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Development of morphologic heterogeneity of hepatocyte mitochondria in the mouse.

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THE ANATOMICAL RECORD 210:315-325 (1984)
Development of Morphologic Heterogeneity of Hepatocyte
Mitochondria in the Mouse
SHINSUKE KANAMURA, KAZUO KANAI, MOTOKO OKA,
JUN WATANABE, AND MAR1 ASADA-KUBOTA
Department of Anatomy, Kansai Medical University, 1 Fumizonecho,
Moriguchi, Osaka, 570 Japan
ABSTRACT
The ultrastructure of mitochondria in periportal and perihepatic hepatocytes from newborn, 5-, lo-, and 20-day-old, and adult male ddY
mice was analyzed by quantitative electron microscopy. In newborn and 5- and
10-day-old animals, the axial ratio (length per diameter), surface to volume
ratio (area of the outer membrane per unit mitochondrial volume), and volume
density were not significantly different between periportal and perihepatic
cells. In 20-day-old and adult animals, the surface to volume ratio was greater
in perihepatic cells than periportal cells, and the volume density was greater
in periportal cells than perihepatic cells. The axial ratio became greater in
perihepatic cells than periportal cells in adult animals. However, there were
no differences in the surface density of the outer membrane, and of the inner
membrane and cristae between the cells of both zones in all age groups
examined. When the data were expressed as volume and area per cell, the
patterns of subacinar distribution and age-related changes differed from the
patterns seen in the volume and surface density data mainly in adult animals.
This difference was generally caused by the marked increase in hepatocyte
volume between 20 days of age and adulthood, especially in perihepatic cells.
The results show that differences between mitochondria in periportal cells
and those in perihepatic cells in the shape (the axial and surface to volume
ratios), volume density, and area of the outer membrane per cell, evident in
adult animals, are not present in newborn animals but arise during postnatal
development.
The hepatocyte mitochondrion is concerned
with urea formation in addition to general
functions of oxidative phosphorylation in the
cell, and, therefore, it is a n important cell
organelle. In adult animals, morphologic heterogeneity has been shown among hepatocyte mitochondria. The volume density is
greater in periportal cells than in perihepatic
(around the terminal hepatic venule) cells
(Loud, 1968; Schmucker et al., 1978; AsadaKubota et al., 1982). The area of the outer
membrane per unit mitochondrial volume is
greater in perihepatic cells than in periportal
cells (Loud, 1968). The shape is round in periportal cells, whereas it is elongated and slender in perihepatic cells (Novikoff, 1959;
Asada-Kubota et al., 1982).
It is of interest to know whether the morphologic heterogeneity of hepatocyte mito-
0 1984 ALAN R. LISS, INC.
chondria arises during postnatal development as a part of the process of formation
of adult liver lobulation. However, there
have been no reports in the literature that
answer this question, except for our paper
on postnatal development of the volume density (Asada-Kubota et al., 1982). Papers describing the quantitative morphology of the
hepatocyte mitochondrion during postnatal
development have been published (Rohr et
al, 1971; Lange and Herbener, 1972; Herzfeld
et al., 1973; David, 1979). In these papers,
however, measurements were carried out
without distinction between periportal and
perihepatic hepatocytes, i.e., on the assumption that all hepatocytes are identical.
Received October 13, 1982; accepted March 27,1984.
316
S. KANAMURA ET AL.
In the present study, therefore, we examined the axial ratio (length per diameter;
Weibel and Gomez, 1962) and surface to volume ratio (area of the outer membrane per
mitochondrial volume; Loud, 1968; Weibel et
al., 1966; Weibel, 1979) as indices of the
shape, volume density, and other stereological parameters of mitochondria in periportal
and perihepatic hepatocytes from growing
and adult mice to study the postnatal development of morphologic heterogeneity of hepatocyte mitochondria.
MATERIALS AND METHODS
Newborn, 5-, lo-, 20-day-old, and adult
(about 3 months old) male ddY mice were
used. In each age group, there were three
animals for estimation of the axial ratio and
five animals for that of all other data. Newborn animals were used immediately after
birth. The 10-day-oldage was chosen because
the subacinar distribution of glucose 6-phosphatase and ornithine carbamoyltransferase
activities becomes similar to the adult type
at this age (Kanamura, 1975; Kanamura and
Asada-Kubota, 1980). The 5-day-old mice
were selected as representing the middle
point between newborn and 10-day-old animals. The 20-day-old animals are weanling.
The adult animals were fed standard laboratory chow (Oriental NMF) and water ad libitum. These animals, kept under a 12:12 hour
light/dark cycle, were killed by cervical dislocation a t 10 A.M.
Pieces (smaller than 1 mm3) from the left
lobe were fixed in 2.5% glutaraldehyde in 0.1
M sodium cacodylate, pH 7.3, a t 4°C for 3
hours and rinsed in 0.1 M sodium cacodylate
containing 8%sucrose at 4°C for 30 minutes.
The pieces were postfixed in buffered 1%osmium tetroxide for 3 hours, dehydrated in
ethanol, and embedded in Spurr medium.
For estimation of all data except for the
axial ratio, eight pieces, 4 including the periportal area and 4 including the terminal hepatic venule, were selected randomly from
each animal.
A 1-pm-thick section was cut from each
piece and stained with a mixture of methylene blue and azure 11. One light micrograph
at a final magnification of x 1,000 was taken
at random from each section. These micrographs were used to estimate average hepatocyte volume, average nuclear volume, and
average volume of hepatocyte cytoplasm according to the method described by Weibel
(1979).Hepatocytes within a three-cell radius
of the portal area and the terminal hepatic
venule were considered as periportal and
perihepatic hepatocytes, respectively.
For measurement of the volume density of
mitochondria and the surface densities of the
outer membrane and the inner membrane
and cristae, a thin section of silver interference color was cut from the surface portion
of each piece with a glass knife on a n LKB
Ultrotome, stained with uranyl acetate and
lead citrate, and examined in a Hitachi H
500 electron microscope. Seven randomly
chosen electron micrographs of portions of
the cytoplasm, located in the upper right corner of the supporting copper grid (200-mesh)
and occupying 50% or more of area of the
micrographs, were taken at a n original magnification of x 10,000 from each thin section.
Thus, 1,400 photographs enlarged to a final
magnification of x 50,000, which contained
portions of the cytoplasm of hepatocytes
within a three-cell radius of the portal area
or the terminal hepatic venule, were prepared. The volume density (cubic micrometer
per cubic micrometer of hepatocyte cytoplasm) of the mitochondrion and the surface
density (square micrometer per cubic micrometer of hepatocyte cytoplasm) of “the
outer membrane” or “the inner membrane
and cristae” were estimated using a multipurpose test system (24 x 24 cm) containing
168 test points spaced at 2.0 cm on each photograph according to the point counting
method described by Weibel et al. (1969) and
Weibel (1979). For measurement of the volume density of mitochondria, however, photographs of final magnification of about
~ 2 0 , 0 0 0are suitable (Weibel et al., 1969;
Weibel, 1979). However, we estimated both
the surface and volume densities from photographs of a magnification of ~ 5 0 , 0 0 0the
,
same group of photographs. “The area of the
outer membrane per unit mitochondrial volume” or “the area of the inner membrane
and cristae per unit mitochondrial volume”
was calculated by dividing “the surface density of the outer membrane” or “the surface
density of the inner membrane and cristae”
by “the volume density.” “The area of the
inner membrane and cristae per unit area of
the outer membrane” was calculated by dividing “the surface density of the inner
membrane and cristae” by “the surface density of the outer membrane.”
The area of the outer membrane per unit
mitochondrial volume (surface to volume ratio) (Loud, 1968; Weibel et al., 1966; Weibel,
DEVELOPMENT OF MITOCHONDRIA
1979)can be used as a n approximate index of
the shape and size of mitochondria. Greater
value in the surface to volume ratio shows that
mitochondria are more elongated, smaller,
or both.
Data were also expressed as volume and
area per average mononuclear hepatocyte in
periportal and perihepatic zones. Volume or
area per cell (biological unit) has been known
to be sometimes more meaningful for morphological comparison than the surface and
volume densities (geometrical unit) (Loud,
1968).
For estimation of the axial ratio (length per
diameter), two Spurr pieces, one including
the periportal area and one containing the
terminal hepatic venule, were selected randomly from each animal. A thin section was
cut from the surface portion of each piece,
and five randomly chosen electron micrographs a t a n original magnification of
~ 2 , 5 0 0were taken from each thin section.
Thus, 150 photographs enlarged to a final
magnification of x 10,000, which contained
one whole hepatocyte within a three-cell radius of the portal area or terminal hepatic
venule, were prepared. Estimations were
done on the photographs. The length and
diameter of curved or branched mitochondria
were considered as shown in Figure 2.
The shape of mitochondria was presumed
by both the axial ratio and the surface volume ratio. All of the data were subjected to
statistical analysis using Student’s t-test.
RESULTS
Immersion fixation of liver pieces less than
1mm3 and cutting of thin sections from surface portions of the pieces resulted in satisfactory preservation of hepatocyte ultrastructure (Figs. 1,2).
Hepatocyte Volume
The values were not significantly different
between periportal and perihepatic zones
from birth to 20 days of age (Table 1).In
adult animals, however, perihepatic hepatocytes were 61% larger than the cells of periportal zones. In the two zones, the values in
5-day-old animals were greater than the values in newborn and 10-day-old animals. Between 20 days of age and the adult, the
values increased markedly both in periportal
(220%) and perihepatic (339%) zones. The
changes in the volume of hepatocyte cytoplasm were similar to those of hepatocyte
volume.
317
Axial Ratio
In newborn and 5-, lo-, and 20-day-old animals, the values did not significantly differ
between periportal and perihepatic cells. The
value became greater in perihepatic cells
than in periportal cells in adult animals (P
< 0.05) (Fig. 3, Table 2). Between birth and
the adult, the values did not change significantly in the cells of the two zones except for
a decrease between newborn and 5-day-old
animals in periportal cells (P < 0.05). Thus,
in periportal cells, the value in newborn animals was highest.
Surface to Volume Ratio
In newborn and 5- and 10-day-oldanimals,
there were no significant differences in the
values between periportal and perihepatic
hepatocytes (Fig. 4,Table 2). However, the
values were greater in perihepatic cells than
in periportal cells in 20-day-old (P < 0.01)
and adult (P < 0.05) animals. In the cells of
both zones, the values did not change significantly from birth to 20 days of age, and then
increased.
Comparison of Pattern of Changes in the
Axial Ratio to That in the Surface to Volume
Ratio
There were two differences between patterns of age-related changes in the axial ratio
and surface to volume ratio, although the
general pattern in one was basically similar
to that in the other (Figs. 3, 4). First, in
periportal cells, the axial ratio was significantly highest in newborn animals, whereas
the surface to volume ratio was low and unchanged between birth and 20 days of age.
Second, in the cells of the two zones between
20 days of age and the adult, the surface to
volume ratio increased, although the axial
ratio remained unchanged.
Volume Density
In newborn and 5- and 10-day-oldanimals,
the values were not significantly different
between periportal and perihepatic hepatocytes (Fig. 5, Table 2). The values became
greater in periportal cells than in perihepatic
cells in 20-day-old(P < 0.05) and adult (P <
0.001) animals. Between birth and 10 days of
age, the values increased in the cells of both
zones. Then, the values remained unchanged
in periportal cells but decreased in perihepatic cells between 10 and 20 days of age,
and 20 days of age and the adult.
Figs. 1, 2. Electron micrographs of portions of mouse
hepatocytes. Liver pieces, smaller than 1 mm3, were
fixed by immersion in buffered 2.5%glutaraldehyde and
then in buffered 1%Os04. Thin sections were cut from
surface portions of the fixed pieces. Preservation of fine
structure is satisfactory. X 9,000.
Fig. 1. Portions of periportal hepatocytes from a 5-day
old animal. Mitochondria are large and round to somewhat slender.
Fig. 2. Portions of perihepatic hepatocytes from a 20day-old animal. Mitochondria are small and round to
slender. Length ( -1 and diameter ( ---- ) of curved
and branched mitochondria.
3 19
DEVELOPMENT OF MITOCHONDRIA
T A B L E 1. Morphometric data of hepatocytes from growing and adult mice
Newborn
Periportal
Perihepatic
Hepatocyte
Volume pm3
Volume of nucleus pm3
Volume of cytoplasm pm3
5 Days
Periportal
Perihepatic
1229 +101.8 1237 288.4 2582 *255.0t'
150.9+ 7.13 145.5k 6.67 232.32 14.09'"
1078 5102.1 1092 k94.7 2349 i242.7vt
2379 k230.4"
211.9+ 10.62"'
2167 +222.8+'
Values represent means SE. *P < 0.05, **P < 0.01, significantly different from value in perihepatic cells. tP < 0.05,
ttP < 0.01, tttP < 0.001, significantly different from value in adjacent and younger age group. Student's t-test.
T A B L E 1. Morphometric data of hepatocytes from growing and adult mice (continued)
10 Days
Periuortal
Periheuatic
1836 +144.5'
168.4+ 5.04''
1668 +141.2'
20 Days
Periuortal
PeriheDatic
Adult
Periuortal
Peri hpnnt ir
1720 k72.0t
2321 k99.4' 2436 k337.7 5114 +467.7'Itt 8252 +887.4'''
161.02 4.67'+ 196.2k16.32 219.6k 27.90 321.2k 34.37**' 478.12 26.41"'
1559 k69.7'
2125 286.0' 2217 k310.6 4792 k454.0*ttt 7774 +873.Zttt
Values represent means i SE. *P < 0.05, **P 0.01, significantly different from value in perihepatic cells. 'P < 0.05,
ttP < 0.01, tttP < 0.001, significantly different from value in adjacent and younger age group. Student's t-test.
0-0
PERIPORTAL
*---•
PERIHEPATIC
0-0
5
PERIPORTAL
I
--- PERIHEPATIC
,4c
T
I
I
I
*
4
(r
I
4
I
I
6
D
z
P
I(
0
e3 3
U
0
3
A
7
0
5
10
20
DAYS AFTER BIRTH
-1
2
0
0
5
10
20
DAYS AFTER BIRTH
-1
Fig. 3. The axial ratio (1engtWdiameter) of mitochondria in hepatocytes from growing and adult mice. A,
adult. *P < 0.05, significantly different from value in
periportal celIs.
Fig. 4. The surface to volume ratio (area of the outer
membrane per unit mitochondria1 volume) of mitochondria in hepatocytes from growing and adult mice. A,
adult. *P < 0.05, "'P < 0.01, significantly different from
values in periportal cells.
Surface Density of the Outer Membrane
for an increase between 20 days of age and
the adult in periportal cells.
There were no differences in the values
between periportal and perihepatic cells in
all age groups studied (Table 2). Further, no
age-related changes in the values were observed for the cells of the two zones except
Surface Density of the Inner Membrane and
Cristae
The values were not different between periportal and perihepatic cells in all age groups
+573.21
2075.1
f86.57
4.84 f 0.293
169.7 a11.44
433.3 f21.73
0.411
4.92
212.8 f 27.53
632.6 f131.38
3037.4
12.42 f 0.895
1.769
13.94 f
+
1.979k 0.237
0.358
2.603+
0.413f 0.0501
1.8 + 0.24
2.58 f 0.152
0.161+ 0.020
Perihepatic
1.205
0.144
0.0208
6557.2
+660.35++
5.25 f 0.232
566.7 f 65.45"
1265.6 +150.68'
11.84 f
2.809f
0.535 5
f
0.15'
2.26 f 0.222
0.243f 0.017t
1.4
Periportal
5 Days
1.486
0.273'
0.0517
6528.4
5941.34'+
4.88 k 0.214
521.8 f 46.48t+t
1317.9 _+128.50ttt
12.63 k
3.013f
0.618f
+
Perihepatic
1.6 f 0.09
2.58
0.250
0.243k 0.012tt
Values represent means ?r SE. Axial ratio 1engtMdiameter. Surface to volume ratio, area of outer membraneiunit mitochondria1 volume. Volume density, @m3/pm3of cytoplasm.
Surface density, pm2/pm3 of cytoplasm. Aredunit mitochondria1 volume, prn2ipm3 of mitochondria. Aredunit area of outer membrane pm2/pm2 of outer membrane. Volumeicell,
pm3/average mononuclear hepatocyte. Aredcell, pm2/average mononuclear hepatocyte. *P < 0.05, **P < 0.01,***P < 0.001,significantly different from value in perihepatic cell.
'P c 0.05, ttP 0.01,
< 0,001,significantly different from value in adjacent and younger age group. Student's t-test.
Area of outer membranehell
Area of inner membrane + cristaei
cell
+
+
0.0836
0.546f
+
0.396
0.016
2.89 k
0.187f
0.12
Surface to volume ratio
Volume density
Surface density of
outer membrane
Surface density of inner
membrane cristae
Area of inner membrane cristaei
unit mitochondrial volume
Area of inner membrane cristaei
unit area of outer membrane
Volumeicell
f
1.9
Newborn
Axial ratio
Periportal
TABLE 2. Morphometric data of mitochondria in hepatocytes from growing and adult mice
1.5 f 0.13
2.53 k 0.157
0.277f
1.3 f 0.05
2.08 f 0.134
0.005
0.0408
0.321t
1.025
0.282f
0.588f
3.553f
12.56 f
f332.08
5721.5
5868.3
0.297
0.0714
0.026*
5969.4
f466.65
5.26 f 0.661
588.2 f 52.31
1175.9 f118.91
10.26 f 0.604
2.843f
0.560f
0.278f
12.52 k
2.569f
0.635k
0.205f
0.819
0.261''
0.0353
0.014tt
1.8 f 0.07
3.13 + 0.158
Perihepatic
5450.1 +422.55
4.02 f 0.221'
440.9 k 43.47
1387.3 f 175.13
20 Days
1.5 f 0.12
2.06 + 0.242**
Periportal
Adult
0.986
0.197
0.0647'
0.012***
0.10*
0.296*+
11252.7 f1421.34't
2.95 f 0.368'
1253.5 f 143.40''
3883.0 f 330.05*t''
9.10 &
2.351f
0.822f
0.261f
1.3 f
3.18 f
Periportal
0.992
0.232
0.0636
0.009'
14843.7
k2049.93"
2.68 +
0.359'
1196.2 f 103.63'"
5563.5 f 478.97+"
12.20 k
1.921+
0.736f
0.156f
0.10
0.367''
Perihepatic
1.7 f
4.72 f
Values represent means k SE. Axial ratio lengtbldiameter. Surface to volume ratio, area of outer membranelunit mitochondrial volume. Volume density, pm3/pm3 of cytoplasm.
Surface density, pm21pm3 of cytoplasm. Aredunit mitochondrial volume, pm2ipm3 of mitochondria. Aredunit area of outer membrane pm2/pm2 of outer membrane. Volume/cell,
pm3/average mononuclear hepatocyte. Aredcell, pm'laverage mononuclear hepatocyte. *P < 0.05, **P < 0.01,***P < 0.001, significantly different from value in perihepatic cell.
tP < 0.05, trP < 0.01,tttP < 0.001, significantly different from value in adjacent and younger age group. Student's t-test.
f644.60
5.33 f 0.394
431.7 f 19.22
1093.6 f 88.71
0.283
0.153
6.05 f 0.402
471.2 + 41.25
981.7 f106.68
13.23 k
3.676f
0.697 f 0.0329
0.007t
Perihepatic
Periportal
10 Days
TABLE 2. Morahometric data o f mitochondria i n heaatocvtes from crowing and adult mice fcontinuedJ
8
3!a
s!
0
5
E
5
2r
8
322
S. KANAMURA ET AL.
patterns seen in the volume and surface density data mainly in adult animals (Table 2).
1
0.3
Mitochondrial Volume or Area of the Inner
Membrane and Cristae per Average
Mononuclear Hepatocyte
The values were not significantly different
between periportal and perihepatic cells in
all age groups studied (Table 2). Between
birth and 5 days of age, and 20 days of age
and the adult, the values increased in the
cells of both zones.
"17 0
0
/ I
5
10
20
'/
A
DAYS AFTER BIRTH
Fig. 5. The volume density of mitochondria in hepatocytes from growing and adult mice. A, adult. "P <
0.05, '**P < 0,001,significantly different from values in
perihepatic cells.
examined (Table 2). However, the values increased between 5 and 10 days of age in periportal cells, and in perihepatic cells were significantly greater for the 5- and 10-day-old
animals than newborn, 20-day-old,and adult
animals.
Area of the Inner Membrane and Cristae per
Unit Mitochondrial Volume
The values did not differ between periportal and perihepatic hepatocytes in all age
groups examined (Table 2). No age-related
changes in the values were seen for the cells
of the two zones. However, the value of newborn animals was significantly greater than
that of adult animals in periportal cells.
Area of the Inner Membrane and Cristae per
Unit Area of the Outer Membrane
There were no differences in the values
between periportal and perihepatic cells in
all age groups studied (Table 2). The values
decreased between 20 days of age and the
adult in periportal cells, and between 10 and
20 days of age and 20 days of age and the
adult in perihepatic cells.
When the data were expressed as volume
and area per average mononuclear hepatocyte, the patterns of subacinar distributions
and age-related changes differed from the
Area of the Outer Membrane per Average
Mononuclear Hepatocytes
There were no differences in the values
between the cells of both zones in newborn
and 5, lo-, and 20-day-old animals. In adult
animals, differences in the values, greater in
perihepatic cells than in periportal cells, appeared. Between birth and 5 days of age, and
20 days of age and the adult, the values increased in the cells of both zones (Table 2).
The difference between the data expressed
per hepatocyte and the volume and surface
density data is generally caused by the
marked increase in hepatocyte volume between 20 days of age and adulthood, especially in perihepatic cells (Table 1). For
comparison of the ultrastructure of periportal cells with that of perihepatic cells during
postnatal development, the volume and surface densities appear more appropriate than
volume and area per cell.
DISCUSSION
Hepatocytes of the adult mouse, rat, rabbit,
dog, monkey, and man reveal functional and
morphologic differences depending on their
positions within the liver acinus (Rappaport,
1963). For example, periportal and perihepatic hepatocytes not only differ ultrastructurally (Loud, 1968; Schmucker et al., 1978;
Asada-Kubota et al., 1982) but also differ in
various enzyme activities (Novikoff, 1959),in
fasting glycogen content (Corrin and Aterman, 1968; Kanamura et al., 1980), in the
rate of protein synthesis (LeBouton, 1968),in
response to cell injury after carbon tetrachloride treatment (Wilson and Williams, 1969),
and in the proliferation of the smooth endoplasmic reticulum after phenobarbital
administration (Jones and Fawcett, 1966;
Kanamura and Ogawa, 1977).
The morphologic heterogeneity has been
shown also among hepatocyte mitochondria.
The volume density is greater in periportal
DEVELOPMENT OF MITOCHONDRIA
cells than in perihepatic cells (Loud, 1968;
Schmucker et al., 1978; Asada-Kubota et al.,
1982). The area of the outer membrane per
unit mitochondrial volume is greater in perihepatic cells than in periportal cells (Loud,
1968). The shape is round in periportal cells,
whereas it is slender in perihepatic cells (Novikoff, 1959; Asada-Kubota et al., 1982).
As revealed in the present study, differences between mitochondria in periportal
hepatocytes and those in perihepatic hepatocytes in the shape (the axial ratio and surface
to volume ratio), volume density, and area of
the outer membrane per hepatocyte, evident
in adult animals, are not present in newborn
animals but arise during postnatal development. It is likely that this change is related
to the sequence of formation of adult lobulation that occurs during the postnatal period.
LeBouton and Marchand (1970) and LeBouton (1974) suggested that postnatal
change observed in the subacinar distribution of labeled nuclei after 3H-thymidine incorporation is caused by the drastic alteration
a t birth in the amount and composition of
blood circulating through the liver. The fetal
liver receives a relatively large volume of
well-oxygenated blood rich in nutrients via
the umbilical vein. In this situation, the microenvironment along the sinusoids might be
constant throughout the acinus, and the hepatocytes of three zones might be able to
synthesize DNA equally. After birth the bulk
of the blood circulating through the liver derives from the portal vein, and this blood is
lower in amount, pressure, oxygen saturation, and nutrient content (Fisher, 1963;
Young, 1966). Such a change in blood supply
could conceivably alter the microenvironment along the sinusoids and produce a difference in hepatocyte function, depending on
the position within the acinus. The postnatal
development of morphologic heterogeneity of
hepatocyte mitochondria, observed in this
study, is probably also related to the change
of hepatic blood supply at birth.
Generally, so-called “functional and structural heterogeneity among hepatocytes” is
not present in newborn animals but arises
during postnatal development. Activities of
glucose 6-phosphatase (Kanamura, 1975;
Katz et al., 19761, ornithine carbamoyltransferase, succinate dehydrogenase, NADH dehydrogenase (Kanamura and Asada-Kubota,
19801, bile canalicular alkaline phosphatase
(DeWolf-Peeterset al., 19721, and labeled hepatocytes after 3H-thymidine injection (Le-
323
Bouton and Marchand, 1970) are evenly
distributed throughout the acinus within a
few days after birth. The zonal distribution
of these enzyme activities and of cells synthesizing DNA, which is characteristic of adult
liver, appears gradually during postnatal development. Similarly, differences in the volume density of the smooth endoplasmic
reticulum and mitochondria between periportal and perihepatic hepatocytes are not
present in newborn animals but are evident
in adult animals (Asada-Kubota et al., 1982).
Postnatal changes have been reported in
the volume density and briefly in the shape
of mitochondria. Rohr et al. (1971) showed
that the volume density remains unchanged
during the early postnatal period after a n
increase between birth and 1day of age. Similarly, Herzfeld et al. (1973) found that the
volume density attains its maximal level by
the second day after birth. However, Lang
and Herbener (1972) observed that the volume density is greater in weanling rats than
in newborn animals, and there is no difference in the length and diameter between animals of the two age groups. David (1979)
reported that the volume density increases
during postnatal development. The reason
for these disagreements is probably that no
distinction was made between periportal and
perihepatic hepatocytes in these reported
studies. We have found considerable differences between hepatocytes of the two zones
in the pattern of age-related change for the
volume density, axial ratio, and surface to
volume ratio.
As the index representing mitochondrial
configuration, the eccentricity (Baudhuin and
Berthet, 19671, the diameter and length
(Lang and Herbener, 19721, the axial ratio
(Asada-Kubota et al., 19821, and “partition
coefficient,” i.e., the volume of the matrix or
external compartment per unit surface area
of internal mitochondrial membrane (Cieciura et al., 19791, have been used. The assumption, which is the basis for the
calculation of all these indices except for
“partition coefficient,” is that mitochondria
are a right circular cylinder, a sphere, or a n
ellipsoid. Therefore, these indices are merely
approximations calculated from mitochondrial images on photographs.
In the present study, the surface to volume
ratio was measured in order to cover up the
fault of the axial ratio. Because the surface to
volume ratio is calculated by dividing the surface density by the volume density, it is ob-
324
S. KANAMURA ET AL.
tained regardless of the shape of the cell
organelle being measured. Therefore, the surface to volume ratio does not contain the uncertainty originating from the assumption
that mitochondria can be likened to a single
geometric shape. In the present results, the
pattern of changes in the axial ratio appeared
basically similar to that in the surface to volume ratio. However, in periportal cells, the
axial ratio was greatest in newborn animals,
whereas the surface to volume ratio was low
and unchanged between birth and 20 days of
age. Stempak (1968)showed by serial section
reconstructions that hepatocyte mitochondria of newborn rats often appear disc-shaped
and branched. It is likely that the shape of
such mitochondria on electron micrographs is
more slender than the original configuration.
For this reason, data of the surface to volume
ratio are probably more reliable than those of
the axial ratio as the index of mitochondrial
shape of newborn animals. Further, between
20 days of age and the adult in the cells of both
zones, the surface to volume ratio increased,
but the axial ratio remained unchanged. Considering that mitochondria appear mostly
round in electron micrographs of the cells at
these ages, the result of the axial ratio may be
correct, and the increase in the surface to volume ratio is probably related to decrease in
the volume. In conclusion, it is probable that
the shape of mitochondria shows no age-related changes.
There were no differences between periportal and perihepatic cells in all age groups
examined in the surface density of the outer
membrane or the inner membrane and cristae, the area of the inner membrane and
cristae per unit mitochondrial volume, and
the area of the inner membrane and cristae
per unit area of the outer membrane. This
indicates considerable uniformity in the surface area and in the area of the inner membrane and cristae among hepatocyte mitochondria in all age groups examined.
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