/ . Embryol exp. Morph. Vol. 19, 3, pp. 311-18, May 1968 3 1 1With 4 plates

Printed in Great Britain

Fine structure ofegg envelopes and the activation changes of corticalalveoli in the river lamprey, Lampetra fluviatilis

BJORN A. AFZELIUS,1 LENNART NICANDER1

& INGER SJODEN1

The Wenner-Gren Institute, University of Stockholm, and theDepartment of Anatomy and Histology and the Laboratory of Electron

Microscopy, Royal Veterinary College, Stockholm

Most eggs are surrounded by several prominent envelopes which have beengiven names depending on their origin, structure or chemical composition. Asour present knowledge of these envelopes is very fragmentary, the results ofattempts to homologize the different layers between different animal groups arestill open to debate. The nomenclature in this field is quite confusing.

According to Raven (1961) the.egg membranes may be divided into 'primaryegg 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 primaryegg membranes, although there is no certainty on this point. At least threedistinct layers can be distinguished in the egg envelope of this species. Commonto two of them is the presence of radial striations, which justifies the name'zona radiata'. In the present study a provisional terminology will be employedwhich makes use of the same names as have been employed for the trout, forinsects 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 implythat there is a basic similarity between the envelopes of the lamprey egg andthose of insect or fish eggs in either morphological, structural, or chemical terms.

The fertilization process is accompanied by several visible and invisiblechanges in the egg. Perhaps the most spectacular of these changes is the corticalreaction such as can be observed in eggs of echinoderms, teleosts, and lampreys.In these animals the activation of the egg at fertilization involves the expulsionof'cortical granules' (echinoderms) or 'cortical alveoli' (fish, lampreys). Micro-scopically 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 alveolito break down are those close to the fertilizing spermatozoon. Neighbouringones then disrupt in a sequence—the fertilization wave—which is reminiscentof an irregular chain reaction.

Accompanying this breakdown of the cortical granules, in sea-urchins there isa 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. Thiswave of contraction is observed in eggs of many animal species, and is notrestricted to those which have a visible cortical reaction. It is particularly promi-nent 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). Formore detailed accounts of cortical reactions the reader is referred to reviewarticles by Rothschild (1956), Runnstrom, Hagstrom & Perlmann (1959) andMonroy (1965). Reviews dealing primarily with these events in fish have beenpublished by Kusa (1956) and in fish and lampreys by Rothschild (1958) andYamamoto (1961). The size and shape of the lamprey egg, its easily identifiableanimal pole, and the presence of a long acrosomal filament in the penetratingspermatozoa make this species a favourable one for electron-microscope studieson fertilization. The purpose of the present investigation has been to gain furtherinsight into the ultrastructure of the egg envelopes and of the cortex of thelamprey egg during fertilization.

MATERIAL AND METHODS

River lampreys, Lampetra fluviatilis, caught in the spawning season wereobtained from the Royal Fishery Board, Alvkarleby, Sweden. The authorsgratefully acknowledge the aid of Mr N. Steffner in providing us with theanimals. The eggs were stripped into porcelain jars containing river water, spermwas added and samples for fixation taken after \, \, 1 and 2 min. The eggs ofthis study were fixed in 3 % glutaraldehyde in cacodylate buffer for 4 h followedby 2 % osmium tetroxide in phosphate buffer for 1-5 h (Sabatini, Bensch &Barrnett, 1963). Dehydration was performed with ethanol and embedding wasin Epon. Sections for light microscopy were stained with 0-2 % toluidine blueor with the PAS technique. Sections for electron microscopy were contrasted withlead acetate, followed by uranyl acetate for 20 min at 30 °C. A Siemens ElmiskopI 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 eitherfrom light microscopy of stained sections or from electron microscopy. Frominside, 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 chorionover other areas of the egg (Plate 2, fig. A).

The inner chorion is recognized with the light microscope as the layer stainingmost intensely with toluidine blue. With the PAS technique both this and theother envelopes are unstained. With the electron microscope the inner chorionis seen to be the most homogeneous of the three layers (Plate 1). It is perforatedby radial pore canals, however, which are about 0-1 fi in width and have aspacing in the sections of about 0-5 /u,. The canals appear empty. At highermagnifications and after intense staining the substance of the inner chorion isseen to be finely fibrillar (Plate 2, fig. D). The majority of the fibrils run parallelto the egg surface but there are indications that fibrils close to the pore canalsare parallel to the latter, and those at a small distance from the pore canalsdeviate at increasingly greater angles from them. This organization thus leads toa fan-shaped pattern similar to that described by Miiller & Sterba (1963) in thechorion of bony fishes and by Bouligand (1965) in a variety of biologicalstructures.

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 verydense, finely granular material scattered among the fine fibrils except in the porecanals (Plate 1). These fibrils have about the same dimensions as those of theinner chorion, but their predominating orientation seems to be in the radialdirection, and they seem to be wavy. Dense streaks are often found in the outerchorion both towards the exterior and towards the inner chorion. Evidentlythese streaks contain more granular material than the rest of the chorion andhence are more electron-dense. The radial pore canals from the inner chorioncontinue through the outer chorion (Plate 2, fig. C), where they are much widerand contain fibres and sometimes what appear to be membrane remnants (Plate2, fig. B).

The tuft or the 'animal tuft' is an incomplete layer over the outer chorioncovering the egg only over the animal pole. It has an irregular configurationwhich may indicate that it undergoes swelling and deformation in water. In theelectron microscope it is distinguished from the underlying chorion by itselectron density, which is distinctly lower than that of the outer chorion butapproximately equal to that of the inner chorion. The tuft appears to containthin, wavy, radial fibrils continuous with those in the pore canals of the outerchorion, but the structural organization is not distinct (Plate 1). It also containssome groups of small, spherical granules and a few patches of membraneremnants.

The periphery of the unfertilized egg contains a single layer of prominentbodies, the cortical alveoli. They are 5-12 /i in diameter and stain intenselyboth with the PAS-technique and with toluidine blue. Although the great majorityof these cortical alveoli border on the egg surface, some bodies with similarstaining 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 foundto have homogeneous contents irrespective of whether only osmium tetroxideor glutaraldehyde followed by osmium tetroxide had been used (Plate 3). Mostof the cortical alveoli within a given egg have the same density but some are lesselectron-dense than the others. It is evident from both light and electron micro-scopy that the cortical alveoli form a single layer around the entire egg with theexception 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 characteristicallythrown into numerous small wrinkles, which at places give the impression thatit has two separate layers (Plate 3, fig. B, to the left). This appearance is due tothe 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 onthe 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 openedtowards the exterior and expelled part of its contents.

The cortex of the fertilized egg has a different appearance. The corticalalveoli have opened up and their contents have been expelled into the peri-vitelline space. The positions of the ruptured cortical alveoli are still visible2 min after the addition of spermatozoa, because numerous small pockets, aboutthe 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 timeof discharge the membrane of the cortical alveoli becomes continuous with theplasma membrane of the egg. However, the pocket membrane is smooth andshows none of the minute wrinkles so characteristic of the undischarged corticalalveoli, 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 cortexwith ruptured cortical alveoli, forming the so-called perivitelline space betweenthe 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 finelygranular 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 contentsof unruptured cortical alveoli, and it seems most likely that they have undergoneswelling.

There are no fibrillar bands under the cortical alveoli of the unfertilized eggor 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 theband of contraction which passes around the egg on activation. On the contrary,these regions consist of a mixture of yolk granules, mitochondria and vacuolesin 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 theanimal pole of the lamprey egg orient the approaching spermatozoa normallyto the chorion over the animal pole. It is otherwise difficult to assign a specificfunction 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 theegg envelopes, except that the acrosome filament and head appear to penetratethrough the pore canals to the egg plasma membrane. The canals are presumablyformed by pseudopodial connexions between the oocyte and the follicle cells, asevidenced by the membranous remnants in them.

The above description of the disappearance of the cortical alveoli from thelamprey egg at fertilization conforms to descriptions made by light-microscopistssuch as Herfort (1901), Okkelberg (1914), Montalenti (1936), Yamamoto (1944,1961) and Kille (1960). The process also shows some resemblances to the explo-sion 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 andthe egg membrane makes the membrane of the alveoli part of the egg surfacemembrane. Another similarity is the relationship between the discharge of thecortical alveoli and the separation of the chorion from the egg surface. Theswollen remnants of the alveolar contents seen in the perivitelline space supportthe opinion of Yamamoto (1961) and others that the contents of the corticalalveoli bring about the separation of the chorion from the egg surface by raisingthe osmotic pressure of the perivitelline fluid. This results in water passingthrough the chorion, and perhaps also from the egg cytoplasm, into the peri-vitelline space.

Accompanying the discharge of the cortical alveoli and separation of theegg membrane from the surface of the egg, a wave of contraction passes overthe surface of many eggs. This wave of contraction is particularly striking in thelamprey and, according to Okkelberg (1914), leads to a 13 % reduction in thevolume of the egg. Since no fine fibrils were found beneath its egg surface, itseems likely that the deformation and contraction of the egg are due to thedischarge of the alveolar contents, and perhaps, as mentioned above, a loss ofwater 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 offish, lampreys and frogs may be 5 /i or more, while those of the other typesmentioned 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 finestructure, while those of the other species mentioned are homogeneous. Theyall closely resemble one another in chemical composition and function. Thecortical granules of all the animals mentioned contain mucopolysaccharide, asconfirmed for lampreys by Kusa (1957). In part at least, they all perform muchthe same function when the egg is activated. Their contents are discharged fromthe egg surface into the perivitelline space, which leads to an influx of water andthe separation of the egg membrane from the cytoplasmic surface. The corticalgranules 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 struc-ture. In the outermost layer, the 'tuft', which is present only over the animalpole, the fibrils are very fine and wavy and run a mainly radial course. Someof 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 theouter and inner borders of the layer. The wide pore canals lack particles butcontain some longitudinal fibrils.

3. The inner chorion has a denser texture of fibrils running parallel to theegg surface except in the vicinity of the narrow, indistinct pore canals, whichare continuous with the wider ones in the outer chorion. Some membranousremnants are seen in all layers.

4. The cortical alveoli of unfertilized eggs have homogeneous contents and athin, wrinkled membrane appearing different from the triple-layered plasmamembrane of the egg.

5. In the newly fertilized egg the contents of the cortical alveoli are foundto be expelled from the egg into the forming perivitelline space. The membraneof the cortical alveoli is continuous with the rest of the plasma membrane andshows a slightly modified structure.

RESUME

Structure fine des enveloppes ovulaires etrevolution 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 onttoutes 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 tresfines et ondulees et sont surtout orientees dans un sens radial. Certaines d'entreelles 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 bienquelques fibres longitudinales.

3. Le chorion interne est d'une texture plus dense, constituee par des fibresparalleles a la surface sauf au voisinage d'etroits et peu distincts canaux (pores)qui sont en continuity des canaux plus larges du chorion externe. Quelquesresidus membraneux se voient dans toutes les couches.

4. Les alveoles corticaux des oeufs vierges ont un contenu homogene et unefine membrane crenelee qui apparait distincte du plasmolemme a trois couchesde l'oeuf.

5. Dans les oeufs fraichement fecondes le contenu des alveoles corticaux seretrouve expulse de l'oeuf et on en voit des restes gonfles dans l'espace peri-vitellin en formation. La membrane des alveoles corticaux est a present encontinuity avec la membrane plasmatique et presente une structure quelquepeu modifiee.

REFERENCES

AFZELIUS, B. A. (1956). The ultrastructure of the cortical granules and their products in thesea urchin egg as studied with the electron microscope. Expl Cell Res. 10, 257.

AUSTIN, C. R. (1956). Cortical granules in hamster eggs. Expl Cell Res. 10, 533.BAXANDALL, J. (1966). The surface reactions associated with immuno-electron methods.

J. Ultrastmct. Res. 16, 158.BOULIGAND, Y. (1965). Sur une disposition fibrillaire torsadee commune a plusieurs structures

biologiques. C. r. hebd. Seanc. Acad. Sci., Paris 261, 4864.HERFORT, K. (1901). Die Reifung und Befruchtung des Eies von Petromyzon fluviatilis.

Arch, mikrosk. Anat. EntwMech. 57, 54.HUMPHREYS, W. J. (1967). The fine structure of cortical granules in eggs and gastrulae of

Mytilus edulis. J. Ultrastmct. Res. 17, 314.KILLE, R. A. (1960). Fertilization of the lamprey egg. Expl Cell Res. 20, 12.KUSA, M. (1956). Studies on cortical alveoli in some teleostean eggs. Embryologia 3, 105.KUSA, M. (1957). Occurrence of a neutral mucopolysaccharide in the cortical alveoli of

lamprey eggs. / . Fac. Sci. Hokkaido Univ. (Ser. VI), 13, 455.LILLIE, F. R. (1911). Studies of fertilization in Nereis. I. The cortical changes in the egg.

/ . Morph. 22, 361.MONROY, A. (1965). Chemistry and Physiology of Fertilization. New York: Holt, Rinehart

and Winston.MONTALENTI, G. (1936). Analisi citologica della fecondazione e della attivazione artificiale

della uova di lampreda. Mem. R. Accad. Ital. (Classe di Sci. Fis. Mat. Nat.), 7, 191.MOTOMURA, I. (1952). Cortical granules in the egg of the frog. Annotnes zool. jap. 25, 238.MULLER, H. & STERBA, G. (1963). Elektronenmikroskopische Untersuchungen iiber Bildung

und Struktur der Eihullen bei Knochenfischen. II. Die Eihiillen jiingerer und alterer Oozytenvon Cynolebias belotti Steindachner. Zool. Jb. Anat. 80,469.

OKKELBERG, P. (1914). Volumetric changes in the egg of the brook lamprey Entosphenus(Lampetra) wilderi (Gage) after fertilization. Biol. Bull. mar. biol. Lab., Woods Hole 26, 92.

PASTEELS, J. J. (1966). Les mouvements corticaux de l'oeuf de Barnea Candida (MollusqueBivalve) 6tudie au microscope electronique. / . Embryol. exp. Morph. 16, 311.

PASTEELS, J. J. & DE HARVEN, E. (1962). Etude au microscope electronique du cortex del'ceuf de Barnea Candida (Mollusque Bivalve), et son evolution au moment de la feconda-tion, de la maturation et de la segmentation. Archs Biol., Liege, 73, 465.

318 B. A. AFZELIUS, L. NICANDER & I. SJODEN

RAVEN, C. P. (1961). Oogenesis: the Storage of Developmental Information. Oxford: PergamonPress.

ROTHSCHILD, LORD. (1956). Fertilization. London, Methuen and Co. Ltd.ROTHSCHILD, LORD. (1958). Fertilization in fish and lampreys. Biol. Rev. 33, 372.RUNNSTROM, J. (1966). The vitelline membrane and cortical particles in sea urchin eggs and

their function in maturation and fertilization. Adv. Morphogen. 5, 221.RUNNSTROM, J., HAGSTROM, B. E. & PERLMANN, P. (1959). Fertilization. In The Cell, ed.

J. Brachet and A. Mirsky. New York: Academic Press Inc.SABATINI, D. D., BENSCH, K. & BARRNETT, R. J. (1963). Cytochemistry and electron micro-

scopy. The preservation of cellular ultrastructure and enzymatic activity by aldehydefixation. J. Cell Biol. 17, 19.

SzoLLOsr, D. (1962). Cortical granules: a general feature of mammalian eggs? / . Reprod.Fert. 4, 223.

YAMAMOTO, T. (1944). On the excitation-conduction gradient in the unfertilized egg of thelamprey, Lampetra planeri. Proc. imp. Acad. Japan 20, 30.

YAMAMOTO, T. (1961). Physiology of fertilization in fish eggs. Int. Rev. Cytol. 12, 361.

{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 thetuft (t) with some granules (g) is visible. The outer chorion (och) shows many slightly obliquesections 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 surfaceof 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 filamentsmainly 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 theplasma membrane and the highly wrinkled alveolar membrane (arrows). Note the homo-geneous 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 probablybeen formed by swelling of the extruded contents of cortical alveoli. The surface depressionbetween the arrows may represent an empty cortical alveolus, ly, Lipid yolk; ich, innerchorion.

B. A. AFZELIUS, L. NICANDER & I. SJODEN