/ . Embryol. exp. Morph. Vol. 34, 2, pp. 419-438, 1975
Printed in Great Britain
419
The ultrastructure of the polar lobe of Crepidula
fornicata (Gastropoda, Prosobranchia)
ByM. R. DOHMEN 1 AND D. LOK 1
From the Zoological Laboratory,
University of Utrecht
SUMMARY
Some novel structural features of the polar lobe of the egg of Crepidula are described. The
significance of two kinds of aggregate is discussed in relation to the morphogenetic factors
present in polar lobes generally. Surface configurations of the polar lobe are also described.
INTRODUCTION
Since the discovery of the vegetal body in the polar lobe of Bithynia (Verdonk,
1973; Dohmen & Verdonk, 1974) we were faced with the problem of its uniqueness. Such a large structure can hardly be overlooked, but nothing like it has
ever been found in other polar lobes. Other polar lobes may contain the same
kind of structure, i.e. an aggregate of small vesicles, but in a much smaller and
inconspicuous form, so that it is likely to escape attention, particularly in big
polar lobes. As the search for this kind of structure is rather laborious in a big
polar lobe, we started gathering evidence for a more general occurrence of
vesicular aggregates by studying the polar lobe of Crepidula, which is even
smaller than that of Bithynia (Fig. 1). The development of Crepidula has been
thoroughly studied by Conklin (1897) and Moritz (1939). Conklin (1897, 1902)
reported the existence of a polar lobe at first and second cleavage and was of
opinion that 'this lobe is probably homologous with the yolk lobe of Chaetopterus, Ilyanassa, Fulgur, etc.'.
In all species so far investigated the polar lobe proved to be essential, in a
specific way, for the normal development of the egg, even in Bithynia, the only
species with a very small polar lobe investigated thus far (Verdonk & Cather,
1973; Cather & Verdonk, 1974). So there seems to be no fundamental difference
between large and small polar lobes. The centrifugation experiments of Clement
(1968) and Verdonk (1968) present strong evidence that the displaceable components of big polar lobes are not of primary importance in the regulation of
development. Most substances in polar lobes are probably displaced by a
1
Authors' address: Zoological Laboratory, University of Utrecht, Padualaan 8, Utrecht,
The Netherlands.
420
M. R. DOHMEN AND D. LOK
centrifugal force of 300-600 g, at least lipid droplets and yolk particles (Conklin,
1917; Clement, 1968; Verdonk, 1968), suggesting that there is a small component, probably attached to the cortex, responsible for the lobe's morphogenetic effects. Even in small polar lobes the morphogenetic cytoplasm may
be small in proportion to the lobe's volume. Bithynia, with its lobe-filling
vegetal body, will probably prove to be an exception in this respect.
We describe below, among other structures, small vesicular aggregates in the
polar lobe of Crepidula. We believe that these aggregates are of the same kind
as the vegetal body of Bithynia. These structures are less conspicuous than the
vegetal body of Bithynia and may represent a more general form in which
morphogenetic substances occur in polar lobes.
MATERTALS AND METHODS
Crepidula fornicata is a marine prosobranch snail which is collected in the
Oosterschelde at Yrseke, The Netherlands. The European C. fornicata is
descended from specimens which were imported with American oysters about
1880 (Orton, 1909). Crepidula is in many respects peculiarly favourable for
embryological research. These molluscs are abundant and eggs can be obtained
in large numbers throughout the year after transferring the animals to the
aquarium. A female C. fornicata may lay more than 13000 eggs several times
a year (Conklin, 1897). Decapsulating the eggs is very easy, for an egg-mass
consists of about 50 thin-walled and easily disruptable capsules, each containing
about 200 eggs. The eggs do not possess a vitelline membrane, which makes
them particularly suitable for scanning electron microscopy. A difficulty is that
the egg-masses are kept under tb^ non-motile female until development has
proceeded through the fully grown veliger stage. Development of eggs outside
this special environment is generally abnormal (Conklin, 1897), but considerable
progress is being made in this respect in our laboratory.
Fixation. The eggs were removed from their capsules and fixed for 3 h at
4 °C in a mixture of equal parts 3 % glutaraldehyde in double concentrated
sea water and 2 % osmium tetroxide in 0-1 M sodium 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 lOmin in a saturated solution of uranyl acetate in
70 % methanol, followed by 1 min in a lead solution according to Reynolds
(1963).
Preparation for scanning electron microscopy (SEM). The eggs were fixed as
described above, then washed in buffer and dehydrated through an acetone
series. The eggs were dried according to the critical-point drying technique
(Anderson, 1951). After drying, the eggs were dispersed at random on to doublecoated tape on specimen studs and then coated with gold.
The polar lobe of Crepidula
421
Fig. 1. Scanning micrograph of first cleavage stage with polar lobe, x ca. 500.
Fig. 2. Scanning micrograph of first polar lobe; cleavage has not yet begun at the
vegetal pole, x ca. 2000.
422
M. R. DOHMEN AND D. LOK
The polar lobe of Crepidula
423
RESULTS
The surface. In sections of the polar lobe area (Fig. 6) the most striking feature
is the abundance of extremely twisted surface projections, which can often be
seen to constitute cytoplasmic bridges between the polar lobe and the blastomeres. In SEM pictures, on the other hand, these surface projections appear to
be rather smooth surface folds running over the polar lobe (Fig. 2). From these
two kinds of picture it is evident that we should distinguish between a rather
straight primary structure and a very tortuous secondary structure. The origin
of the secondary structure may be explained by assuming that, when the
surface folds are being formed, the short villi which cover the surface of the
egg (Figs. 3, 4) remain as such on the folds. When the polar lobe is being
constricted, the folds are partly pinched off from the surface of the egg and thus
form cytoplasmic bridges between the polar lobe and the blastomeres. High
magnification of the egg surface reveals the existence of a fibrous layer outside
the membrane (Fig. 4). The fibres seem to emanate from the short villi which
cover the surface.
The interior. The most interesting finding in the polar lobe was a number of
aggregates consisting of vesicles filled with a moderately electron-dense material
(Figs. 5-7). These vesicles strikingly resemble the vesicles constituting the
vegetal body of Bithynia (Dohmen & Verdonk, 1974). In both species the
diameter of the vesicles is 50-100 nm and they contain in both cases an amorphous electron-dense substance. The only difference is that in Crepidula the
vesicles are elongated, while in Bithynia they are round. This is not an essential
difference, however, because the vesicles of Bithynia are also elongated before
they become round in the ripe egg (to be published). Because of this resemblance
the vesicular aggregates of Crepidula are also thought to contain morphogenetic
substances, responsible for the lobe's effects on development. The polar lobe
of Crepidula appears to contain about five of these aggregates. They have no
apparent connexion with each other or with the cortex and, just as in the vegetal
body of Bithynia, there is no sign of a structure holding the vesicles of an
aggregate together.
Another structure which was found exclusively in the polar lobe and which
FIGURES 3-5
Fig. 3. Scanning micrograph of a detail of the blastomere surface next to the polar
lobe with a few surface folds. The blastomere surface is covered with numerous
short villi. x 18800.
Fig. 4. Transmission micrograph of a detail of the blastomere surface with a
surface fold, forming a cytoplasmic bridge between the blastomere and the polar
lobe. Note the fibrous layer apparently emanating from the short villi (arrow),
x 18800.
Fig. 5. Detail from Fig. 6 with two vesicular aggregates, x 35400.
27
E M B 34
424
M. R. DOHMEN AND D . LOK
Fig. 6. First polar lobe with three vesicular aggregates (arrows). No complex
aggregates are present in this section, mvb, multivesicular body, x 5800.
The polar lobe o/Crepidula
Fig. 7. Detail of vesicular aggregate, x 72700.
Fig. 8. Complex aggregate, x 56300.
27-2
426
M. R. DOHMEN AND D. LOK
has not been previously described, to our knowledge, was called 'complex
aggregate' (Fig. 8). It consists of a mass of small vesicles intermingled with
granular bodies. The vesicles vary in diameter and their contents are slightly
electron-dense. The granular bodies are mostly rounded, but irregular forms
also occur. The diameter of such a granular body is about 0-2 jum, but irregular
forms up to 0-75 jum have been found. Their component granules measure
about 13 nm. As in the vesicular aggregates, an outer membrane is lacking and
no other structure holding the complex together could be detected.
Other organelles to be found in the polar lobe are: large multivesicular bodies
(Fig. 6), mitochondria, small yolk platelets and many undefined vesicles. The
egg of Crepidula, including the polar lobe, is densely filled with glycogen-rosettes
in certain preparations. If one uses the fixation we described, most of the
glycogen dissolves away. By thus removing the bulk of electron-dense material,
the remaining structures can be more easily distinguished. Glycogen can be
preserved by applying a special fixation, such as the one devised by de Bruyn
(1973). This fixation proved to be disastrous, however, for the structures
described above.
DISCUSSION
The finding of aggregates in the polar lobe of Crepidula, consisting of vesicles
which are similar to those of the vegetal body of Bithynia, reinforces our hypothesis that these vesicles contain morphogenetic determinants.
The complex aggregates in the polar lobe have not previously been described,
to our knowledge. At first sight they seemed to be disintegrating yolk platelets,
but we never found a trace of an outer membrane. The granular bodies of a
complex aggregate resemble the perinuclear corpuscles in oocytes of Ilyanassa
(Gerin, 1971), although they differ in size: the perinuclear corpuscles have a
diameter of about 1 /*m and they are composed of filaments measuring between
15 and 45 nm. The granular bodies of Crepidula measure about 0-2 fim and the
granules about 13 nm. The granular bodies also resemble the polar granules
of insect eggs and the germinal granules in amphibian eggs. These granules,
although having a different appearance in different species (Mahowald, 1962;
Balinsky, 1966;Williams & Smith, 1971 ;Mahowald &Hennen, 1971 ;Czolowska,
1972), and even in the same species at different stages (Mahowald, 1968;
Schwalm, Simpson & Bender, 1971; Schwalm, 1974; Mahowald, 1975), can
generally be described as particulate or fibrous electron-dense bodies without a
limiting membrane. Germ-cell determinants have never been found in spiralian
eggs, so it should be interesting to investigate the final destination of the complex aggregates in Crepidula.
The significance of the impressive array of surface folds on the polar lobe of
Crepidula is not clear. The folds constitute cytoplasmic bridges between the polar
lobe and the blastomeres. In Bithynia we found the same kind of cytoplasmic
bridges, but far less developed. As the polar lobe is never completely pinched
The polar lobe of Crepidula
427
off, it is not clear why there should be additional cytoplasmic bridges between
the lobe and the blastomeres. The formation of a polar lobe is a process which is
overtly similar to cytokinesis. In cleavage furrows surfacefolds radiating from the
line of the constriction (Bluemink, 1970; Bluemink & de Laat, 1973) or parallel
to it (Arnold, 1969,1974) may be caused by a contraction of bands of underlying
microfilaments, structures which are also present in polar lobes (Conrad et al.
1973). In Crepidula the surface folds of the cleavage furrow (Fig. 1) as well as
the folds on the polar lobe may be caused by the same mechanism, but for some
unknown reason the folds on the polar lobe are far more developed.
We thank Dr P. F. Elbers and his Biological Ultrastructure Research Unit for making
available the SEM facilities. The skilful help of Mr J. Pieters and Mr C. van der Meij in
preparing specimens and operating the SEM is gratefully acknowledged. We also wish to
thank Mr W. van Veenendaal for preparing the prints and Mr M. J. van Oosterum and
collaborators for collecting and taking care of the snails. We are grateful to Dr N. H. Verdonk
and Dr V. Labordus for reading and commenting upon the manuscript.
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{Received 26 March 1975, revised 4 June 1975)