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Increase in zymogen granule volume accounts for increase in volume density during prenatal development of pancreas.

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THE ANATOMICAL RECORD 207:487-501(1983)
Increase in Zymogen Granule Volume Accounts for
Increase in Volume Density During Prenatal
Development of Pancreas
Laboratory of Cellular Dynamics, Department of Physiology, School of
Medicine, University of California, San Francisco, CA 94143
The sudden increase in volume density of zymogen granules
in acinar cells of the fetal rat pancreas was examined with particular attention
to the respective roles of granule size and number in this event. Volume density
increased some twelvefold, from about 3%of cytoplasmic volume a t 17 days to
about 45%at 20 days, following a sigmoidal pattern in which the greatest rate
of increase occurred during day 18. This increase in volume density was
primarily the result of a n increase in granule volume. Zymogen granule
diameter increased from 0.55 pm at 17 days to 1.20 pm at 20 days, a n order of
magnitude increase in average granule volume. The total number of granules
in the tissue increased in proportion to the increase in organ weight (cell
number and size), but changes in the number of granules per unit cytoplasmic
volume were minor (+ 40%) in comparison to the increase in volume density.
The distribution of granule diameter was roughly normal and unimodal a t
each time interval, and the increase in average diameter over time was marked
by a n increase in the upper limit of the size distribution and a n increased
percentage of large granules. The size of condensing vacuoles also increased
during this period, and their distributions were roughly coextensive with those
seen for zymogen granules a t the same time. The potential origins of changes
in granule size are discussed, as well as the important effect that size has on
the number of granules observed in “two-dimensional” tissue sections viewed
in the electron microscope. If size is not considered in our estimates, then we
underestimate the numerical density in cells with small granules compared to
those with large granules. The results indicate the central role of granule size,
as opposed to number, in determining granule volume density in the embryonic pancreas.
The zymogen granule content of the pancreatic acinar cell can potentially be changed
by varying either the size or number of granules, or both. Until recently, however,
changes in the volume density of secretion
granules, i.e., the percentage of cytoplasmic
or total cell volume occupied by granules,
have been thought to occur primarily as a
result of changes in the number of granules
(Palade, 1959, 1975; Jamieson and Palade,
1977). This is because granules have been
considered to be of constant size once formed
and because digestive enzymes have been
thought to be secreted by the loss of granules
by fusion of granule membrane with the apical cell membrane (Palade, 1959).Within this
context, changes in granule volume density
have been conceived of in terms of changes
0 1983 ALAN R. LISS, INC.
in number as a result of the formation of new
granules or loss of old granules through exocytosis. However, recent studies indicate that
a major portion of the alterations in granule
volume density seen during active secretion
can be due to changes in the size of granules
(Ermak and Rothman, 1980, 19811, and,
moreover, that these changes involve fluctuations in the size of individual, formed
zymogen granules. These size changes are
significant in light of physiological evidence
that pancreatic proteins may not be secreted
by exocytosis but by a diffusion-based mechanism involving transport of enzymes across
the zymogen granule membrane (Rothman,
1975, 1980).
Received August 2, 1982; accepted May 25, 1983.
In this article, we examine the respective
roles of granule size and number as volume
density increases for the first time during
prenatal development of the rat pancreas.
This period is unique in that the rate of enzyme accumulation far surpasses the rate of
enzyme secretion (Doyle and Jamieson, 1978;
Larose and Morisset, 1977; Werlin and
Grand, 1979). During this time, the concentration of digestive enzymes in the tissue
increases over a thousandfold (Rutter et al.,
1968; Sanders and Rutter, 19741, and zymogen granules accumulate within the tissue
and increase greatly in size (Parsa et al.,
1969; Pictet et al., 1972). This situation produces the largest zymogen granules normally found in the gland, such that granules
in the newborn rat average about six times
the volume of granules seen in fasted adult
animals (Ermak and Rothman, 1980).
The volume density, size distribution, and
number of zymogen granules in the tissue
were determined by stereology and quantitative electron microscopy over a 4-day period of rapid granule accumulation (17-20
days of gestation). This time interval immediately follows the first appearance of granules in cells (Pictet et al., 1972)when enzyme
specific activity increases most rapidly
(Sanders and Rutter, 1974). During this period, granule size increased to higher mean
values by a shift in a roughly normal distribution, and average granule volume increased by about one order of magnitude.
Granule number increased in proportion to
the increase in tissue mass, but despite the
dramatic visual appearance of increasing
granule number in electron micrographs, increases in granule size accounted for virtually the entire measured increase in
volume density.
bryos were chosen at 17 days, four a t 18 days,
and three at 19 and 20 days. More animals
were sampled at early time intervals because
some cells at these times lacked zymogen
granules altogether. The whole pancreas of
embryos from other litters was extirpated
and then weighed on a n electrobalance to
measure organ mass.
Electron Microscopy
Tissue samples were fixed for 1.5 hr in 1.5%
glutaraldehyde and 1%formaldehyde in 0.08
M cacodylate buffer (pH 7.2), postfixed for 1.5
h r in 1% OsO4 in the same buffer, stained en
bloc with uranyl acetate, embedded in English araldite, and viewed in a JEM-100B electron microscope as previously described
(Ermak and Rothman, 1981).
Volume density was determined by point
counting analysis according to Weibel(1969)
using a transparent overlay with 1-cm spacings. The volume density (VV) of zymogen
granules and condensing vacuoles was determined in relation to cytoplasmic volume (total cell volume minus nuclear volume), i.e.,
VV =VzgNcyt.The volume density of the cytoplasm was determined in relation to total
The method of samcell volume, V, tNcell.
pling was standiardized by using only acinar
cells sectioned through the cell apex and nucleus (a sagittal section). Such a procedure is
commonly used to examine changes of state
for polarized epithelial cells where random
sampling cannot be performed without a n
extremely large sample size (Ermak and
Rothman, 1980, 1981; Geuze and Kramer,
1974; Helander, 1978). Sections were photographed at X4,OOO and printed at x 10,000.
In order to account for acinar cells without
granules at 17 and 18 days, samples were
further standardized by selecting only those
Animal Procedures
sagittally sectioned cells in the upper left
Fetal rats were obtained from mated fe- corner of grid squares (Weibel, 1969). Two
male Sprague-Dawley (Simonsen) rats on 17, tissue blocks per animal (13-18 electron mi18, 19, and 20 days of embryonic develop- crographs per animal) were examined at 17
ment. Day 0 was defined as the day a vaginal and 18 days, and one block per animal (10
plug was detected. Females were sacrificed electron micrographs per animal) a t 19 and
by spinal section (after light etherization). 20 days. Some electron micrographs containThe body cavity and uterus were opened and ing two cells were included.
individual embryos placed in a petri dish
The number of granules per cell profile (N,)
containing Earle’s balanced salt solution. was determined by counting the total numPancreatic rudiments from randomly chosen ber of granules in the plane of section
embryos were dissected with the aid of a through a single ceII. The numericaI profile
binocular microscope, and a portion of the density (NA; number per 100 test points or
pancreas adjacent to the stomach and spleen approximately 100 pm2 of surface area
was fixed for electron microscopy. Five em- counted at X10,OOO) was determined by di-
viding the number of granules per cell profile
by the number of test points over the cytoplasm times 100. The numerical density, or
number of granules per unit volume of cytoplasm (Nv), was determined from NA by two
independent methods. First:
where NA and Vv are as defined above, 1.38
is a shape constant for spheres (&)
, and
K is a proportionality constant that depends
upon the size distribution of the granules
(Weibel, 1963; Weibel and Gomez, 1962).The
larger the standard deviation of the mean
size, expressed as a percent, the larger K is.
For each time interval during the prenatal
period, K was determined from the size distribution using the equation
where D1 is the arithmetic mean diameter
(D) and D3 is the cube root of the third moment about the origin of the distribution. D3
was estimated from each size distribution by
the following equation:
D3 =
+ (d213 + . . .
+ (dn)3)111’
where dl through d, equal the individual
granule diameters in the distribution and n
equals the total number of granules in the
population (Williams, 1977). The same value
for K (K = 1.1)was used for all time inter2% from
vals, because K differed by only
population to population, and because the
standard deviation was a constant proportion
of the mean diameter at all times prenatally
(Fig. 9). Second:
N v =NA
3 ,
sectioned near the equator, i.e., the granule
membrane was nearly vertical to the plane
of section. Thus, granules sectioned through
the poles (or granules with “halos”) were excluded. This method is particularly convenient when comparing different physiological states and has given results similar t o
those obtained by other techniques (for a discussion of the method, see Ermak and Rothman, 1980, 1981). Some granules at 19 and
20 days were elliptical or had angular outLines; they were measured by taking the average value of the major and minor axes [(a
b)/2)]. For 19 and 20 days, the same electron micrographs were used for measuring
granule diameter and volume density
(~10,000).For 17 and 18 days, granule diameter was measured on different micrographs taken at higher magnification,
because their diameter was smaller. At 17
days, micrographs were taken at x 10,000
and magnified to ~25,000,and at 18 days
they were taken at ~ 7 , 0 0 0
and magnified to
x 17,500. Fifteen electron micrographs were
examined from each animal at these two time
intervals, and granules from adjacent cells
were measured in order to increase the sample size of granules. The precise magnification of each set of micrographs was
determined with the aid of a carbon calibration grating.
Analysis of Data
Data were collected separately from each
animal and mean values combined to give an
average value for the time interval
standard error (where N = the number of
animals). For zymogen granule size, data
from all animals in a time period were combined to give a mean value for the whole
population f the standard deviation of the
mean. The size distributions shown in Figures 8 and 12 include data from all animals.
When necessary, the ratio of experimental
variances was determined comparing intravs interanimal variance (F test; Snedecor and
Cochran, 1980).In general, the observed variance for N = the number of cells (or electron
micrographs) was not different from the observed variance for N = the number of animals. However, all standard errors are given
as N = the number of animals.
where is the mean diameter of the population (DeHoff and Rhines, 1968). These two
methods have given similar results under
other circumstances (for a comparison of
these methods, see Mayhew, 1972) and do
Organ Growth and Cell Structure
here as well.
At 17 days of gestation, the embryonic panThe diameter of zymogen granules was determined (to the nearest 0.5 mm on electron creas was transparent, pink, and weighed
micrographs) by measuring only granules about 1.0 mg. During the following 3 days,
contained numerous zymogen granules, and
by 20 days acinar cells were packed full of
granules (Fig. 51, giving a visual appearance
similar to that seen in the newborn (Ermak
and Rothman, 1980).
(days postcoitum 1
Fig. 1. Increase in weight of the embryonic pancreas
from 17 to 20 days of gestation. Data are mean values f
SEM. The number of animals included for each point is
given in parentheses.
the tissue became opaque and white in color,
and organ weight increased exponentially,
roughly doubling every day (Fig. 1).By 20
days, the pancreas was a rich white, similar
to that seen in the newborn, and weighed a n
average of 15 mg.
At all time intervals, the gland was composed of well-formed acini, although a t 17
days and, t o a lesser extent, 18 days, sections
of many cells and several acini lacked zymogen granules altogether (Fig. 2). At 17 days,
about 35%of the acinar cells had no granules
at all, and by 18 days that value had decreased to about 13%(Table 1).
Zymogen granule content increased on each
day. At 17 days, several small granules were
seen adjacent to the apical cell membrane
(Fig. 3). A few granules were occasionally
observed near the Golgi complex or in the
basolateral region of the cells. By 18 days of
gestation, granules occupied a larger proportion of the cell apex (Fig. 4) and, in addition,
were observed more frequently in the basolateral region. By 19 days, all cells surveyed
Morphometric Analysis
The size of the pancreatic acinar cell increased over twofold during the late prenatal
period as estimated from the number of test
points per cell (Table 1; Figs. 2-5). This was
primarily due to a n increase in the nonnuclear cell volume (see cytoplasmic volume
density, Table 11, and the increase in volume
density of granules was responsible for the
majority of that increase (about 68%).
The percentage of cytoplasm occupied by
zymogen granules increased by more than 12
times in a sigmoidal pattern during this period (Fig. 6). At 17 days, zymogen granules
occupied about 3% of the cytoplasmic volume. By 18 days, volume density had increased slightly to 7%. Between 18 and 20
days of gestation, volume density increased
exponentially to about 24% at 19 days and
about 45% a t 20 days, approximately the
same value seen a t birth (Ermak and Rothman, 1980).
Condensing vacuole volume density likewise increased during this period from less
than 1% at 17 days to about 5% by 20 days
(Fig. 6), slightly higher than that seen at
birth (Ermak and Rothman, 1980).
Zymogen Granule Size
The increase in volume density of zymogen
granules was primarily due to a n increase in
granule size rather than granule number.
The mean volume (calculated from mean diameter) increased ten-fold during the 4-day
prenatal period (Table 2; 0.09 vs 0.90 pm3).
This volume change corresponded to a n in-
Figs. 2-5. Increase in zymogen granule content during prenatal development of the rat pancreas. The size
and number of granules per cell profile increases on each
day until the entire cytoplasm is filled with granules.
Compare cell size in each figure; it roughly doubles over
the 4-day interval. The cells shown do not necessarily
depict the average morphometric values (see also Tables
1-31, x 8,000.
Fig. 2. Day 17; cells lack zymogen granules altogether.
Fig. 3. Day 17; granules are small and are seen primarily below the apical plasmalemma.
Fig. 4. Day 18; granules fill a greater portion of the
Fig. 5. Day 20; granules have a wide range of sizes and are now seen in the
basolateral portions of the cell. x 8,000.
TABLE 1. Cytoplasmic and cellular features during prenatal development
No. of
cells sampled
Increase in
to zymogen
granules (%Y
61.0 f 0.7
62.8 k 0.9
85.2 f 0.9
'Total test points per cell (cytoplasm + nucleus) relative to 100 test points ( = total test points per cell at 3 weeks
postnatally; Ermak and Rothman, 1980).
'Percentage of total cell volume (cytoplasm + nucleus) SEM where N = number of animals.
'Relative to 17 days.
crease in granule diameter from an average
of 0.55 pm at 17 days to 1.20 pm at 20 days
of gestation. The greatest change in size, as
with volume density, occurred between 18
and 19 days with over a four-fold increase in
mean volume being observed, or a doubling
in the original volume every 6 hr. On 17 and
19 days there was approximately a 50%daily
increase in mean volume.
The increase in mean volume accounted for
virtually all of the increase in volume density over this 4-day period, and the increases
in volume that occurred from day to day were
accompanied by corresponding increases in
volume densit-y (Fig. 7). A plot of the two
parameters, V and Vvzg, formed a linear
function that intercepted the y axis near the
origin and whose slope was such that doubling the volume of granules roughly accounted for a doubling of volume density.
The fact that granule volume is related to
volume density in a linear fashion indicates
that the relationship between average diameter
and volume density is described
by an exponential function that intercepts
the axes at or near the origin and not by a
linear function that intercepts the diameterNB axis at a substantial distance from the origin
(Fig. 7).
20 21
(days postcoitum)
Fig. 6. Zymogen granule (ZG) and condensing vacuole
(CV) volume density during prenatal development of the
rat pancreas. Volume density is expressed as percentage
cytoplasmic volume. Data are mean values
SEM for
N = 5 (17 days), N = 4 (18 days), and N = 3 animals (19
and 20 days). Condensing vacuole SEM Q 0.8 units for
all points. Newborn WB) added for comparison, from
Ermak and Rothman (1980).
Zymogen Granule Number
Since increases in the volume of zymogen
granules accounted for the majority of the
increase in volume density, and volume density is the product of mean volume and the
number of granules per unit volume, an increase in granule number could have contributed little to the increase in volume density.
When the number of granules per unit cyto-
TABLE 2. Mean diameter and volume of zymogen granules during prenatal development
Time period
(days postcoitum)
Magnitude of
volume increase
Mean diameter
Calculated mean
volume (pm3)
0.55 f 0.20
N = 480
0.65 k 0.20
N = 595
1.05 f 0.30
N = 688
1.20 k 0.35
N = 1098
1.50 f 0.40
1.5 x
7 X
10 x
20 x
'Arithmetic mean to nearest 0.05 pm
differ from interanimal variance.
'Relative to 17 days.
3From Ermak and Rothman (1980).
number of granules. For all samples, intraanimal variance did not
plasmic volume was actually measured, it
was found to have increased by only about
40% (numerical density; Table 3) during a
period in which volume density increased
some twelvefold. This does not mean that the
number of granules in the tissue was not
greatly changed during this period, but
rather that the increase that occurred was
roughly in proportion to the increase in tissue mass (approximately a fifteenfold increase; Fig. 1).
Size Distribution
At each time interval, the distribution of
granule size was roughly normal and unimodal (Fig. 8). As the mean diameter increased, the absolute range of granule size
also increased due to an increase in the size
of the largest granules (from 1.10-2.60 pm).
For the distributions seen at 17, 18, and 20
days, the mean diameter was slightly smaller
than the mode, and for the broader distribution seen at 20 days there were several size
classes near the mean diameter (0.90-1.40
pm) each of which accounted for approximately the same proportion of the population. The smallest granules (0.20-0.70 pm)
accounted for most of the population at 17
days (89%), but were only a minor component
(about 8%)by 20 days.
Both the mean diameter and standard deviation around the mean increased proportionately during the 4-day period, i.e., the
two variables were related to each other in a
linear fashion, and the line intercepted the
axes near the origin (Fig. 9). Thus, the standard deviation of granule size remained a
constant proportion of the mean size as size
increased, i.e., about 30%. In other words,
whether for the broad distribution of granule
sizes seen at 20 days or the comparatively
MEAN VOLUME ( p m 3 1
Fig. 7. Calculatd mean granule volume @) or mean
granule diameter (D) vs granule volume density during
prenatal development. Data are absolute values for mean
diameter, or derived, therefrom rather than rounded.
Granule volume vs volume density is a linear function
that regresses through the axes near the origin. Notice
that the slope of this function is such that increases in
granule volume lead to roughly proportional increases
in volume density. Because volume is a cubic function of
diameter. the points for diameter vs volume density were
fitted to a power function 07 = 25x3). Both functions
were significant at less than the 1%level and had correlation coefficients of greater than 0.998.
TABLE 3. Zymogen granule number during prenatal development
Numerical density4
No. per
cell profile'.'
7.0 rf10.4
37.6 rf65.2 rf-
K . NA~''
15.1 i-3.0
19.5 1.9
43.4 k 4.9
45.0 ? 3.2
k SEM where N = number of animals.
'Per section of single cell.
3Number per unit test area = 100 test points = 100 pm' acinar cell cytoplasm.
4Numbcr per unit volume (100 pm3 acinar cell cytoplasm).
narrow distributions seen at 17 or 18 days,
the range of sizes remained a constant percentage of mean size. A similar relationship
is also seen during the 3-week postnatal period as granule size decreases (Fig. 9) (Ermak
and Rothman, 1980), although in this case
the observed standard deviation of granule
size is somewhat smaller (18-27%) and decreases slightly (as a percentage) as size decreases (viz., the line does not intercept the
axes a t the origin).
Condensing Vacuole Size
Condensing vacuoles were only observed
occasionally a t 17 and 18 days but became a
salient feature of the acinar cells by 19 and
20 days of gestation (Fig. 10). They increased
in diameter from a n average of 0.65 pm a t 17
days to 1.45 pm at 20 days, over a tenfold
increase in average volume. During the first
2 days, condensing vacuoles increased in size
more rapidly than zymogen granules, and
thereafter their size remained roughly constant even though zymogen granules continued to increase in size (Fig. 11).At birth,
condensing vacuoles were smaller than at 19
and 20 days and were also smaller than zymogen granules seen at the same time (Ermak and Rothman, 1980).Although condensing vacuoles prenatally had a larger mean
diameter than zymogen granules for the
same time periods, their distributions encompassed roughly the same range of sizes, i.e.,
condensing vacuoles had a n equally broad, if
not a broader, range of sizes than zymogen
granules (Fig. 12).
The pattern of development and population
dynamics of the pancreatic zymogen granule
from the onset of its appearance in the secretory cell at approximateIy 17 days of gestation to its full expression as the dominant
cytologic feature of the cell by birth can be
summarized as follows: 1)The volume density of zymogen granules increases by one
order of magnitude from days 17 to 20 of
gestation following a sigmoidal pattern with
the greatest increase occurring during the
18th day. 2) The number of granules per unit
cytoplasmic volume changes little (+ 40%)
between days 17 and 20 despite the magnitudinal increase in volume density. 3) The
volume of the average granule increases by
one order of magnitude and accounts in great
part for the observed increase in volume density. 4)The number of granules in the tissue
increases in proportion to the increase in organ mass (increased cell size and cell num-
Fig. 9. Mean granule diameter vs standard deviation
of the mean at each time interval during prenatal development 0 . Data are absolute values for mean diameter
rather than rounded. The calculated linear regression
was significant at less than the 1%level and had a
correlation coeficient of 0.990. The relationship between
size and variability seen during the 3-week postnatal
period 0 is given for comparison (from Ermak and Roth-
man, 1980). Prenatal points in ascending order are: 17,
18,19, and 20 days. Postnatal points in descending order
are: newborn, 1, 0.5, 2, and 3 weeks. The relationships
differ in two ways. First, zymogen granules are more
variable in size prenatally, and second, the standard
deviation prenatally remains a constant proportion of
the mean diameter, whereas postnatally it becomes a
decreasing proportion of the mean as diameter decreases.
Fig. 10. Condensing vacuoles (CV) from acinar cells at 19 days of gestation have a wide
range of sizes and electron densities. Because condensing vacuoles are more numerous in the
supranuclear region, this section overestimates their volume density and number. x 6,500.
ber). 5) Granule size conforms to a roughly
normal distribution at all times. 6) The increase in mean diameter of granules is due
to an increase in the upper limit of granule
size and an increase in the proportion of large
granules in the population. 7) As granule size
increases so does the absolute variance
around mean size, i.e., the range of granule
size increases as average size increases. 8)
This increase in variance is proportional to
the increase in mean size and hence the p r o
portional variance remains a constant percentage of mean diameter as size increases.
9) The volume of the average condensing vacuole increases by one order of magnitude. 10)
Although the average size of condensing vacuoles is slightly larger than for zymogen
granules, their size distributions are nevertheless roughly coextensive. 11) The maximum size of condensing vacuoles appears t o
be reached by day 19, whereas zymogen
granule size continues to increase until birth
(at day 22).
Granule Size and Volume Density
The increase in volume density of enzymecontaining zymogen granules which fill the
acinar cells as birth approaches might be
expected to be attributable to changes in the
concentration of granules within the developing cell. However, a dramatic increase is
not seen, and the increase in volume density
during this period is primarily due to increases in the volume of individual granules
and not their numerical density.
In other circumstances, the concentration
of granules in the cytoplasm can even vary
inversely with volume density. For example,
during the first 3 weeks of postnatal life, the
volume density of zymogen granules in the
rat pancreas decreases by about 60%, while
numerical density doubles (Ermak and Rothman, 1980).Conversely, in the parotid gland,
volume density appears to increase as numerical density decreases during recovery
after maximal stimulation (Cope and Wil-
5 I.o
0 cv
(days postcoitum)
Fig. 11. Increase in condensing vacuole size (0)
during prenatal development of the rat
pancreas. Increase in zymogen granule size ( 0 )given for comparison. Condensing vacuoles
increase in size more rapidly than zymogen granules and then remain at a constant size as
granule size continues to increase. Data are mean values & SEM for N = the number of
animals. Newborn (NB) added from Ermak and Rothman (1980).
liams, 1981). Thus, it is becoming evident
that changes in the size of granules are often
the major cause of large fluctuations in the
volume density of granules in a cell.
Granule Size and Number
Despite the fact that numerical density did
not change substantially during development, a visual examination of tissue sections
under the electron microscope gives the unmistakable impression that a great increase
has occurred in the concentration of zymogen
granules. This appearance of greatly increasing numerical density is in great part delusory. Granules appear more numerous simply
because cell size more than doubles, and the
total number of granules in the cell and organ increases even though their concentration is not dramatically changed. In addition,
larger granules give the appearance of
greater numerical density, because they occupy a greater percentage of the cytoplasmic
Even so, if we measure the number of granules per unit surface area, we find that the
numerical profile density is increased some
threefold during this 4-day developmental
period. The central reason for this disparity
is related t o the fact that we examine twodimensional images and not the actual threedimensional space itself, and such images do
not give us an accurate representation of the
number of granules per unit volume. The
number of objects seen in electron micrographs of sectioned tissue depends on numerous factors such as the size, shape, size
distribution, and plane of section, as well as
the actual number of objects themselves. For
spheres such as the zymogen granule, size or
diameter is particularly important, since
small spheres are sampled in tissue sections
less frequently than large ones (see method
2 [DeHoff and Rhines, 19681in Materials and
Methods); hence fewer small granules are
seen or counted in “two-dimensional” sections. To determine true numerical density,
17 d
W 30
Fig. 12. Distribution of condensing vacuole diameters at 17, 19, and 20 days of prenatal
development. Data are expressed as percentage of total population (N = 56 for 17 days, 178 for
19 days, and 151 for 20 days). Each size category represents a 0.2-pm interval. Distribution of
zymogen granule diameters a t the same time (smooth curves) is given for comparison.
we must correct for this sampling problem.
Thus, at 17 days, our “two-dimensional”
measurement of numerical density underestimates the true value per unit volume relative to 20 days when granules are more than
twice their diameter.
The effect of granule size on the number of
granules counted in tissue sections must also
be considered when conditions produce a decrease in granule size, rather than an increase. For example, decreases in the number
of granules seen in a cell during augmented
secretion are usually thought to reflect real
decreases in granule number. However,
changes in granule size also occur under
these conditions (Ermak and Rothman, 19811,
one for increases in granule size and another
for decreases that are observed under different circumstances (Ermak and Rothman,
1980, 1981). Finally, we did not observe profiles suggestive of the fusion of granules in
electron micrographs from any of the samples studied.
Increases in the size of new or forming
granules appear to be a major cause of the
observed increase in size. New granules are
being added to the organ such that the number more than doubles every day. In addition,
since larger and larger condensing vacuoles
are seen during the developmental period
(until day 19), larger zymogen granules are
apparently being formed as a result. These
new granules reflect the formation of an increasingly broad size range as time advances
Causes of Changes in Granule Size
and are not the summation of several narrow
The increase in zymogen granule size that distributions of larger and larger size formed
occurs during prenatal development may be at different times.
Finally, the increase in zymogen granule
the result of several phenomena, either singly or in combination: 1)fusion of formed size may be attributable to increases in the
granules with each other; 2) the formation of size of formed granules. Decreases in the size
larger granules with time (either accom- of individual formed granules have been obpanied by the discontinuance of the forma- served in the pancreas after feeding (Ermak
tion of smaller granules, or in addition to and Rothman, 1981), and analogous inthem); and 3) an increase in the size of indi- creases might occur during prenatal development. The size distributions seen during
vidual formed zymogen granules.
Granule to granule fusion does not seem to development are unimodal, relatively norbe a dominant feature of the developing pan- mal functions. That is, there is no indication
creas. This conclusion is based on the fact of separate distributions for older (smaller)
that the size of condensing vacuoles in- granules and new granules. If indeed all
creases in parallel with that of zymogen granules, old and new, fit a single distribugranules, indicating that new granules of tion of sizes, as appears to be the case, then
progressively larger size are being formed of course, if the size of the average granule
(that is, of course, if we assume that the size increases with time, then so must the size of
pattern of condensing vacuoles indicates the the old (formed)granules.
In conclusion, the dramatic increase in
size pattern of newly forming granules).
Other factors also argue against a major role granule volume density that occurs during a
for fusion in the observed size changes. In brief period of prenatal development is priother circumstances, e.g., periods of fasting, marily due to an increase in the size of grangranule size remains constant, and, thus, we ules in the cell and not the number per unit
would have to propose special circumstances cytoplasmic volume. Thus, size change can
that permit fusion at one time and prevent it be the central determinant of changes in
at another. In addition, the fusion of gran- granule volume density, and granule numules produces a decrease in surface-to-volume ber may vary much less than is usually
ratio that, assuming the conservation of thought in producing such changes. Furthergranule membrane, would result in a sub- more, because the size of granules can
stantial increase in granule volume (two change, the effect of size on the apparent
granules of equal diameter will increase number of granules in cells must be acabout 40% in volume). The multiple fusion of counted for.
granules necessary to produce the observed
increase in granule diameter would result in
We gratefully acknowledge the excellent
a sizable decrease in granule electron density
which was not observed. Furthermore, we technical assistance of Ms. Sara Nelson. This
would have to propose two mechanisms, viz., study was supported by research grants
and, therefore, the decreased number seen in
electron microscopic sections is at least in
part only an apparent decrease due to the
decreased frequency with which we sample
small granules. This is not to say that
changes in granule number do not occur, but
when they appear to, the number of granules
must by counted and corrected for sampling
probabilities in order to properly draw the
conclusion that number has indeed changed.
Assuming that changes in numerical density
occur based on the visual examination of a
two-dimensional image without considering
this issue may lead to an erroneous conclusion about the existence and extent of
changes in the number of granules contained
within cells during secretion.
AM25664 and AM16990 from the National
Institutes of Health and by a research grant
from the Academic senate
of the university
of California.
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development, volume, increase, prenatal, granules, pancreas, account, zymogen, density
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