/. Embryo!. exp. Morph. Vol. 51, pp. 155-J64, 1979
Printed in Great Britain © Company of Biologists Limited 1979
155
The ultrastructure of the
dorsal yolk-free cytoplasm and the immediately
surrounding cytoplasm in the symmetrized
egg of Xenopus laevis
By JORGE HERKOVITS 1 AND GEERTJE A. UBBELS 2
From the Hubrecht Laboratory, Utrecht, Netherlands
SUMMARY
Cytoplasmic segregation and subsequent dorsad displacement of the segregated cytoplasm
lead to symmetrization of the egg of Xenopus laevis. At 60 min post-fertilization (p.f.) the
'dorsal yolk-free cytoplasm' (DYFC) is located in the dorso-animal part of the egg. Its
ultrastructure and that of the immediately surrounding cytoplasm have been studied with
transmission electron microscopy (TEM).
The endoplasmic reticulum (ER) within the DYFC consists of single or paired cisternae
and many small vesicles, both with moderately dense contents. Numerous particles, presumably ribosomes and glycogen, are present together with many mitochondria and some
Golgi structures. The fraction of total yolk-free area occupied by mitochondria in the DYFC
is about three times that in the adjacent cytoplasm. The number of cytoplasmic vesicles per
unit area of cytoplasm is far larger in the DYFC than in the surrounding area.
The morphological characteristics of the DYFC at 60 min p.f. suggest that it represents
a region of high metabolic activity. Since it is located in the dorso-animal quadrant of the
uncleaved egg, it may be partly responsible for a difference in metabolism between the dorsal
and the ventral side of the egg, and hence may play an essential role in the determination of
dorso-ventrality.
INTRODUCTION
Previous studies of the ultrastructure of the uncleaved and first cleavage egg
of Xenopus laevis particularly emphasized the cortical regions (Balinsky, 1966;
Van Gansen, 1966a, b\ Hebard & Herold, 1967; Bluemink, 1971; Kalt, 1971;
Singal & Sanders, 1974 and Perry, 1975). The present study was performed
within the framework of our analysis of the origin of dorso-ventrality in anuran
eggs and concentrates on the 'dorsal yolk-free cytoplasm' (Ubbels, 1978).
Animal-vegetal polarity in the unfertilized egg of Xenopus laevis is externally
expressed by the dark pigmentation of the animal cap. The unfertilized and newly
fertilized egg are internally axially symmetrical because of the characteristic
1
Author's address: Instituto de Biologia Celular, Facultad de Medicina, Paraguay 2155,
2e Piso, Buenos Aires, Argentina.
2
Author's address: Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Correspondence and requests for reprints should be sent to the Hubrecht Laboratory.
156
J. HERKOVITS AND G. A. UBBELS
arrangement of yolk granules of various sizes (Ubbels, 1978). In the interior of
the egg the yolk platelets increase in number and size from the animal towards
the vegetal pole.
The first outer manifestation of bilateral symmetry in the fertilized, uncleaved
egg is the appearance of the grey crescent. In the majority of cases this appears
on the side opposite that of the sperm entrance point, in Xenopus laevis (Palecek,
Ubbels & Rzehak, 1978), as a result of a concentration of pigment around this
point. Experimental evidence has shown that this external symmetrization of
the egg is strictly related to the definitive symmetrization of the embryo. Although there are almost no animal species where the egg cortex can be sharply
defined morphologically, much attention has been paid to the structure, composition and role of the cortex in the early development of the anuran embryo
(cf. Brachet, 1977; Nieuwkoop, 1977).
Recent observations in Discog/ossus pictus (Klag & Ubbels, 1975), and Xenopus
laevis (Ubbels, 1978) have shown that upon fertilization the uncleaved egg also
acquires an internal bilateral symmetry as a result of cytoplasmic segregation and
the subsequent displacement of all or part of the segregated cytoplasm towards
the future dorsal side. In Xenopus laevis this cytoplasm is ultimately found in
the dorso-animal quadrant of the still uncleaved egg and is then called 'dorsal
yolk-free cytoplasm' (DYFC). It reacts strongly with PAS for glycogen and
with pyronin for RNA.
The present EM study is a description of the ultra structural morphology of
the DYFC in the symmetrized egg of Xenopus laevis fixed at 60 min postfertilization (p.f.). At this stage the DYFC has reached its most dorsal position.
It is characterized by large numbers of cytoplasmic vesicles and mitochondria in
comparison with the surrounding cytoplasm and shows the general morphological characteristics of a region of high metabolic activity.
MATERIALS AND METHODS
Fertilized eggs of Xenopus laevis were obtained from hormonally stimulated
females by artificial insemination. The jelly coat was removed by a 2-3 min
treatment with 2 % thioglycolic acid in distilled water, adjusted to pH 7-5-8
with Tris buffer (see Palecek et al. 1978).
Decapsulated eggs were kept at room temperature in tap water till 50 min p.f.
For fixation eggs were selected which clearly exhibited (1) a darkly pigmented
spot in the animal hemisphere, marking the sperm entrance point and (2) the
grey crescent on the side opposite this point. Since at 60 min p.f. the dorsal
cytoplasm is always found in the plane running through the sperm entrance
point and the animal-vegetal axis, on the side of the grey crescent (PaleSek et al.
1978), the sperm entrance point and the centre of the grey crescent were used as
landmarks for orientation during embedding.
The fixation fluid consisted of 1 % acrolein and 2-5 % glutaraldehyde in
Ultrastructure of cytoplasm in the egg o/Xenopus
157
0-067 M cacodylate buffer, pH 7-2. The eggs were fixed at room temperature at
60 min p.f. for ca. 2-5 h, and then rinsed and stored in buffer (170 m-osmole).
They were carefully oriented in 5 % agar at 55 °C and when the agar had solidified
the lateral parts were cut away. The specimens were then re-embedded in cubeshaped blocks of agar and postfixed for 2 h in a solution containing 2 % osmium
tetroxide in cacodylate buffer (pH 7-2-7-4). Blocks were stained in 1 % aqueous
uranyl acetate for 1-2 h, dehydrated in increasing concentrations of cellosolve
and embedded in Dow Epoxy Resin (see Bluemink, 1972).
Serial 1 jtim paramedian sections (parallel to the future dorso-ventral plane
running through the animal-vegetal axis) were cut with glass knives. Each
tenth section was stained with 1 % toluidine blue and examined for the presence
of the DYFC with a phase contrast microscope. Silver-reflecting ultrathin
sections were then cut of the part of the egg containing the DYFC and some of
the surrounding cytoplasm, using an LKB ultratome equipped with glass knives.
The sections were picked up on coated copper grids, stained with lead citrate
(Reynolds, 1963) and studied with a Zeiss EM 10 electron microscope.
The numbers of cytoplasmic vesicles and mitochondria were counted in nine
randomly selected areas within each of three regions: (a) the central part of the
DYFC, (b) the more peripheral parts of the DYFC, and (c) the yolky cytoplasm
outside but adjacent to the DYFC. To obtain numbers of inclusions per 100 /tm2
of cytoplasm (without yolk), the total surface area of the yolk granules present
in the area concerned was determined with a Wang XY digitizer coupled to a
Wang 2200 minicomputer, using a programme written for the determination of
areas. This was then subtracted from the total area examined. The fractional
yolk-free surface area occupied by mitochondria was also determined with the
digitizer.
RESULTS
Light microscopy
The axial symmetry found in the newly fertilized egg and expressed in the
distribution and size of the yolk granules, changes upon fertilization into a
bilateral symmetry as a result of cytoplasmic shifts. At 60 min p.f. this internal
symmetrization is expressed in the changed distribution of the yolk granules
and in the presence of the DYFC in the animal part of the egg (Fig. 1 a). The
DYFC is variable in shape (diameter ca. 100-150 ^m) and contains only a few
rather small yolk granules (Fig. 1 b).
TEM observations on the DYFC and the immediately surrounding cytoplasm
The overall composition of the DYFC, the yolky cytoplasm and the area in
between is shown in Figs. 2-4. The DYFC is mainly composed of a matrix with
densely packed electron-dense granules (200-350 nm) (Fig. 5). The most conspicuous features of the DYFC are the large numbers of mitochondria and
II
EMB
51
158
J. HERKOVITS AND G. A. UBBELS
Ultrastructure of cytoplasm in the egg of Xenopus
159
Table 1. Distribution of subcellular components in the
dorsal yolk-free cytoplasm and adjacent regions
Yolk-free cytoplasm
Cytoplasmic regions
in animal hemisphere
Centre of DYFC
Periphery of DYFC
Yolky area
adjacent to DYFC
Percentage of
total area
occupied by
yolk granules
0-2
13-4
22-5
Mitochondria
K
,
No./100/tm 2
»
% of total
area
Cytoplasmic
vesicles
(no./100/*m2)
113-2
88-9
42-6
4-4
3-6
1-6
153-3
900
69-7
cytoplasmic vesicles, the long cisternae often oriented in the dorso-ventral
plane, and the absence of yolk granules, microtubules and microfilaments.
The mitochondrial profiles are round or oval (Figs. 4, 5) and occasionally
filliform (largest diameter 0-2-1-5 /«n). The cristae are often irregular and the
matrix is deeply stained with osmium. Occasionally the mitochondria show
a tendency to form clusters (Fig. 4). Per 100 /tm2 of cytoplasmic surface area
(corrected for the area occupied by the yolk) more than twice as many mitochondria and cytoplasmic vesicles are present in the central part of the DYFC
than in the yolky area adjacent to the DYFC (Table 1). The fraction of the
cytoplasmic surface area occupied by mitochondria in the centre of the DYFC
is about three times that in the cytoplasm surrounding the DYFC.
The endoplasmic reticulum (Figs. 5, 6) consists of cisternae and cytoplasmic
vesicles, both with a slightly electron-dense content (Figs. 5-8). Short filamentous
projections occasionally extend from the inner membrane surface into the
FIGURES
1-5
Fig. 1. (a) Paramedian 1 /tin section of a Xenopus laevis egg at 60 min p.f., stained
with 1 % toluidine blue. The section just grazes the DYFC, which consequently is
not shown in its maximal dorsal extension. The light area in the central part of the
vegetal moiety is due to restricted osmium penetration. DYFC, dorsal yolk-free
cytoplasm; V, future ventral side; D, future dorsal side, (b) Enlargement of the
DYFC from the same section. Only a few relatively small yolk granules (y) are
present.
Figs. 2-4. Low-power electron micrographs showing the yolky region immediately
surrounding the DYFC (2), and the periphery (3) and centre (4) of the DYFC; (y),
yolk granules. Many small vesicles (v) and mitochondria (m) are observed in the
DYFC.
Fig. 5. Higher magnification of the centre of the DYFC. The cytoplasmic matrix
contains densely packed (200-350 nm) granules (assumed to be glycogen particles
and ribosomes, r), sometimes next to the membranes of vesicles (v) and long
cisternae (c). The cisternae are often paired and oriented dorsal-ventral Iy in the
median plane. Note: slightly electron-dense content in cisternae and vesicles; mitochondria (w) and primitive Golgi structure (g).
160
J. HERKOVITS AND G. A. UBBELS
Fig. 6. Part of a long cisterna, paired over some distance with another one; (c)
both in close relationship to mitochondria (m). In places electron-dense particles
are found next to the membrane (r). /, thin filaments protruding into the cisternal
lumen.
Fig. 7. Enlargement of the dilated end of a cisterna with projections (/) extending
from the inner membrane surface into the lumen (tannic acid post-treatment).
Fig. 8. Well-developed Golgi structure (g) very close to cytoplasmic vesicles (v) and
(dilated) ER cisternae. Electron-dense particles (r) very close to the cisternal membrane. /, thin filaments protruding into the cisternal lumen.
infrastructure of cytoplasm in the egg o/Xenopus
161
lumen (Figs. 6, 7). The numerous round or oval vesicles (Figs. 4, 5; Table 1) are
often seen in clusters.
The cisternae, up to 5 jum in length, are sometimes seen in pairs which
occasionally run over relatively long distances (Figs. 5, 6), and are often oriented
in the dorso-ventral plane. Such paired cisternae may fuse, and in some cases
the ends of the cisternae are dilated (Fig. 6). Occasionally electron-dense
particles, presumably ribosomes, are observed next to the ER membranes
(Figs. 5-8). In the cytoplasmic area close to the DYFC, ER cisternae are often
closely apposed to yolk granules.
Some of the cisternae are situated within 0-3 /an from a Golgi structure (Fig.
8). No, or only very few, electron-dense particles are found next to such cisternae.
Small vesicles are often present in between the ER cisternae and the Golgi body
(Fig. 8).
The simplest Golgi structure in the DYFC is a single fenestrated cisterna
accompanied by vesicles or ER membranes or both. Moderately electron-dense
material is observed in such cisternae, and small (200-400 nm) round or oval
vesicles of similar density are associated with them. A well-developed Golgi
structure has two to four straight cisternae (Fig. 8); Golgi structures with six or
seven cisternae occur only rarely.
In the DYFC at 60 min p.f. yolk granules are rarely seen, particularly in the
centre (Fig. 1 b). They are round or oval and their number increases progressively
towards the cytoplasmic area surrounding the DYFC (Table 1). They have a
crystalline core partially surrounded by a diffuse granular substance. Lipid
droplets as described by Singal & Sanders (1974) are also rarely observed in
the DYFC; they are round, oval or multilobed (0-3-1 /tm), moderately dense
in appearance and devoid of a limiting membrane.
DISCUSSION
The cytoplasmic matrix in the DYFC contains many moderately electrondense granules (200-350 nm). We assume that the smaller ones represent
ribosomes (cf. Perry, 1967) and the larger ones glycogen, since they are darkly
stained after post-treatment of thefixedeggs with a tannic acid solution according
to Simionescu & Simionescu (1976) (Ubbels & Bleumink, unpublished).
With the aid of light-microscopical cytochemistry large amounts of glycogen,
ribonucleoprotein and mitochondria have been demonstrated in the DYFC
(Ubbels, 1978). The present TEM study confirms the most prominent features
of the DYFC as suggested by light microscopy, i.e. the scarcity in yolk platelets
and the presence of relatively large numbers of mitochondria. In addition it
reveals the presence of large numbers of cytoplasmic vesicles (Table 1). The
present results do not explain how this composition comes about, although
they suggest that a true segregation of cytoplasmic components is involved in
the formation of the DYFC.
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J. HERKOVITS AND G. A. UBBELS
Numbers of mitochondria per unit surface area (Table 1) do not necessarily
reflect the total volume of mitochondria. However, since the fraction of the
total area of yolk-free cytoplasm occupied by mitochondria in the centre of the
DYFC is about three times that in the cytoplasm adjacent to the DYFC, we
conclude that the DYFC is indeed an area with a relatively high concentration
of mitochondria. Some hours after the beginning of in vitro maturation of eggs
of Xenopus laevis and some other anuran and urodelan species, a cytoplasmic
region with characteristics similar to those of the DYFC (Ubbels, 1978) originates
on the basal side of the nucleus (Brachet, Hanocq & Van Gansen, 1970). It
probably represents the centrally located yolk-free cytoplasm in the newly laid
egg. A morphological continuity between the 'mitochondrial cloud' found in
perinuclear position in young ovarial oocytes of Xenopus laevis (Billett & Adam,
1976) and the DYFC is unlikely since no yolk-free region is present in the fullgrown but immature egg (Ubbels, unpublished).
The endoplasmic reticulum (ER) in the DYFC consists of single or paired
long cisternae, often oriented in the future dorsal-ventral plane, and numerous
small vesicles (Table 1), both with a moderately dense content. The ER in the
DYFC is morphologically similar to the 'fringed endoplasmic reticulum' (FER)
found in the first-cleavage embryo by Singal & Sanders (1974).
In the case of cytoplasmic vesicles, as in the case of mitochondria, objection
may be raised against relying on numbers rather than amounts of membrane
per unit area of yolk-free cytoplasm. However, since the vesicles form part of
the endoplasmic reticulum, the membrane amount of the relatively long cisternae
should be added, which would lead to even greater differences between the
areas considered than are already suggested by the numbers. Preliminary
observations have shown that such relatively long cisternae are not present in
other regions of the egg.
With a cytochemical stain myoid fibrils consisting of a myosin-like protein
have been shown to be present in the DYFC and to be oriented in the median
or paramedian plane (Ubbels, 1978). In the present study, .microtubules were
not revealed in the DYFC and filamentous material is rather scarce and scattered
in comparison with the cortical region of the oocyte (Franke et al. 1976) and the
egg (Bluemink, 1971; Perry, 1975). However, large numbers of microtubules
are visualized in the DYFC at 60 min p.f., when the fixed eggs are post-treated
with a tannic acid solution (Simionescu & Simionescu, 1976) (Ubbels & Bleumink, unpublished). These microtubules show a non-random orientation and
their exact orientation as well as their possible function in the cytoplasmic
segregation process is being analysed. One might consider a possible role for
the cytoplasmic cisternae in the cytoplasmic segregation as well.
Several studies suggest that the dorsal region of the amphibian egg is metabolically more active than the ventral region and experimental interference
with this difference affects the orientation of the dorso-ventral axis (see Brachet,
1977 and the references cited there). The larger number of mitochondria, together
Ultrastructure of cytoplasm in the egg 0/Xenopus
163
with the presence of 'Fringed ER' in the DYFC, lead us to suggest that the
DYFC in this stage has the morphological characteristics of a region of high
metabolic activity. The location of this region in the dorso-animal part of the
uncleaved egg may in part explain the difference in metabolic activity between
the dorsal and the ventral side of the egg. By functioning as a 'high point' in a
metabolic gradient (Child, 1929) or in a diffusion field (Nieuwkoop, 1967) the
DYFC may play an essential role in the determination of dorso-ventrality.
In Xenopus laevis the segregation and the dorsad movement of the yolk-free
cytoplasm are characteristic features of normal development (Ubbels, 1978).
Hence the DYFC may contain some morphogenetic principle(s). A technique
for the transplantation of cytoplasm taken from well-defined sites in the egg of
Xenopus has been developed (Hengst & Reitsma, 1976) and experiments to
determine the possible morphogenetic significance of the DYFC are in progress.
We want to thank Dr J. G. Bluemink for stimulating discussions and for placing his
technical facilities at our disposal; his technicians L. Tertoolen and P. v. Maurik for their
technical instructions and kind help. We also thank Professor P. D. Nieuwkoop for his
continuous interest and critical reading of the manuscript, Dr J. Faber for discussions and
editorial assistance and Mr L. Boom for the accurate preparation of the illustrations.
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{Received 25 September 1978, revised 24 November 1978)