Fine structure of egg envelopes and the activation changes of cortical alveoli in the river lamprey, Lampetra fluviatilis

According to Raven (1961) the egg membranes may be divided into ‘primary egg membranes’, formed in the ovary by the egg cell, ‘secondary egg mem-branes’ formed in the ovary by the follicle epithelium, and ‘tertiary egg mem-branes’ 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). Micro-scopically large bodies in the cortical layer of the egg are expelled and a new 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 defor-mation 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 re-action is scanty, although there are some papers dealing with the events in sea-urchin 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.

River lampreys, Lampetra fluviatilis, caught in the spawning season were obtained from the Royal Fishery Board, Älvkarleby, 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.

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 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 μ in width and have a spacing in the sections of about 0·5 μ. 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 Müller & 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 μ 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.

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 triple-layered 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 Å) than the membrane. The cortical alveoli may border directly on the plasma membrane, or be separated from it by a very narrow rim of cyto-plasm (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 peri-vitelline 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 invaginat-ing 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 mem-brane 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 ex-truded 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 ex-pected 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.

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 light- and 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 peri-vitelline 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 (Hum-phreys, 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 μ or more, while those of the other types mentioned above are 1 μ or less. The cortical granules of sea-urchins (Afzelius, 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.

1. The true egg envelopes of the river lamprey all have a fibrillar fine struc-ture. 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, electron-dense 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.

1. Les enveloppes proprement dites de l’œuf de la Lamproie fluviatile ont toutes une structure finement fibrillaire. Dans la couche la plus périphérique, la ‘touffe’, qui n’est présente qu’au niveau du pôle animal, les fibrilles sont très fines et ondulées et sont surtout orientées dans un sens radial. Certaines d’entre elles se prolongent dans les canaux du chorion externe.

2. Le chorion externe se caractérise par une grande quantité de petites parti-cules denses aux électrons qui sont éparpillées entre de fines fibres radiaires; elles sont plus nombreuses dans les couches externe et interne de ce chorion. Les larges pores disposés en canaux ne contiennent pas de particules mais bien quelques fibres longitudinales.

3. Le chorion interne est d’une texture plus dense, constituée par des fibres parallèles à la surface sauf au voisinage d’étroits et peu distincts canaux (pores) qui sont en continuité des canaux plus larges du chorion externe. Quelques résidus membraneux se voient dans toutes les couches.

4. Les alvéoles corticaux des œufs vierges ont un contenu homogène et une fine membrane crénelée qui apparaît distincte du plasmolemme à trois couches de l’œuf.

5. Dans les œufs fraîchement fécondés le contenu des alvéoles corticaux se retrouve expulsé de l’œuf et on en voit des restes gonflés dans l’espace péri-vitellin en formation. La membrane des alvéoles corticaux est à présent en continuité avec la membrane plasmatique et présente une structure quelque peu modifiée.