papers recently published by Korr and associates on mouse brain [86-88]. They reported both
nuclear DNA repair, measured as nuclear unscheduled DNA synthesis, and cytoplasmic DNA synthesis
labeled with 3H-thymidine in several types of cells in brains as observed by only 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 mitochondrial DNA without observing the mitochondria by
electron 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 and endothelial cells, did not show such age-related changes without counting the number of
mitochondria in respective cells neither counting the number of labeled mitochondria nor calculating the
labeling indices of mitochondria at respective aging stages. Thus, their result from obtained from the
grain counting seems to be not accurate without observing mitochondria directly by electron microscopy.
5.2. Mitochondrial RNA Synthesis in Various Cells
The present results obtained from the livers of mice revealed that the incorporation of 3H-uridine
indicating RNA synthesis resulted in silver grain localization over the nuclei and cell bodies of almost all
mononucleate and binucleate hepatocytes from perinatal animals at embryonic day 19, postnatal day 1, 2,
9, 14 to adult and senescent stages at postnatal month 1, 2, 6, 12 and 24, which showed the localization
of newly synthesized RNA within the mitochondria of hepatocytes. From the results obtained, the
number of mitochondria in each hepatocytes per cellular profile area showed increases and decreases
reaching the maxima at postnatal month 2 in case of mononucleate cell and at month 6 in case of
binucleate cells. On the other hand, the number of labeled mitochondria with silver grains due to 3Huridine demonstrating intramitochondrial RNA synthesis also showed increases and decreases reaching
the maximum at postnatal month 1 in case of mononucleate cell in contrast that no significant increase
and decrease were noticed in case of binucleate cells. The labeling indices of mitochondria with 3Huridine, however, did not show significant increases and decreases in both mononucleate and binucleate
cells. On the other hand, the radioautograms in the present study showing incorporations of 3H-uridine
into mitochondria indicating mitochondrial RNA synthesis resulted in silver grain localization over the
mitochondria independently from the nuclei whether the nuclei were labeled with many silver grains or
not in both mononucleate and binucleate hepatocytes from perinatal day 1, 3, 9, 14 to adult and senescent
month 1, 2, 6, 12 and 24. With regards to RNA 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 by
biochemical approaches or by morphological approaches [66-70]. Most of these authors observed
mitochondrial ribosomes appeared in the matrix compartment as small particles, 10-15 nm in diameter,
by electron microscopic observation, which were extracted with RNase. The 3 classes of RNA, i. e.,
messenger, transfer and ribosomal, were also found in mitochondria [71](Schatz 1970). Mitochondria of
various cells contained aminoacyl-RNA synthetase and DNA dependent RNA polymerase [71]. The
evidence for RNA synthesis in mitochondria was first demonstrated by means of electron microscopic
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radioautography incorporating 3H-uracil in lower organisms such as Agaricus campestris by Vogel and
Kemper [73] or 3H-uridine in Ochromonas by Gibbs [74], then in animal cells such as chicken and mouse
livers by André [75,76] and Curgy [77], in rat adrenocortical cells by Nussdorfer and Mazzochi [78], in
human cells such as HeLa cells by Nagata [3-15]. Most of these authors, however, used old-fashioned
developers consisting of methol and hydroquinone (MQ-developer) which produced coarse spiral silver
grains resulting in inaccurate localization over cell organelles, especially mitochondria, when observed
by electron microscopy. Thus, most of these authors, except Nagata [3-15], showed photomicrographs
of electron radioautographs with large spiral-formed silver grains (2-3 µm in diameter) localizing not
only over the mitochondria but also outside the mitochondria. In order to obtain smaller silver grains, we
first used elon-ascorbic acid developer after gold latensification [1-7], which produced comma-shaped
smaller silver grains (0.3-0.8 µm in diameter), then later we used phenidon developer after gold
latensification, producing dot-like small silver grains (0.2-0.3 µm in diameter) localizing only inside the
mitochondria showing ultrahigh resolution of radioautograms [21,42,64,65,83]. These papers [1-7] were
the first which demonstrated intramitochondrial RNA synthesis incorporating 3H-uridine with accurate
intramitochondrial localization in avian and mammalian cells.
Likewise, we also demonstrated that increases and decreases were observed by direct observation on
mitochondria at electron microscopic level and obtaining accurate mitochondrial number and labeling
indices labeled with 3H-uridine (38). Thus, this paper should be the first to show the relationship between
the RNA synthesis and aging in hepatocytes of mice in vivo at various ages by means of electron
microscopic radioautography observing the small dot-like silver grains, due to incorporations of 3Huridine, which exactly localized inside the mitochondria. We formerly studied the relationship between
the RNA synthesis and aging in the adrenal glands of mice at various ages from postnatal day 14 to
month 6 and 12 and found that the numbers of total mitochondria and the numbers of labeled
mitochondria with incorporations of 3H-uridine in respective adrenal cortical zones, zona glomerulosa,
fasciculata, reticularis and the adrenal medulla cells increased from postnatal day 14 to month 12 [55].
However, the animals at the perinatal stages from prenatal day to postnatal day 14, as well as the
senescent stage at postnatal year 2, were not yet studied. There was a discrepancy between the results
obtained from the adrenal glands which showed only increases of the total mitochondria and the labeled
mitochondria with aging and the present results from the hepatocytes which showed increases and
decreases with aging. The reason for this difference might be the difference between the cell types
(adrenal cells and hepatocytes).
From the results obtained at present, it was concluded that almost all the hepatocytes of mice at various
ages, from prenatal embryos to postnatal newborn, young, adult and senescent animals, were labeled
with silver grains showing RNA synthesis with 3H-uridine in their mitochondria. Quantitative analysis on
the numbers of labeled mitochondria with 3H-uridine showing RNA synthesis and the labeling indices
increased from prenatal days to postnatal days, reaching the maximum at postnatal month 1, then
decreased to year 2.
6. Concluding Remarks
From the results obtained at present, it was concluded that almost all the hepatocytes of mice at various
ages, from prenatal embryos to postnatal newborn, young, adult and senescent animals, were labeled
with silver grains showing DANN and RNA synthesis either with 3H-thymidine or 3H-uridine in their
mitochondria. Quantitative analysis on the numbers of mitochondria in mononucleate hepatocytes
resulted in an increase from the prenatal day to postnatal month 1, 2, 6, reaching the maximum at
postnatal month 6, then decreased to year 2. We demonstrated that increases and decreases were
observed in the mitochondrial numbers and labeling indices of DNA synthesis with 3H-thymidine
incorporations by direct observation on mitochondria at electron microscopic level. It was also concluded
that almost all the hepatocytes of mice at various ages, from prenatal embryos to postnatal newborn,
young, adult and senescent animals, were labeled with silver grains showing RNA synthesis with 3Huridine in their mitochondria. Quantitative analysis on the numbers of labeled mitochondria with 3H-
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uridine showing RNA synthesis and the labeling indices increased from prenatal days to postnatal days,
reaching the maximum at postnatal month 1, then decreased to year 2. The results indicate that
mitochondria in hepatocytes synthesize nucleic acids independently from the nuclei and cytoplasm of
hepatocytes, but their synthetic activities are affected from the aging of the animals.
Thus, the results obtained from the liver at present should form a part of special radioautographology
[65], i.e., application of radioautography to the liver, as well as a part of special cytochemistry [64], 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 in the future.
Acknowledgments
This study was supported in part by Grants-in-Aids for Scientific Research from the Japan Society for
Promotion of Sciences (No. 18924034, No. 19924024) while the author has been working at Shinshu
Institute of Alternative Medicine since 2005. The author is also grateful to Dr. Kiyokazu Kametani,
Research Center for Instrumental Analysis, Shinshu University, Matsumoto, for his technical assistances
during the course of this study.
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