J. Embryol. exp. Morph. Vol. 27, 1, pp. 1-13, 1972
Printed in Great Britain
Cytological and cytochemical studies of centrifuged
eggs of the slug Avion ater rufus L.
By A. H. SATHANANTHAN 1
From the Department of Zoology, University of Ceylon, Colombo
L
F
SUMMARY
The stratification of various cell organelles and of important chemical substances have
been studied in the eggs of the slug, after moderate centrifugation.
As in other molluscs the egg contents stratify typically into three well-defined zones - lipid,
hyaline and yolky zones - but a distinct equatorial band of inclusions consisting mainly of
phagosomes and associated lysosomes was detected in the most centrifugal region of the
hyaline zone.
The sub-stratification of various cell inclusions in their respective zones was determined
in some detail.
The role of the cell membrane and egg cortex in the redistribution of these inclusions and
the nature of the ergastoplasm are discussed in the light of electron-microscope studies of
eggs of this slug and of the sea urchin.
\
1
i.
fc
INTRODUCTION
The centrifuge has been an extremely valuable tool in cytological and cytochemical research, especially on eggs. Unlike differential centrifugation and
fractionation procedures used to isolate cellular components in artificial media,
by this method the cell inclusions could be concentrated within their cell
membrane and studied in their natural environment.
Such studies have been made of eggs of various other molluscs, including
Limnaea (see Raven, 1958). Recently, centrifuged eggs of the sea urchin have
been examined with an electron microscope (Mercer & Wolpert, 1962). The
main aim of this paper is to determine in some detail the stratification of various
cell organelles and of certain important chemical substances in a molluscan egg.
MATERIALS AND METHODS
Entire egg capsules with ova, 2-cell and 4-cell stages, were subjected to a
centrifugal force of 950 g (at 3750 rev/min) for 6 min using a Cambridge electric
centrifuge. The embryos were then quickly decapsulated in tap-water, rinsed in
isotonic saline (0-28 % NaCl) and examined by vital methods or fixed, sectioned
and studied by cytological and cytochemical methods.
1
I
Author's address: 213, N. Roselake Avenue, Los Angeles, California 90026, U.S.A.
E M B 27
A. H. SATHANANTHAN
Table 1. Summary of important staining and cytochemical reactions
of centrifuged embryos
Stain or test
Sudan black B
Reference
McManus's
method (Pearse,
1961)
Zone
LZ
HZ
YZ
Acid fuchsin
Metzner & Krause
(1928)
HZ
YZ
Mann-Kopsch
McClung Jones
YZ
Stratification
Lipid globules
Lipochondria
Mitochondria, lysosomes
Phagosomes, chromosomes, nucleoli
Golgi
Yolk (E, I)
Mitochondria
Chromosomes, nucleoli
Golgi
Few mitochondria
Golgi
Reaction
++++
++++
+++
++
++
++
+++
+++
+++
+++
+++
+
+, +
+
+
+
+
+
(1950)
PAS
Best's carmine
HZ
McManus's
method (Pearse,
1961)
YZ
Pearse (1961)
HZ
YZ
Hale's reagent
Pearse (1961)
HZ
YZ
Aldehyde
fuchsin/Alcian
blue
Vitamin C
Spicer & Meyer
(1960)
HZ
YZ
Bourne's & Bacchus's methods
(Pearse, 1961)
Kurnick's method
(Pearse, 1961)
YZ
RNA
Feulgen
Iron
Calcium
Melanin
Cytochrome
oxidase
Succinic
dehydrogenase
Pearse (1961)
Pearl's method
(Pearse, 1961)
Sodium rhodizonate
(McGee-Russel,
1958)
Lillie's method
(Pearse, 1961)
Burstone's method
(Pearse, 1961)
Nachlas's method
(Pearse, 1961)
HZ
YZ
HZ
YZ
YZ
HZ
YZ
HZ
HZ
YZ
HZ
YZ
Lysosomes, phagosomes + + + +
Free glycogen
+++
Golgi
++++
Yolk (E, I)
+ + +,+ +
Free glycogen
+++
Phagosomes
++
Golgi
++++
Yolk (E, I)
+ + +,+ +
Lysosomes, phagosomes
+++
Golgi
++++
Yolk (E, I)
+ + +,+ +
Lysosomes, phagosomes
+ + +P
Golgi
+ + + +P
Yolk (E, I)
+ + +P,+B
Free granules
++++
Ribosomal RNA,
nucleoli
Yolk (E, I)
Chromosomes
Free granules, Golgi
Golgi
Yolk (E, I)
Nucleoli
Golgi
Yolk (E, I)
Nucleoli
Mitochondria
Golgi
Mitochondria
Golgi
+++
++,+
+++
+++
+++
+++
+
+++
++
+
+++
+++
++
++
+
+ , ++ +
+
+
+,+ +
+
+
+
+
+
<
Centrifuged slug eggs
Table 1 (cont.)
Stain or test
Reference
Acid phosphatase
Azo dye method
(Pearse, 1961)
Benzidine
peroxidase
Van Duijn's
method (Pearse,
1961)
Novikoff & Goldfischer(1961)
Fredricsson's
method (Pearse,
1961)
Baker (1958)
Thiamine pyrophosphatase,
alkaline
phosphatase
Toluidine blue,
methylene blue
Zone
HZ
YZ
HZ
YZ
HZ
Stratification
Reaction
Lysosomes
Phagosomes
Golgi
Lysosomes
Phagosomes
++++
++
++++
++++
+++
Golgi
Yolk (E, I)
Nucleoli
++++
+ + + +, + +
++
Lysosomes, phagosomes
+ + +P
Golgi
+ + + +B
Yolk (E, I)
+ +, +B
HZ
Neutral red,
Baker (1958)
Lysosomes
+++
Nile blue
Phagosomes
++
YZ
Golgi
+++
Yolk (E, I)
+ +, +
+ + + + = Very intense reaction; + + + = strong reaction; + + = moderate
reaction; + = weak reaction; P = purple; B = blue; E = externum; I = internum;
HZ = hyaline zone; LZ = lipid zone; YZ\ = yolky zone.
HZ
YZ
Vital studies included examination of embryos by phase-contrast microscopy
and after vital staining and incubating them alive for certain enzymes - ' vital
enzymology' (Sathananthan, 1968). For phase-contrast work the embryos were
squashed in isotonic saline and examined under oil immersion with a Wild M 20
phase-microscope. Vital staining was carried out according to Baker (1958).
Embryos were stained in Grubler's neutral red, Nile blue, toluidine blue and
methylene blue for 5-40 min using a 0-01 % solution of the dye in isotonic
saline, rinsed, squashed and examined with an apochromatic oil-immersion
objective. Most of the enzyme tests were carried out successfully by incubating
alive whole stratified embryos in various media and examining them in much
the same way as in the vital staining procedure. Cytochrome oxidase, succinic
dehydrogenase, acid phosphatase, benzidine peroxidase and thiamine pyrophosphatase were the enzymes studied in this manner (Table 1).
Centrifuged embryos were also fixed in a wide variety of fixatives, including
Altmann, Champy, Mann, Helly, Zenker, Ciaccio, Perenyi, Bouin, Carnoy and
acetone/ethanol, impregnated with celloidin and rapidly embedded in ceresin/
wax (56 °C) according to Peterfi's method (Pantin, 1960). Serial sections (4 /*m
thick) of each embryo were cut with a Cambridge rocking microtome and the
distribution of mitochondria, lysosomes, Golgi, yolk and important cytochemical substances such as lipids, nucleic acids, polysaccharides, mucosubstances, vitamin C, iron, calcium, pigment, thiamine pyrophosphatase and
A. H. SATHANANTHAN
LZ
HZ
EB
YZ
ABBREVIATIONS ON FIGURES
EB
ER
G
HZ
L
LG
LZ
M
equatorial band
endoplasmic reticulum
Golgi
hyaline zone
lysosome
lipid globule
lipid zone
mitochondrion
m
N
n
P
PB
R
Y
YZ
microsome
nucleus
nucleolus
phagosome
polar body
ribosome
yolk
yolky zone
Figs. 1-5. Illustrations showing distributions of various cell inclusions in centrifuged
ova.
Fig. 1. General distribution of cell organelles in lipid, hyaline and yolky zones.
Fig. 2. Cytochrome oxidase- and succinic dehydrogenase-positive granules (mitochondria and Golgi).
Fig. 3. Acid phosphatase-positive granules (lysosomes, phagosomes and Golgi).
Fig. 4. Benzidine peroxidase activity (pronuclei are copulating).
Fig. 5. Glycogen (fine stippling) and vitamin C (coarse granules).
Centrifuged slug eggs
5
alkaline phosphatase were determined using appropriate methods (Table 1).
Some embryos were fixed in formaldehyde/saline (10% neutral formalin in
0-28 % NaCl), embedded in 15 % gelatin (Pearse, 1961) and frozen sections (6 /tm
thick) were cut in a cryostat. These were used to study lipids and acid phosphatase activity. All colour reactions were visualized with a Zeiss apochromatic
objective and photomicrographs were taken with a Zeiss photomicroscope
using Ilford Pan-F film.
OBSERVATIONS
Fairly good stratification of various cell inclusions and very sharp demarcations between three well-defined zones (fat cap, hyaline and yolky zones) were
obtained immediately after centrifugation (Figs. 1, 6). Broadly speaking the
relative amounts of lipid andprotein present in an inclusion seems to determine
its stratification. Centrifuged eggs are oval but on standing they round up in a
few minutes and redispersion of certain inclusions begins almost immediately,
especially between the lipid and hyaline zones. The demarcation between the
hyaline and yolky zones, however, persists for about 1 h or more. Prolonged
centrifugation tends to constrict the embryo at the equator and sever it into two
segments. If centrifuged eggs are left to develop in saline or within the capsule
cleavage continues apparently normally though slightly retarded. The cleavage
furrows, when established, prevent further redistribution of visible inclusions.
As in other molluscs (Raven, 1958) the egg contents stratify typically into three
distinct major zones (Fig. 1) according to their specific gravity: (a) centripetal
oil cap zone, which is lightest and consists of lipid globules; (b) intermediate
clear hyaline zone with most of the ergastoplasm, mitochondria, lysosomes,
phagosomes, nuclear inclusions and asters; (c) centrifugal yolky zone, which is
heaviest and consists mostly of yolk spherules and Golgi bodies. The most
remarkable feature in this embryo is that an equatorial band of inclusions consisting mainly of albumen vesicles (phagosomes) and lysosomes was detected in
the most centrifugal region of the hyaline zone.
The axis of stratification is usually more or less parallel or is oblique to the
animal-vegetal axis of the embryo, and the polar bodies are usually attached to
its lighter half. Exceptionally they may be found attached to the yolky half.
Substratification of cell organelles
The distribution of various cell organelles in their respective zones will now
bs dealt with. Most of the important cell constituents stratify in the hyaline
zone (Fig. 1).
Ergastoplasm is the chief component of this zone. It consists of ground
cytoplasm (hyaloplasm), ribosomes and elements of the endoplasmic reticulum
(ER). These observations are supported by cytochemical evidence and also
ultrastructural studies of normal eggs (Sathananthan, 1966). The presence of
A. H. S A T H A N A N T H A N
50 //m
FIGURES
6-8
Photomicrographs of centrifuged embryos
Fig. 6. v.s. mature ovum (Helly/acid fuchsin).
Fig. 7. v.s. ovum at second maturation (Helly/Sudan black B). Note equatorial band
and flecks of lipid in yolky zone.
Fig. 8. v.s. ovum at second maturation (Helly/PAS). Note equatorial band.
Centrifuged slug eggs
7
ribosomes is deduced by the stratification RNA in this zone (Fig. 12). Electron
micrographs of unstratified eggs show that there is an abundance of free ribosomes in ova and that the ER is sparsely developed and consists of vesicular
elements (microsomes) and a few elongate cisternae, sometimes associated with
mitochondria and yolk spherules. Further, strands of ER were also seen after
staining with pyronin and Sudan black and some of these may remain in the
yolky zone on account of their association with yolk spherules.
Mitochondria are easily stainable with acid fuchsin and show cytochrome
oxidase and succinic dehydrogenase activities. They stratify chiefly in the hyaline
zone, forming a very broad band above the equator (Fig. 10). They are more
abundant towards the equator and decrease gradually in number towards the
oil cap zone. There is invariably an almost clear region just below the fat cap
with few visible inclusions, which presumably abounds with ribosomes and
perhaps microsomes. A few mitochondria are always seen in the yolky zone
evenly distributed between the yolk spherules or form rows or chains around
the latter.
Lysosomes stratify among the mitochondria in the hyaline zone and could be
best demonstrated by the acid-phosphatase test and the periodic acid/Schiff's
(PAS) reaction. They also show vital metachromasy with toluidine blue and are
neutral red-positive and contain peroxidase. They are, however, more abundant
towards the equator and together with larger bodies called phagosomes, with
which they are associated as 'satellites', they form a distinct supra-equatorial
band (Figs. 1, 7-9). The phagosomes are, in fact, tiny albumen vesicles which
stratify above the equator in the most centrifugal region of the hyaline zone.
Albumen is ingested at the egg surface from the very onset of development
(Sathananthan, 1968). Perhaps this is the first time that such a layer has been
detected in a molluscan egg.
Nuclear inclusions, etc. Nuclei with nucleoli, maturation and mitotic figures
and associated chromosomes stratify in the hyaline zone. The nuclei are sometimes displaced centripetally towards the oil cap (Fig. 6). The second maturation
figure and chromosomes are often seen on a side associated with the first polar
body (Fig. 7) and are not displaced from their normal site at the animal pole.
The mitotic figures lie freely in the hyaloplasm and usually occupy a more
central position in the hyaline zone (Fig. 9).
The heaviest inclusions, chiefly consisting of yolk and Golgi bodies, stratify
in the yolky zone (Figs. 1, 9, 11).
Golgi bodies exist in a variety of forms and range from tiny spheres (dictyoles),
duplex vesicles, crescents, bean-shaped bodies to rodlets, as revealed in MannKopsch preparations, where they are heavily impregnated with osmium (Fig. 1).
They also show thiamine pyrophosphatase activity and are easily stainable with
the vital dyes used. While the dictyoles and vesicles are independent objects the
other dictyosomes are associated with yolk spherules and are inseparable on
centrifugation. The dictyoles and vesicles which have yolky interna usually
A. H. SATHANANTHAN
10
50/tm
Centrifuged slug eggs
9
stratify in the most centripetal region of this zone just below the equator and a
few may occasionally invade the clear hyaline zone.
Yolk spherules could be best demonstrated by tests for thiamine pyrophosphatase and alkaline phosphatase and by the PAS reaction. They are also stained
by vital dyes but less intensely than Golgi bodies. Each spherule is duplex in
structure, with a membranous externum and a yolky internum. They vary in
size and are sometimes arranged in their zone along a gradient, the smaller ones
being more centripetal while the larger mature ones are displaced centrifugally.
Yolk is chemically very complex, being glyco-lipo-protein in nature, with
proteins predominating.
Stratification of chemical substances
These substances are either found freely or are bound to some cell organelle,
in which case their stratification will conform to that of the latter. The cytochemical methods used to identify these substances are given in Table 1.
Lipids. Free lipids stratify mainly in the oil cap zone (Figs. 1,7). The oil cap
stains very intensely with Fettrot and Sudan black B and consists of numerous
minute globules and also some diffuse fat. Certain lipid inclusions remain in the
yolky zone (Fig. 7) and appear as blobs or flecks in association with a few mitochondria. All these lipid inclusions are composed of both neutral fats and
phospholipids. There are also discrete membrane-bound lipid bodies (lipochondria) which stratify close to the oil cap zone or are associated with asters
or are found in polar bodies. They are probably overgrown lysosomes impregnated with lipid (Sathananthan, 1968). They have a strong affinity for Sudan
black and are stained by vital dyes such as neutral red and Nile blue and hence
seem to be predominantly phospholipid. Sudan black also stains the membranebound lipids of various cell organelles (Fig. 7). Thus mitochondria, lysosomes,
Golgi, yolk membranes, plasma and vitelline membranes, and even strands of
the ER appear in these preparations. These protein-bound or masked lipids are
essentially phospholipid in nature. The phagosomes that stratify at the equator,
chromosomes and nucleoli too have appreciable amounts of lipid.
Oxidative enzymes. When centrifuged eggs are incubated for cytochrome
FIGURES
9-12
Photomicrographs of centrifuged embryos
Fig. 9. v.s. 2-cell stage (Helly/PAS). Note phagosomes and lysosomes at the equator.
Fig. 10. v.s. ovum at second maturation showing mitochondria in hyaline zone
(Helly/acid fuchsin). Note redispersion of lipid has begun.
Fig. 11. v.s. ovum fixed in acetone/ethanol and incubated for thiamine pyrophosphatase.
Fig. 12. v.s. ovum showing distribution of RNA (Helly/pyronin Y). Note diffuse
staining in hyaline zone and pyroninophily of yolk granules.
10
A. H. SATHANANTHAN
oxidase and succinic dehydrogenase, the sites of enzyme activity closely correspond to the distribution of mitochondria (Fig. 2). The hyaline zone gives an
intense positive reaction, the region above the equator being most intense. On
closer examination, the mitochondria are the chief sites of enzyme activity.
Those in the yolky zone and the Golgi bodies there are also intensely positive.
The results obtained on incubation for succinic dehydrogenase, however, are
less striking than those obtained for cytochrome oxidase (see Sathananthan,
1970).
Acid-phosphatase activity. Lysosomes, phagosomes and Golgi bodies are
strongly acid phosphatase-positive (Fig. 3). When incubated by the azo dye
method the hyaline zone is intensely stained and there appears the supraequatorial band which consists of phagosomes and associated lysosomes. The
Golgi bodies are found distributed among the yolk spherules, which are acidphosphatase-negative. Acid-phosphatase-rich granules, chiefly lysosomes and
a few lipochondria, are also seen around nuclei and asters in the hyaline zone
and in polar bodies.
Benzidine peroxidase activity. A remarkable reaction is seen when centrifuged
eggs are tested for this enzyme (Fig. 4). A sharply defined, intense blue, supraequatorial band appears in about 20 min of incubation, similar to that observed
in Limnaea (Raven, 1958). The picture resembles very much the reaction seen in
embryos incubated for acid phosphatase. When examined closely the blue band
is seen to consist of fine irregular granules (lysosomes) and larger rounded bodies
(phagosomes). A few of these inclusions are also found in the hyaloplasm,
especially around nuclei and asters. A diffuse cytoplasmic reaction in the band
region is also evident and needle-like spicules are sometimes seen among the
inclusions, which is probably an artifact. The identification of benzidine peroxidase as an intrinsic component of lysosomes and phagosomes, perhaps for
the first time, is rather significant (see Sathananthan, 1968).
Thiamine pyrophosphatase activity is almost exclusively limited to the yolky
zone (Fig. 11). The Golgi bodies and yolk membranes (externa of yolk spherules)
are intensely positive while the yolk interna also show some activity. Golgi are
probably involved in vitellogenesis and the yolk membranes are very likely
derived from Golgi membranes (Sathananthan, 1966). Nucleoli in the hyaline
zone are also positive.
Alkaline-phosphatase activity. Results are exactly similar to those obtained for
thiamine pyrophosphatase activity.
Nucleic acids. Free RNA stratifies in the hyaline zone (Fig. 12) and presumably
corresponds to the distribution of ribosomes in the hyaloplasm. A few strands
of ER that remain in the yolky zone are RNA-positive and these seem to be associated with yolk spherules. Bound RNA is mainly found in the yolk membranes.
The mitochondria in the hyaline zone are also feebly positive while the nuclear
RNA is also stainable. The presence of RNA was confirmed with ribonuclease.
DNA is found in the nuclei that stratify in the hyaline zone but is rather difficult
Centrifuged slug eggs
11
to demonstrate in ova. The chromosomes, however, are readily DNA-positive
and could be easily stained with acid fuchsin in mitochondrial preparations
(Fig. 10).
Polysaccharides and mucosubstances. Glycogen appears finely granular and
stratifies in the hyaline zone (Fig. 5). It could be stained with Best's carmine or
PAS reagent and removed with saliva. Traces of galactogen could also be
demonstrated in the phagosomes by the same methods, by predigesting with
pectinase.
The PAS reaction also reveals larger polymorphic granules (lysosomes) in the
hyaline zone and a fairly distinct equatorial band consisting of phagosomes and
associated lysosomes (Figs. 8, 9) corresponding to that seen after incubation for
acid phosphatase and benzidine peroxidase. Some of these inclusions are also
seen, as usual, around nuclei and asters and in polar bodies. In the yolky zone,
the yolk membranes and Golgi are also intensely PAS-positive, while the flecks
of lipids give a weak reaction. Various mucosubstances and glycolipids are
known to answer the PAS test.
The distribution of acid mucopolysaccharides (AMP) in the hyaline zone
also conforms to that of the phagosomes and lysosomes but the results are not
as striking as those obtained with the PAS reaction due to a weaker reaction
and a diffuse staining of the hyaloplasm. Aldehyde fuchsin, Alcian blue and
Hale's reagent were used to demonstrate AMP. More striking results were
obtained using vital metachromasy. Lysosomes and phagosomes stain purple
with toluidine blue, methylene blue and sometimes with Nile blue. The hyaline
zone as a whole, however, stains diffuse purple while a more intense metachromatic band is seen above the equator corresponding to that seen earlier.
The diffuse staining could be due to some free AMP and also RNA that stratifies
here, which is feebly metachromatic. AMP in bound form is also present in the
Golgi bodies, yolk membranes (externa), nucleoli, plasma and vitelline membranes. All results indicate that most inclusions have a higher content of
sulphated AMP, the exceptions being the yolky interna and vitelline membrane
which have more non-sulphated AMP.
Vitamin C exists freely as sharply defined coarse granules (1-2 /an. in diameter),
strongly resembling Golgi bodies, and stratifies in the yolky zone (Fig. 5). The
substance, however, is not bound to Golgi or any other inclusion.
Iron. Ferric iron is found distributed diffusely as fine irregular particles (0-31 /tm in diameter), which stratify in the yolky zone. It seems to be also bound
to Golgi bodies and to a lesser extent to nucleoli.
Calcium is bound to Golgi bodies and yolk spherules that stratify in the
yolky zone. The yolk membranes are more intensely positive than their interna.
Nucleoli are also calcium-positive. Golgi seem to be involved in the metabolism
of calcium.
Pigment. Golgi bodies and yolk spherules have a yellowish-brown pigment
which has been identified as melanin. Its distribution is similar to that of calcium.
12
A. H. SATHANANTHAN
The fat cap is also pigmented brown, but this pigment has not been confirmed
as melanin.
DISCUSSION
The results show that by moderate centrifugation one could obtain a fairly
sharp stratification of cell inclusions. This undoubtedly has been most valuable
in subcellular studies of this egg. Although stratification is sharp, it is by no
means complete. A few mitochondria and elements of the ergastoplasm are
always seen in the yolky zone. Likewise some lipid inclusions are retained in
the yolky half or may invade the hyaloplasm. Exceptionally a few Golgi bodies
or yolk spherules may be found in the hyaline zone. This confirms the view that
complete stratification cannot be achieved by the moderate centrifugation
commonly used in embryological studies.
The centrifugal force causes an inevitable stretching of the cell membrane but
it is capable of regaining its original form when the force ceases to act. This
indicates that the membrane is fairly elastic in nature. The tension developed
in it probably causes the egg to round up soon after centrifugation. In Aplysia
the elastic recovery of the spherical shape by the elongated egg after centrifugation is thought to play an important part in the redistribution of its cytoplasmic
contents (see Raven, 1958). This seems to be the case in Arion.
It has been also postulated that in certain molluscs the egg cortex may play a
part in the redistribution of inclusions (ooplasmic re-segregation) after centrifugation, and that it is the seat of polarity of the egg (Raven, 1958). It is believed
that this cortex is a gel that cannot be displaced by moderate centrifugation.
This is also the commonly accepted view in the case of the sea urchin. There is
no structural evidence of a cortical gel layer in the egg of this slug. Electron
micrographs of normal eggs (Sathananthan, 1966) show a well-defined 'unit'
cell membrane and a zone usually devoid of larger granules (about 1 jam thick)
below it, corresponding to a clear agranular cortical region seen with the light
microscope. This zone has an abundance of ribosomes and also vesicular
elements of the ER and those of micropinocytotic origin. Occasionally larger
inclusions like mitochondria and lysosomes invade it and are found very close
to the cell membrane, indicating the absence of a cortical gel layer. Further,
ultrastructural studies of both normal and centrifuged eggs of the sea urchin
(Mercer & Wolpert, 1962) have failed to detect a gel-like cortex, beneath the
cell membrane. These authors have, however, shown that the small particulate
cytoplasmic fraction (probably ergastoplasm) appears to be relatively unaffected
by centrifugation and forms a continuous 'ground substance' in which are
found the larger mobile inclusions, such as mitochondria and yolk granules.
This, indeed, is the impression gained by the present study. The incomplete
stratification of the ER and undoubtedly of the hyaloplasm which suspends the
granules even in the yolky zone is evidence that the ergastoplasm is relatively
unaffected. A similar electron-microscope investigation of centrifuged eggs,
however, needs to be carried out for further confirmation of these views.
Centrifuged slug eggs
13
I am most grateful to Professor Alastair Graham and Dr Vera Fretter for providing the
facilities for my work at the University of Reading, U.K., and for their supervision and
encouragement.
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{Manuscript received 10 February 1971)