/ . Embryol. exp. Morph. Vol. 31, 2, pp. 423-433,1974
Printed in Great Britain
423
The structure of a morphogenetic
cytoplasm, present in the polar lobe of Bithynia
tentaculata (Gastropoda, Prosobranchia)
By M. R. DOHMEN 1 AND N. H. VERDONK 1
From the Zoological Laboratory, University of Utrecht, The Netherlands
SUMMARY
In the first polar lobe of the egg of Bithynia tentaculata a cup-shaped mass of small vesicles
is described, which fills the greater part of the lobe. It is named the 'vegetal body'. With
methyl green-pyronin the vegetal body stains clearly, but after treatment with RNase no
staining occurs, thus indicating the presence of RNA. The first polar lobe of Bithynia is of
great importance for further development of the embryo and it is argued that the vegetal body
could be a morphogenetic cytoplasm, responsible for the developmental effects of the polar
lobe.
INTRODUCTION
In the eggs of many annelids and molluscs the first cleavages are characterized
by the appearance of polar lobes. The contents of these lobes are of great importance for further development, as is shown by experiments involving removal
of the lobe or deletion of cells receiving the lobe contents. In Ilyanassa, for instance, lobe-dependent structures are: foot, eyes, operculum, statocysts, shell,
heart and intestine (Crampton, 1896; Clement, 1952, 1956, 1962; Cather, 1967;
Atkinson, 1971). The successively appearing lobes do not all have the same
effect. In Dentalium (Wilson, 1904), Sabellaria (Hatt, 1932; NovikofT, 1938a, b)
and Mytilus (Rattenbury & Berg, 1954) development of the apical tuft of the
larva is dependent on the presence of the first polar lobe, but not on the second
one.
Attempts to identify polar lobe factors have given very poor results up till now.
In many cases differences in composition can be demonstrated between the
cytoplasm of the polar lobe and the rest of the egg (Pitotti, 1947; Clement &
Lehmann, 1956; Pasteels & Mulnard, 1957; Reverberi, 1958, 1970; Berg &
Kato, 1959; Collier, 1960a, b; Dalcq & Pasteels, 1963; Crowell, 1964). But
centrifugation experiments (Clement, 1968; Verdonk, 1968) have established
that in Ilyanassa and Dentalium the morphogenetic factors are not located in the
displaceable components of the polar lobe cytoplasm. Unfortunately ultrastructural and histochemical studies on displacement of cytoplasmic components
in polar lobes of centrifuged eggs are lacking.
1
27
Authors'1 address: Zoology Laboratory, Janskerkhof 3, Utrecht, The Netherlands.
E MB 31
424
M. R. DOHMEN AND N. H. VERDONK
Accumulation of morphogenetic substances in special areas of the egg and
segregation of these accumulations into special blastomeres are not restricted to
polar-lobe-forming organisms, but can be demonstrated in many other animals.
In spite of such a widespread occurrence of the segregation phenomenon,
identification of the morphogenetic substances in the segregated cytoplasm has
not been successful. Even in the polar granules of insect eggs, 'the only case
known where developmentally significant information is localized in organelles
that can be seen and followed during development' (Mahowald, 1971), it is not
known whether it is the protein or the RNA component which is developmentally
significant. We describe a structure in the polar lobe of Bithynia tentaculata
which could be a second case of an organelle storing morphogenetic substances.
METHODS
Bithynia tentaculata is a freshwater prosobranch snail which can be collected
in ditches around Utrecht, Holland. In aquaria in the laboratory, egg masses are
soon deposited on plant leaves. These egg masses consist of capsules which
cannot be separated from each other. The tough capsule membrane is first
perforated with a very sharp knife in order not to compress the egg. Then the egg
can be removed from the capsule with a hair-loop and the viscous capsule fluid
can be washed off in tap-water.
Fixation and staining for light microscopy. Eggs fixed in Zenker's fluid were
stained with Heidenhain's iron haematoxylin and eosin for general study or with
methyl green-pyronin for nucleic acids (Brachet, 1942,1953). For the latter stain
eggs usually were fixed in ethanol-acetic acid (3:1) for a more vivid stain. For the
Feulgen method, eggs werefixedin ethanol-acetic acid (3:1) and either stained as
sections or in toto according to the method of van den Biggelaar (1971).
Fixation and staining for electron microscopy. Eggs were fixed for 3 h at 4 °C in
a mixture of equal parts 2 % glutaraldehyde and 2 % osmium tetroxide, both in
0-1 M Na-cacodylate buffer at ph 7-4. The eggs were then washed in buffer,
oriented in agar, dehydrated in a graded series of ethanol, followed by propylene
oxide, and embedded in Epon 812. Sections were stained for 10 min in a saturated
solution of uranyl-acetate in 70 % methanol, followed by 1 min in a lead solution
according to Reynolds (1963).
RESULTS
I. Observations with the light microscope
At the vegetal pole of freshly laid eggs of Bithynia, in which the germinal
vesicle is still present, a densely staining cytoplasm is present. At this stage the
dense plasm is intimately connected with the surface of the egg (Fig. 1). After the
germinal vesicle has disappeared, the dense cytoplasm rises from the surface
(Fig. 2) and soon assumes the shape of a cup with the open side towards the
vegetal pole. Because of its position we call it the 'vegetal body'. It stays at the
The polar lobe o/Bithynia
425
• • •
k
*
.
Figs. 1-5. Light micrographs of eggs of Bithynia tentaculata stained with iron
haematoxylin and eosin. x 350.
Fig. 1. Egg just after oviposition with the germinal vesicle still present. Arrow
indicates dense cytoplasm at the vegetal pole.
Fig. 2. Egg after breakdown of the germinal vesicle. Dense cytoplasm (arrow) free
from the surface.
Fig. 3. Egg at first cleavage. Arrow indicates dense cytoplasm now present as cupshaped vegetal body in the polar lobe.
Fig. 4. Two cell stage with the vegetal body (arrow) at the vegetal side of the CDblastomere.
Fig. 5. Section through the CD-blastomere at the beginning of second cleavage.
Arrow indicates the second polar lobe, filled with clear cytoplasm but missing the
vegetal body.
Fig. 6. Light micrograph of an egg at first cleavage, stained with methyl greenpyronin. The vegetal body (arrow) is densely stained.
27-2
426
M. R. DOHMEN AND N. H. VERDONK
Fig. 7. Electron micrograph of the first polar lobe with vegetal body. Arrow points to
a dense body. AZ, Attachment zone; L, lipid; M, mitochondrion, x 9700.
vegetal pole, surrounded by clear cytoplasm, until first cleavage, when a polar
lobe is formed. Compared with other polar lobes, described in molluscs, this
lobe is remarkable in several respects: it is extremely small (diameter 20-30 /an)
and it is usually nearly free from yolk granules.
The polar lobe of Bithynia
427
The vegetal body is incorporated in the first polar lobe (Fig. 3). After first
cleavage the vegetal body is transferred with the lobe to the CD-blastomere.
During the whole 2-cell stage it remains at the vegetal side of the CD-blastomere
(Fig. 4). At second cleavage the vegetal body suddenly disappears at about the
start of anaphase. A second polar lobe is formed at this stage, which, however, is
never as completely separated as the first polar lobe and remains broadly connected with the CD-blastomere. The second lobe contains a clear cytoplasm but
the vegetal body is no longer present (Fig. 5). In order to study the chemical
nature of the vegetal body, we stained it with the Feulgen method for the presence
of DNA. Both in sections and in whole mounts the staining was negative. With
methyl green-pyronin the body stains clearly (Fig. 6). After pre-treatment of the
sections with RNase (Worthington Biochem. Corp.) no staining of the vegetal
body occurs, indicating the presence of RNA.
II. Observations with the electron microscope
The vegetal body is a cup-shaped mass of small vesicles and among them some
ribosomes, mitochondria, large vesicles and dense bodies can be found (Fig. 7).
The vesicles have a diameter of 50-100 nm and most of them are filled with a
dark-staining substance (Fig. 8), probably RNA, as RNase treatment of thick
sections makes the vegetal body almost completely disappear. The vesicles are
not homogeneously distributed, but they are arranged in an irregular network,
with vesicle-free areas in between (Fig. 7). The same type of vesicles also forms a
thin layer under the membrane of the stalk which connects the polar lobe to the
egg and this layer extends to a limited distance (about 15 /im) under the neighbouring egg-membrane (Fig. 9). Occasionally a small cluster of vesicles can be
found outside the polar lobe in the egg cytoplasm, close to the stalk. In view of
their distribution it seems possible that a few vesicles will come to lie in the ABblastomere at first cleavage.
Preliminary centrifugation experiments indicate that the vegetal body is hard
to displace. Even when part of the uncleaved egg is centrifuged off, the vegetal
body stays in most cases in its original place and when the egg is left to cleave after
centrifugation, the vegetal body is found in the polar lobe at first cleavage. These
results indicate that the vegetal body is firmly attached to the vegetal pole and
that there is a strong cohesion of the body itself. Still, no special devices responsible for attachment and cohesion can be found. As attachment is likely to take
place by means of the cytoplasmic zone lying between the vegetal body and the
vegetal pole, we called this zone the 'attachment zone' (Fig. 7).
In the polar lobe there is also to be found a concentration of multivesicular
bodies. They often contain densely coated vesicles, which can easily be distinguished from the vesicles making up the vegetal body. The multivesicular
bodies are often ruptured and their coated vesicles may be seen lying in strings in
the attachment zone (Fig. 10).
M. R. DOHMEN AND N. H. VERDONK
Fig. 8. Detail of the vegetal body, showing the small vesicles of which it consists.
Most vesicles are completely or partlyfilledwith a dark-staining substance, probably
RNA. x 105000.
Fig. 9. Superficial layer of the stalk (ST) of the polar lobe and its implantation
region on the egg, showing a layer of small vesicles under the membrane, x 27 700.
The polar lobe o/Bithynia
429
Fig. 10. Multivesicular bodies {MVB) in the polar lobe. One of them is ruptured
(double arrow), releasing coated vesicles (arrow) into the attachment zone {AZ).
x 27 700.
430
M. R. DOHMEN AND N. H. VERDONK
DISCUSSION
The facts that are known at present about the vegetal body are all in agreement
with the view that the vegetal body could contain the morphogenetic factors
involved in determining the lobe-dependent structures.
In Bithynia the significance of the polar lobe for development has been established by removing the lobe at first cleavage. No adult organs like eyes, tentacles,
shell, etc., develop in lobeless embryos (Verdonk & Cather, 1973). The vegetal
body fills the greater part of this polar lobe so it is obvious to assume that the
developmental information is located in the vegetal body and most probably
inside the vesicles. After first cleavage the vegetal body is transferred with the
polar lobe to the CD-blastomere. In isolation experiments (Verdonk & Cather,
1973) the AB-blastomere forms hardly any adult structures, whereas the CDblastomere, containing the polar lobe, forms most adult structures. At second
cleavage the vegetal body disappears rather suddenly between meta- and anaphase, some time after the disappearance of the nuclear membrane. After
second cleavage the C- and D-blastomeres have the same morphogenetic capacities (Verdonk & Cather, 1973). As the vegetal body disappears before the
cleavage furrow separates the C- and D-blastomeres, the substances contained
in the vegetal body can spread over both cells and in this way give them the same
developmental potentialities.
Other arguments in favour of a morphogenetic role are the fact that the
vesicles forming the vegetal body are typical storage vesicles, which could keep
the morphogenetic factors inactive until their time has come to act, and the fact
that the vegetal body contains RNA, which is an obvious candidate for the role
of morphogenetic factor.
A large structure such as the vegetal body of Bithynia cannot easily be overlooked, still nothing like it has been found in thoroughly investigated species like
Ilyanassa (Pucci-Minafra, Minafra & Collier, 1969; Gerin, 1972), Mytilus
(Humphreys, 1964) and Dentalium (Reverberi, 1970). This probably means that
the vegetal body is an exceptional structure, but the vesicles composing it could
well be a general carrier of developmental information. If in other animals these
vesicles should not be as numerous as in Bithynia and scattered over a larger
area, they could probably escape attention.
In all species investigated thus far, several larval or adult structures are dependent on one lobe, so it is to be expected that this lobe contains several kinds
of morphogenetic substances which become separated during cleavage until at
last each one reaches its own target cell. Such a mechanism might be deduced
from experiments with Dentalium. Here the apical tuft and the post-trochal
region are both dependent on the first polar lobe. If 60 % of the lobe is removed,
however, larvae develop with an apical tuft and a reduced post-trochal region
(Geilenkirchen, Verdonk & Timmermans, 1970). This result points to the
possibility of at least two different factors: an apical tuft factor located in the
The polar lobe of Bithynia
431
animal region of the lobe, which is not removed, and a post-trochal region factor
in the vegetal region. The finding in Bithynia of a mass of apparently identical
vesicles, whose contents probably determine several adult organs, points to
another possible mechanism: a polar lobe might contain one kind of morphogenetic factor only, which in some way, maybe by unequal distribution over the
cells, determines the development of several organs.
The lack of any visible mechanical structure holding together the vegetal body
and attaching it to the vegetal pole is another problem. Special cytoplasms
accumulated under a particular area of the cell periphery are a common feature
in many eggs, but they do not seem to react in the same way to centrifugal force.
The vegetal body of Bithynia and the polar lobe factors of Ilyanassa and Dentalium
are not displaced by centrifugation, while the animal pole plasm and the subcortical accumulations of Lymnaea, for instance, are easily disrupted and dispersed (Raven, 1945; Raven & Brunnekreeft, 1951; Raven & van der Wai, 1964;
Raven, 1967). In all cases there is probably some kind of attraction exerted by
particular areas of the egg periphery upon certain cytoplasmic components, but
we can only speculate about their nature.
The last problem to be discussed is the concentration of multivesicular bodies
in the polar lobe of Bithynia. Very little is known about multivesicular bodies.
In eggs they seem to be formed by transformation of yolk granules (Pasteels & de
Harven, 1963) and they contain a high concentration of phosphatases (Dalcq,
1962; Dalcq & Pasteels, 1963). Reverberi noted multivesicular bodies in the
polar lobe of Dentalium, and the polar lobe of Sabellaria shows a high concentration of phosphatase (Dalcq & Pasteels, 1963), which could also result from a high
concentration of multivesicular bodies. No multivesicular bodies have been
reported in the polar lobes of Ilyanassa and Mytilus. Too little is known as yet to
enable us to suggest a function for the multivesicular bodies in polar lobes.
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