/. Embryol exp. Morph. Vol. 19, 3, pp. 311-18, May 1968
With 4 plates
Printed in Great Britain
311
Fine structure of
egg envelopes and the activation changes of cortical
alveoli in the river lamprey, Lampetra fluviatilis
BJORN A. AFZELIUS,1 LENNART NICANDER 1
& INGER SJODEN1
The Wenner-Gren Institute, University of Stockholm, and the
Department of Anatomy and Histology and the Laboratory of Electron
Microscopy, Royal Veterinary College, Stockholm
Most eggs are surrounded by several prominent envelopes which have been
given names depending on their origin, structure or chemical composition. As
our present knowledge of these envelopes is very fragmentary, the results of
attempts to homologize the different layers between different animal groups are
still open to debate. The nomenclature in this field is quite confusing.
According to Raven (1961) the.egg membranes may be divided into 'primary
egg membranes', formed in the ovary by the egg cell, 'secondary egg membranes ' formed in the ovary by the follicle epithelium, and ' tertiary egg membranes' formed in the genital ducts after ovulation.
The egg envelopes in the river lamprey, as in fish, are supposed to be primary
egg membranes, although there is no certainty on this point. At least three
distinct layers can be distinguished in the egg envelope of this species. Common
to two of them is the presence of radial striations, which justifies the name
'zona radiata'. In the present study a provisional terminology will be employed
which makes use of the same names as have been employed for the trout, for
insects and for some other animal groups and have also been used by Kille
(1960) in a study of the lamprey egg. The choice of these terms does not imply
that there is a basic similarity between the envelopes of the lamprey egg and
those of insect or fish eggs in either morphological, structural, or chemical terms.
The fertilization process is accompanied by several visible and invisible
changes in the egg. Perhaps the most spectacular of these changes is the cortical
reaction such as can be observed in eggs of echinoderms, teleosts, and lampreys.
In these animals the activation of the egg at fertilization involves the expulsion
of'cortical granules' (echinoderms) or 'cortical alveoli' (fish, lampreys). Microscopically large bodies in the cortical layer of the egg are expelled and a new
1
Authors' address: Wenner-Grenn Institute, Stockholm 23, Sweden.
312
B. A. AFZELIUS, L. NICANDER & I. SJODEN
surface is formed in a few minutes. The first cortical granules or cortical alveoli
to break down are those close to the fertilizing spermatozoon. Neighbouring
ones then disrupt in a sequence—the fertilization wave—which is reminiscent
of an irregular chain reaction.
Accompanying this breakdown of the cortical granules, in sea-urchins there is
a separation of the vitelline membrane from the egg surface, followed by deformation of the egg by a wave of contraction which passes over its surface. This
wave of contraction is observed in eggs of many animal species, and is not
restricted to those which have a visible cortical reaction. It is particularly prominent in the lamprey egg.
Information on the ultrastructural changes responsible for the cortical reaction is scanty, although there are some papers dealing with the events in seaurchin fertilization (Afzelius, 1956; Baxandall, 1966; Runnstrom, 1966). For
more detailed accounts of cortical reactions the reader is referred to review
articles by Rothschild (1956), Runnstrom, Hagstrom & Perlmann (1959) and
Monroy (1965). Reviews dealing primarily with these events in fish have been
published by Kusa (1956) and in fish and lampreys by Rothschild (1958) and
Yamamoto (1961). The size and shape of the lamprey egg, its easily identifiable
animal pole, and the presence of a long acrosomal filament in the penetrating
spermatozoa make this species a favourable one for electron-microscope studies
on fertilization. The purpose of the present investigation has been to gain further
insight into the ultrastructure of the egg envelopes and of the cortex of the
lamprey egg during fertilization.
MATERIAL AND METHODS
River lampreys, Lampetra fluviatilis, caught in the spawning season were
obtained from the Royal Fishery Board, Alvkarleby, Sweden. The authors
gratefully acknowledge the aid of Mr N. Steffner in providing us with the
animals. The eggs were stripped into porcelain jars containing river water, sperm
was added and samples for fixation taken after \, \, 1 and 2 min. The eggs of
this study were fixed in 3 % glutaraldehyde in cacodylate buffer for 4 h followed
by 2 % osmium tetroxide in phosphate buffer for 1-5 h (Sabatini, Bensch &
Barrnett, 1963). Dehydration was performed with ethanol and embedding was
in Epon. Sections for light microscopy were stained with 0-2 % toluidine blue
or with the PAS technique. Sections for electron microscopy were contrasted with
lead acetate, followed by uranyl acetate for 20 min at 30 °C. A Siemens Elmiskop
I was used at 60 kV and with primary magnifications between 1500 and 20000.
RESULTS
The envelopes of the lamprey egg consist of distinct layers, as is evident either
from light microscopy of stained sections or from electron microscopy. From
inside, these layers are the inner chorion, the outer chorion and, over the animal
Lamprey egg envelopes
313
pole, the tuft. There are also irregular strands of material outside the chorion
over other areas of the egg (Plate 2, fig. A).
The inner chorion is recognized with the light microscope as the layer staining
most intensely with toluidine blue. With the PAS technique both this and the
other envelopes are unstained. With the electron microscope the inner chorion
is seen to be the most homogeneous of the three layers (Plate 1). It is perforated
by radial pore canals, however, which are about 0-1 fi in width and have a
spacing in the sections of about 0-5 /u,. The canals appear empty. At higher
magnifications and after intense staining the substance of the inner chorion is
seen to be finely fibrillar (Plate 2, fig. D). The majority of the fibrils run parallel
to the egg surface but there are indications that fibrils close to the pore canals
are parallel to the latter, and those at a small distance from the pore canals
deviate at increasingly greater angles from them. This organization thus leads to
a fan-shaped pattern similar to that described by Miiller & Sterba (1963) in the
chorion of bony fishes and by Bouligand (1965) in a variety of biological
structures.
The outer chorion is the most heavily staining layer of the surrounding coat.
The greater density is apparently due to the presence of large amounts of very
dense, finely granular material scattered among the fine fibrils except in the pore
canals (Plate 1). These fibrils have about the same dimensions as those of the
inner chorion, but their predominating orientation seems to be in the radial
direction, and they seem to be wavy. Dense streaks are often found in the outer
chorion both towards the exterior and towards the inner chorion. Evidently
these streaks contain more granular material than the rest of the chorion and
hence are more electron-dense. The radial pore canals from the inner chorion
continue through the outer chorion (Plate 2, fig. C), where they are much wider
and contain fibres and sometimes what appear to be membrane remnants (Plate
2, fig. B).
The tuft or the 'animal tuft' is an incomplete layer over the outer chorion
covering the egg only over the animal pole. It has an irregular configuration
which may indicate that it undergoes swelling and deformation in water. In the
electron microscope it is distinguished from the underlying chorion by its
electron density, which is distinctly lower than that of the outer chorion but
approximately equal to that of the inner chorion. The tuft appears to contain
thin, wavy, radial fibrils continuous with those in the pore canals of the outer
chorion, but the structural organization is not distinct (Plate 1). It also contains
some groups of small, spherical granules and a few patches of membrane
remnants.
The periphery of the unfertilized egg contains a single layer of prominent
bodies, the cortical alveoli. They are 5-12 /i in diameter and stain intensely
both with the PAS-technique and with toluidine blue. Although the great majority
of these cortical alveoli border on the egg surface, some bodies with similar
staining properties can be found deeper in the egg cytoplasm.
314
B. A. AFZELIUS, L. NICANDER & I. SJODEN
When observed under the electron microscope the cortical alveoli were found
to have homogeneous contents irrespective of whether only osmium tetroxide
or glutaraldehyde followed by osmium tetroxide had been used (Plate 3). Most
of the cortical alveoli within a given egg have the same density but some are less
electron-dense than the others. It is evident from both light and electron microscopy that the cortical alveoli form a single layer around the entire egg with the
exception of the animal pole.
The membrane of the cortical alveoli appears to be thinner than the triplelayered plasma membrane of the egg (Plate 3, fig. B). It is also characteristically
thrown into numerous small wrinkles, which at places give the impression that
it has two separate layers (Plate 3, fig. B, to the left). This appearance is due to
the fact that the membrane is cut obliquely in a section, which is much thicker
(about 800 A) than the membrane. The cortical alveoli may border directly on
the plasma membrane, or be separated from it by a very narrow rim of cytoplasm (Plate 3, fig. A). Occasionally a cortical alveolus seems to have opened
towards the exterior and expelled part of its contents.
The cortex of the fertilized egg has a different appearance. The cortical
alveoli have opened up and their contents have been expelled into the perivitelline space. The positions of the ruptured cortical alveoli are still visible
2 min after the addition of spermatozoa, because numerous small pockets, about
the same shape and size as undischarged cortical alveoli, can be seen invaginating at the egg surface (Plate 4). These pockets strongly suggest that at the time
of discharge the membrane of the cortical alveoli becomes continuous with the
plasma membrane of the egg. However, the pocket membrane is smooth and
shows none of the minute wrinkles so characteristic of the undischarged cortical
alveoli, and its dimensions are more in agreement with those of the egg membrane than the alveolar membrane.
The egg periphery has separated from the chorion in sectors of the cortex
with ruptured cortical alveoli, forming the so-called perivitelline space between
the egg surface and the chorion. In this perivitelline space remnants of the extruded contents of cortical alveoli can be seen as rounded masses of a finely
granular substance trapped between egg cell and chorion. Characteristically,
these masses are more voluminous than the pockets from which, presumably,
they have been expelled (Plate 4). They also appear looser than the contents
of unruptured cortical alveoli, and it seems most likely that they have undergone
swelling.
There are no fibrillar bands under the cortical alveoli of the unfertilized egg
or under the cell membrane pockets of fertilized eggs, as might have been expected from some theories which have been put forward to account for the
band of contraction which passes around the egg on activation. On the contrary,
these regions consist of a mixture of yolk granules, mitochondria and vacuoles
in what seems to be a low state of organization.
Lamprey egg envelopes
315
DISCUSSION
Kille (1960) has suggested that the radially directed fibres of the tuft over the
animal pole of the lamprey egg orient the approaching spermatozoa normally
to the chorion over the animal pole. It is otherwise difficult to assign a specific
function to any of the different structures of the envelopes of the lamprey egg.
Events during fertilization, as will be described in this and a subsequent paper,
have not been informative with respect to the roles of the structures within the
egg envelopes, except that the acrosome filament and head appear to penetrate
through the pore canals to the egg plasma membrane. The canals are presumably
formed by pseudopodial connexions between the oocyte and the follicle cells, as
evidenced by the membranous remnants in them.
The above description of the disappearance of the cortical alveoli from the
lamprey egg at fertilization conforms to descriptions made by light-microscopists
such as Herfort (1901), Okkelberg (1914), Montalenti (1936), Yamamoto (1944,
1961) and Kille (1960). The process also shows some resemblances to the explosion of the cortical granules of sea-urchin eggs, as described by several lightand electron-microscopists (see Introduction), and several other species (starfish,
Monroy, 1965; polychaetes, Lillie, 1911; Rothschild, 1958; Yamamoto, 1961).
For example, the apparent fusion of the membrane of the cortical alveoli and
the egg membrane makes the membrane of the alveoli part of the egg surface
membrane. Another similarity is the relationship between the discharge of the
cortical alveoli and the separation of the chorion from the egg surface. The
swollen remnants of the alveolar contents seen in the perivitelline space support
the opinion of Yamamoto (1961) and others that the contents of the cortical
alveoli bring about the separation of the chorion from the egg surface by raising
the osmotic pressure of the perivitelline fluid. This results in water passing
through the chorion, and perhaps also from the egg cytoplasm, into the perivitelline space.
Accompanying the discharge of the cortical alveoli and separation of the
egg membrane from the surface of the egg, a wave of contraction passes over
the surface of many eggs. This wave of contraction is particularly striking in the
lamprey and, according to Okkelberg (1914), leads to a 13 % reduction in the
volume of the egg. Since no fine fibrils were found beneath its egg surface, it
seems likely that the deformation and contraction of the egg are due to the
discharge of the alveolar contents, and perhaps, as mentioned above, a loss of
water to the perivitelline space.
There is no good reason for making a distinction between 'cortical alveoli'
of fish and lampreys, and ' cortical granules' of echinoderms, mussels (Humphreys, 1967), mammals (Austin, 1956; Szollosi, 1962) and frogs (Motomura,
1952). These two kinds of cortical body overlap in size and structure. Those of
fish, lampreys and frogs may be 5 /i or more, while those of the other types
mentioned above are 1 /i or less. The cortical granules of sea-urchins (Afzelius,
21
J EEM 19
316
B. A. AFZELIUS, L. NICANDER & I. SJODEN
1956; Baxandall, 1966) and Mytilus (Humphreys, 1967) have a complex fine
structure, while those of the other species mentioned are homogeneous. They
all closely resemble one another in chemical composition and function. The
cortical granules of all the animals mentioned contain mucopolysaccharide, as
confirmed for lampreys by Kusa (1957). In part at least, they all perform much
the same function when the egg is activated. Their contents are discharged from
the egg surface into the perivitelline space, which leads to an influx of water and
the separation of the egg membrane from the cytoplasmic surface. The cortical
granules of Mytilus differ from the others as, according to Humphreys (1967),
they are not discharged from the egg surface.
SUMMARY
1. The true egg envelopes of the river lamprey all have a fibrillar fine structure. In the outermost layer, the 'tuft', which is present only over the animal
pole, the fibrils are very fine and wavy and run a mainly radial course. Some
of them continue into the pore canals of the outer chorion.
2. The outer chorion is characterized by large numbers of small, electrondense particles scattered among fine, radial fibres and most numerous near the
outer and inner borders of the layer. The wide pore canals lack particles but
contain some longitudinal fibrils.
3. The inner chorion has a denser texture of fibrils running parallel to the
egg surface except in the vicinity of the narrow, indistinct pore canals, which
are continuous with the wider ones in the outer chorion. Some membranous
remnants are seen in all layers.
4. The cortical alveoli of unfertilized eggs have homogeneous contents and a
thin, wrinkled membrane appearing different from the triple-layered plasma
membrane of the egg.
5. In the newly fertilized egg the contents of the cortical alveoli are found
to be expelled from the egg into the forming perivitelline space. The membrane
of the cortical alveoli is continuous with the rest of the plasma membrane and
shows a slightly modified structure.
RESUME
Structure fine des enveloppes ovulaires et
revolution des alveoles corticaux a la suite de Vactivation chez
la Lamproie de riviere, Lampetra fluviatilis.
1. Les enveloppes proprement dites de l'oeuf de la Lamproie fluviatile ont
toutes une structure finement fibrillaire. Dans la couche la plus peripherique, la
'touffe', qui n'est presents qu'au niveau du pole animal, les fibrilles sont tres
fines et ondulees et sont surtout orientees dans un sens radial. Certaines d'entre
elles se prolongent dans les canaux du chorion externe.
2. Le chorion externe se caracterise par une grande quantite de petites parti-
Lamprey egg envelopes
317
cules denses aux electrons qui sont eparpillees entre de fines fibres radiaires;
elles sont plus nombreuses dans les couches externe et interne de ce chorion.
Les larges pores disposes en canaux ne contiennent pas de particules mais bien
quelques fibres longitudinales.
3. Le chorion interne est d'une texture plus dense, constituee par des fibres
paralleles a la surface sauf au voisinage d'etroits et peu distincts canaux (pores)
qui sont en continuity des canaux plus larges du chorion externe. Quelques
residus membraneux se voient dans toutes les couches.
4. Les alveoles corticaux des oeufs vierges ont un contenu homogene et une
fine membrane crenelee qui apparait distincte du plasmolemme a trois couches
de l'oeuf.
5. Dans les oeufs fraichement fecondes le contenu des alveoles corticaux se
retrouve expulse de l'oeuf et on en voit des restes gonfles dans l'espace perivitellin en formation. La membrane des alveoles corticaux est a present en
continuity avec la membrane plasmatique et presente une structure quelque
peu modifiee.
REFERENCES
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AUSTIN, C. R. (1956). Cortical granules in hamster eggs. Expl Cell Res. 10, 533.
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{Manuscript received 3 July 1967, revised 30 November 1967)
/. Embryol. exp. Morph., Vol. 19, Part 3
PLATE 1
Section cut radially to the envelopes at the animal pole. Only the innermost layer of the
tuft (t) with some granules (g) is visible. The outer chorion (och) shows many slightly oblique
sections of pore canals (pc) with radial filaments, and also dense, granular material (dm)
accumulated near the inner border and membranous remnants (m). The inner chorion (ich)
shows only a faint radial striation caused by the indistinct pore canals leading to the surface
of the egg (e).
B. A. AFZELIUS, L. NICANDER & I. SJODEN
facing p. 318
J. Embryol. exp. Morph., Vol. 19, Part 3
PLATE 2
B
Fig. A. Except for the 'tuft' area the outer chorion (och) is covered by some strands (s)
of moderately electron-dense material with ill-defined structure.
Fig. B. Part of outer chorion with fibrils (/) and membranous remnants (m) in a pore canal,
and much granular material.
Fig. C. Markedly oblique section of the outer chorion (och) to show pore canals (arrows).
/, Tuft; ich, inner chorion.
Fig. D. Radial section of part of the inner chorion to show the fine, densely packed filaments
mainly oriented parallel to the egg surface.
B. A. AFZELIUS, L. NICANDER & I. SJODEN
/. Embryo/, exp. Morph., Vol. 19, Part 3
PLATE 3
Fig. A. Survey of egg cortex, with cortical alveoli (ca), protein yolk (py) and lipid yolk (ly),
villi (v) at the surface, and part of inner chorion (ich).
Fig. B. High magnification showing a depression of the egg surface with juxtaposition of the
plasma membrane and the highly wrinkled alveolar membrane (arrows). Note the homogeneous character of the alveolar contents (ca). v, Villi.
B. A. AFZELIUS, L. NICANDER & I. SJODEN
/. Embryol. exp. Morph., Vol. 19, Part 3
PLATE 4
Egg cortex near the periphery of the animal pole, just after the start of the cortical reaction.
The wide perivitelline space (pvs) contains two granular masses (x), which have probably
been formed by swelling of the extruded contents of cortical alveoli. The surface depression
between the arrows may represent an empty cortical alveolus, ly, Lipid yolk; ich, inner
chorion.
B. A. AFZELIUS, L. NICANDER & I. SJODEN