J. Cell Sci. 12, 163-173 (1973)
Printed in Great Britain
163
AN ULTRASTRUCTURAL STUDY OF THE
PANCREATIC ACINAR CELL IN MITOSIS,
WITH SPECIAL REFERENCE TO CHANGES IN
THE GOLGI COMPLEX
R. N. MELMED, CAROL J. BENITEZ AND S. J. HOLT
Department of Cytochemical Research, Courtauld Institute of Biochemistry, Middlesex
Hospital Medical School, London, WiP 5PR, U.K.
SUMMARY
Changes occurring in the rat pancreatic acinar cell during mitosis have been studied by
electron microscopy. Special attention has been given to those occurring in the Golgi complex.
There is an initial increase in the number of small vesicles between the rough-surfaced endoplasmic reticulum and the saccules of the Golgi apparatus. These persist throughout the
mitotic cycle. The Golgi apparatus becomes considerably smaller in metaphase. In anaphase
the normal stacked appearance of its saccules is lost. In telophase the Golgi complex is restored
to its normal interphase appearance and the small vesicles may contribute both to the reformation of its saccules and to the cleft forming between the daughter cells. Condensing vacuoles
and zymogen granules appear to form throughout mitosis, although they have a much greater
heterogeneity of size than in the interphase cell. The possible significance of some of these
changes is discussed.
INTRODUCTION
There have been few descriptions of ultrastructural changes that occur in cytoplasmic organelles of differentiated mammalian cells in situ during the mitotic cycle,
presumably because such cells generally have a low mitotic activity, making their
study by electron microscopy a tedious and exacting task. Even in regenerating liver,
with the notable exception of the study by Kent et al. (1965) on the behaviour of the
lysosomal system during mitosis, ultrastructural observations on cytoplasmic organelles of dividing cells in mammalian tissues have been rare (Dougherty, 1964; Murray,
Murray & Pizzo, 1965; Kimura & Onoe, 1970).
We now report some details of organelle changes in the pancreatic acinar cell during
mitosis, observed in the pancreas of a rat injected with alloxan and fed a diet containing soybean trypsin inhibitor. In addition, a limited number of dividing acinar
cells were studied in the pancreas of normal suckling rats, in which mitotic activity
is higher than in the adult gland (Enesco & Leblond, 1962).
MATERIALS AND METHODS
All materials used for electron microscopy were obtained from TAAB Laboratories, 52 Kidmore End Road, Emmer Green, Reading, England.
The rat pancreases used in this study were from a 70-g young adult male Wistar rat which
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R. N. Melmed, C. J. Benitez and S. J. Holt
had been injected subcutaneously with alloxan in a dose of 20 mg/100 g body weight, and from
25-30 g suckling rats of both sexes. The adult animal had been starved for 24 h before the
injection, after which it was fed on a mixture of equal weights of powdered Oxoid 41B diet
(Wm. Lillico & Son Ltd., Wonham Mill, Betchworth, Surrey, U.K.) and unheated soya flour
which contains active trypsin inhibitor (Soya Foods Ltd., 30 Mincing Lane, London, E.C. 3,
U.K.). The adult rat, 8 days after the alloxan injection, and the suckling rats were killed by
cervical dislocation, and small fragments of the tail of each pancreas were fixed for 4 h at
room temperature in a mixture of 2 % glutaraldehyde and 3 % formaldehyde buffered at
pH 7-2 with 67 mM cacodylate buffer (after Karnovsky, 1965). After rinsing in the same buffer,
the tissues were postfixed for 2 h at room temperature in 1 % osmium tetroxide buffered at
pH 7 2 with o-i M phosphate buffer. They were then dehydrated in graded ethanols and passed
via propylene oxide into Epon 812 (Luft, 1961). Ultrathin sections of the polymerized blocks
were cut with a diamond knife, collected on uncoated TAAB 5o-/wn grids, stained with 4 %
uranyl acetate (1 min) and lead citrate (Reynolds, 1963) (3 min) and examined in a Philips
E M 200 electron microscope at 60 kV.
RESULTS
Successive sectioning of a number of blocks permitted the study of the acinar cell
in the different phases of the mitotic cycle and the generalizations made in this article
relate to observations made on 7 acinar cells in prophase, 14 in metaphase, 5 in
anaphase and 13 in telophase. The progress of mitosis appeared normal as far as
changes in the fine structure of the mitotic apparatus were concerned. Changes in the
acinar cell cytoplasmic organelles during each phase of mitosis were similar in both
the alloxan-treated and suckling rats and are described in detail below; the major
changes are summarized diagrammaticaHy in Fig. 1.
Prophase
In early prophase, the Golgi complex has an appearance similar to that of the
normal interphase acinar cell (Fig. 2). Later in prophase, however, but before complete disappearance of the nuclear membrane, a striking increase in the number of
small coated and uncoated vesicles between the rough-surfaced endoplasmic reticulum
and the forming face of the Golgi complex was frequently observed (Fig. 3). Numerous
continuities exist between some of these vesicles and the membranes of the roughsurfaced endoplasmic reticulum or those of the Golgi saccules.
The rough-surfaced endoplasmic reticulum retains its normal morphology during
prophase.
Metaphase
In early metaphase, vesiculation adjacent to some of the Golgi saccules may be
very marked and the saccules themselves much reduced in size (Fig. 4). In addition to
normal condensing vacuoles and zymogen granules, numerous small granules are
seen in the region of the Golgi complex. The content of these granules is homogeneous and has a density varying between that of condensing vacuoles and zymogen
granules (Fig. 4). Some of the metaphase cells contain large numbers of zymogen
granules located in the Golgi region of the cell, rather than in their interphase position
Pancreatic acinar cell in mitosis
^*
-».
Incerphase
Zymogen granule
Condensing vacuole
Peripheral vesicles
Rough surfaced
endoplasmic reticulum
Fig. 1. Diagram summarizing the changes occurring in and around the Golgi complex
of the pancreatic acinar cell during mitosis.
in the cell apex. In such cases, the luminal surface membrane is devoid of microvilli
(Fig. 5)There is some alignment of mitochondria on the periphery of the spindle at this
stage.
Anaphase
During anaphase, small vesicles are very numerous and are largely present in the
region between the 2 groups of separating chromosomes, where large numbers of
single ribosome-like particles are also seen (Fig. 6). Golgi saccules are also present in
this region but have a convoluted tubular conformation rather than their normal
stacked appearance. They are intermixed with the small vesicles and with zymogen
granules of various sizes (Fig. 6). Normal-looking rough-surfaced endoplasmic reticulum forms concentric layers around the periphery of the dividing cell.
Telophase
During telophase, the Golgi saccules begin to recover their elongated shape and to
realign themselves into stacks. Numerous small and normal-sized zymogen granules
are present around the condensing chromosomes (Fig. 7) but condensing vacuoles
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R. N. Melmed, C. J. Benitez and S. jf. Holt
are not prominent at this stage. In late telophase large numbers of small vesicles may
still be seen close to some regions of the Golgi apparatus, although, at this stage, there
was no evidence of budding from the rough-surfaced endoplasmic reticulum (Fig. 8).
In some cells, groups of vesicles and single ribosome-like particles were close to the
developing cleft between the 2 telophase nuclei (Fig. 9). The development of the
cleavage furrow is accompanied by the presence of microfilaments and microtubules
and by the formation of interdigitations between the plasma membranes of the daughter
cells and the adjacent cells (Fig. 10). The unusual peri-Golgi vesicular activity and
the considerable variation in the size of the zymogen granules persist into early
interphase.
DISCUSSION
A wave of mitosis occurs in the exocrine pancreas of the rat within 24 h after a
diabetogenic dose of alloxan (Duff & Starr, 1944) or within 72 h after addition of
soybean trypsin inhibitor to the diet (R. N. Melmed, A. A. El-Aaser & S. J. Holt,
unpublished observations). The combined use of these agents has now been found
to produce a considerably enhanced mitotic activity.
The fact that similar morphological changes were observed in dividing acinar cells
in the pancreas of both suckling rats and rats treated with alloxan plus soybean
trypsin inhibitor indicates that the present findings are valid for normal acinar cells
and are not the consequence of metabolic changes induced by the mitogenic agents.
In addition to the expected changes in the mitotic apparatus, a well-defined sequence
of events occurred in and around the Golgi complex. The most striking change, seen
first in prophase, is the increase in the number of small peripheral vesicles situated
between the forming face of the Golgi saccules and the rough-surfaced endoplasmic
reticulum. The numerous images showing continuities between some of these vesicles
and the membranes of the adjacent rough-surfaced endoplasmic reticulum or of the
Golgi saccules suggest that they are the vesicles normally responsible for conveying
newly synthesized protein from the cisternae of this reticulum to the Golgi complex
(Jamieson & Palade, 1967a). Furthermore, their prominence in the presence of the
extensive Golgi saccules of both prophase and late telophase cells suggests that the
vesicles are unlikely to be formed simply by fragmentation of Golgi cisternae. The
reduction in size of the Golgi saccules that occurs during metaphase may well be
analogous to the shrinking of Golgi saccules in enucleated amoebae (Flickinger, 1968),
since normal nuclear functions, such as RNA synthesis, are largely in abeyance
during mitosis (Mitchison, 1971). However, Golgi saccules were present in the acinar
cell throughout mitosis, just as has been found in other normal cell types, such as
dividing insect spermatocytes, plant cells and amoebae (Roth, Wilson & Chakraborty,
1966), although the converse has been reported for dividing liver parenchymal cells
(Dougherty, 1964; Kimura & Onoe, 1970).
The accumulation of zymogen granules around the Golgi complex during metaphase, rather than at the cell apex, and the flattening of the apical cell membrane
suggest a probable suspension of secretory activity at this time. However, this loss of
Pancreatic acinar cell in mitosis
167
polarity is most striking in anaphase, where even the stacked appearance of the Golgi
cisternae is disturbed.
With a complete lack of precise information concerning the metabolic state of the
pancreatic acinar cell in mitosis, interpretation of the observed morphological changes
is necessarily speculative. However, they are consistent with a fundamental alteration
in the normal inter-relationships (Jamieson & Palade, 1967 a, b; 1971) of the components of the intracellular transport and secretory system of the acinar cell. For
example, the increased number of small vesicles observed in the mitotic cell may
reflect a change in their function from one largely concerned with transportation to
one in which they provide a reservoir of membrane for use in the restoration of the
Golgi saccules during telophase, and possibly for the formation of the partition membranes between the daughter cells.
No obvious changes occurred in the elements of the extensive rough-surfaced
endoplasmic reticulum, which forms concentric layers around the mitotic apparatus
during all phases of the cycle. However, there was a striking accumulation of single
ribosome-like particles between the separating chromosomes during anaphase and in
early telophase. The accumulation of RNA-containing material in this area during
anaphase has been well documented by light microscopists for several cell types
(Jacobson & Webb, 1952; Davies, 1952; Boss, 1955; Rustad, 1959) and the present
finding of many ribosome-like particles in the same region probably represents the
first ultrastructural observation of this phenomenon.
We thank the Fleming Memorial Fund for Medical Research for providing a Philips EM 200
electron microscope and the Cancer Research Campaign for their support of this work.
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KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use
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ROTH, L. E., WILSON, H. J. & CHAKRABORTY, I. (1966). Anaphase structure in mitotic cells
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(Received 17 May 1972)
Fig. 2. Rat pancreatic acinar cell in early prophase, showing part of the nuclear
membrane (nm) still intact adjacent to chromosomal material (chr). The Golgi complex (g) is normal in appearance and is associated with several condensing vacuoles
(vac), a zymogen granule (2), and a number of small coated (cv) and uncoated
vesicles (v) having a dark content, x 25 000.
Fig. 3. Acinar cell in later prophase. Abnormally large numbers of small vesicles (v)
and some coated vesicles (cv) are situated between the saccules of the Golgi complex (g)
and the cisternae of the rough-surfaced endoplasmic reticulum (rer), from which
vesicles of both types appear to be budding (arrows). A condensing vacuole (vac) is
present to the left of the Golgi saccules and nuclear material (n) is present at the
bottom of the field, x 30000.
Fig. 4. The Golgi complex (g) of an acinar cell in early metaphase. The saccules are
considerably reduced in size (cf. Fig. 2) and are surrounded by large numbers of small
vesicles. In addition to normal-sized condensing vacuoles (vac) and zymogen granules
(z), there are many smaller granules (arrows) of comparable density. Numerous
ribosome-like particles (r) are scattered throughout the field, x 30000.
Fig. 5. A portion of a metaphase acinar cell showing the luminal surface denuded of
microvilli (arrows). Most of the numerous secretory granules present in the cell are
unusually distant from this surface. A part of a metaphase chromosome (chr) is
present at the right-hand edge of the field, x 13 500.
Pancreatic acinar cell in mitosis
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Fig. 6. Part of a pancreatic acinar cell in anaphase showing the region between the
separated chromosome plates (chr). Large numbers of closely packed small vesicles (v)
are seen both above, and interspersed with, the lower group of Golgi saccules (g), which
at this stage of mitosis, have a convoluted and tubular appearance. A similar association
of Golgi saccules (g') and vesicles (v) is seen at the top of the field. An accumulation of single ribosome-like particles (r) is present between the two groups of chromosomes. Secretory granules showing a considerable variation in size are also present
(arrows), x 24000.
Pancreatic acinar cell in mitosis
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R. N. Melmed, C. J. Benitez and S. J. Holt
Fig. 7. Part of an acinar cell in early telophase showing numerous secretory granules
of variable size (arrows). The majority of them have a density comparable to that of
mature zymogen granules (2). A small group of transversely cut spindle microtubules
(circle) is seen on the periphery of the condensing chromosomes (chr). x 40000.
Fig. 8. An acinar cell in late telophase showing a short length of partly reformed
nuclear membrane (nm) around condensed chromatin. A large number of small vesicles
(v) are seen in the centre of the field between the rough-surfaced endoplasmic reticulum
(rer) and a partially reformed Golgi complex (g). Images suggestive of fusion of such
vesicles with Golgi saccules are indicated by arrows. A membrane-bound body (mbb)
containing a few peripheral vesicles and a matrix of comparable density to the
zymogen granules (z) is present in the lower right of the field, x 32500.
Fig. 9. The beginning of cleft formation (cl) in a late telophase acinar cell. There are
numerous vesicles (v) in this region and some of them (arrows) show continuities
with the surface membrane. Groups of single ribosome-like particles (r) are still
present at this stage of mitosis. Zymogen granules (2) are seen on the right of the field,
x 56000.
Fig. 10. A later stage in cleft formation, showing numerous interdigitations that have
formed with an adjacent cell (asterisk). Note the presence of 2 apparently newly
formed symmetrically placed desmosomes (arrows), and the filamentous structures
(/) and microtubules (nit) associated with the developing cleft, x 35000.
Pancreatic acinar cell in mitosis