The formation of the second maturation spind le in the ...dev.biologists.org/content/develop/12/4/805.full.pdfThe formation of the second maturation spind le in the eggs of various

J. Embryol. exp. Morph., Vol. 12, Part 4, pp. 805-823, December 1964Printed in Great Britain

The formation of the second maturation spind lein the eggs of various Limnaeidae

by CHR. P. RAVEN1

From the Zoological Laboratory, University of Utrecht

WITH TWO PLATES

I N a number of papers (Raven, 1949; Raven et al, 1958; Raven, 1959) I havereported on egg maturation, especially the formation of the second maturationspindle, in various pulmonate snails. It appeared that this early and quitefundamental process of development exhibits a surprising variation.

In broad outline, three main modes of formation of the second maturationspindle can be distinguished among the species studied up to the present. Thefirst of these can be observed most clearly in the eggs of Agriolimax reticulatusand Limaxflavus. After the extrusion of the first polar body, the centrosphere ofthe inner aster of the first maturation spindle contains a pair of dark bodies(' centrioles') which move apart to opposite poles of the centrosphere. The latterelongates and is directly transformed into the second maturation spindle. Itdevelops an aster at both ends, taking their origin from the 'centrioles'. Thedyads lie at first as a compact group on one side against the equatorial region ofthe developing spindle. They penetrate into the spindle when the latter has beencompletely formed (Raven et al, 1958).

In Physa acuta the second maturation spindle is formed in a similar way. InSuccineaputris, the process shows some complications but presumably it belongsto the same category (Raven, 1959).

The second type is found in Limnaea stagnalis. Here the deep centrosphere ofthe first maturation amphiaster contains only one 'centriole', which remainsundivided. When the centrosphere elongates after the extrusion of the firstpolar body, the 'centriole' moves as a whole to its outer pole. The centrosphereis then transformed to a spindle, which is asymmetric and egg-shaped. An asteris formed at its pointed outer end, starting from the' centriole' lying at this place.The blunt inner end of the spindle fuses secondarily with the sperm aster, whichbecomes the inner aster of the second maturation spindle. The dyads moveasunder into an irregular ring, sliding along the surface of the transforming

1 Author's address: Zoologisch Laboratorium der Rijksuniversiteit, Janskerkhof 3, Utrecht,Netherlands.

806 CHR.P.RAVEN

centrosphere until they have reached the equatorial region of the spindle; thenthey penetrate into the latter (Raven et ah, 1958).

The third mode of formation of the second maturation spindle has beenobserved in Planorbis planorbis and PL comeus. Here, as in Limnaea, the ' cen-triole' of the inner aster of the first maturation spindle remains undivided.After the extrusion of the first polar body, this aster flattens against the surface.Without great changes it becomes the outer aster of the second maturationspindle. The latter's inner aster is again provided by the sperm aster, which inthese species often reduplicates. The second maturation spindle proper is notformed by transformation of a centrosphere, but by the 'spinning' of proto-plasmic fibres between the 'centriole' of the maturation aster and one or both'centrioles' of the sperm aster. The dyads, first forming a narrow ring aroundthe centre of the maturation aster, then move centrifugally along its astral rays,and are conveyed by the latter bending inwards toward the outer surface of thesecond maturation spindle, into which they then penetrate (Raven, 1959).

It is evident that there are fundamental differences in the course of the secondmaturation division among the pulmonates. Representatives of even ratherclosely related families of snails, as the Limnaeidae, Planorbidae and Physidae,may differ markedly in this respect. In this connexion it is of some interest tostudy to what extent this variability respects the boundaries between taxonomicgroups. In other words, we may ask whether the variations within a same family,or within a genus, are of a lower order of magnitude than those between families.

To this end, five species belonging to the family Limnaeidae have been studied:Limnaea peregra (Mull.), L. ovata (Drap.), L. palustris (Miill.), L. stagnalis (L.)and Myxas glutinosa (Miill.). Of these, L. stagnalis has already been dealt within a previous paper (Raven et ah, 1958).

MATERIAL AND METHODS

The eggs of Limnaea palustris, L. ovata and Myxas glutinosa were from snailscaught in the neighbourhood of Utrecht. Those of Limnaea peregra were froma stock of animals of Scottish origin received from the Institute of AnimalGenetics in Edinburgh, and cultured for some time in the laboratory. (I amgreatly indebted to Prof. C. H. Waddington in Edinburgh for his kindness insending these snails.)

In order to obtain eggs, the snails were, as a rule, stimulated to oviposit byraising the water temperature. A freshly laid egg-mass was observed under amicroscope, and the time of formation of the first polar body in about half theeggs was noted. This was taken as zero time, and all subsequent observationswere related to this moment. Batches of eggs were fixed at regular intervalsduring the second maturation division. The age of the eggs provides only arough estimate of their stage of development, however, owing to considerablevariations among eggs of the same batch.

Egg maturation in various Limnaeidae 807

The eggs of all species were fixed in Bouin's fluid, sectioned at 5 or 7/JL, andstained with iron haematoxylin and erythrosin.

The following numbers of eggs have been studied in sections: L. peregra, 122;L. ovata, 100; L. palustris, 115; Myxas glutinosa, 50.

RESULTS

1. Limnaea peregra

The eggs of Limnaea peregra are very suitable for a study of the maturationdivisions, giving images of great clarity. The formation of the second maturationspindle in this species takes place in a quite simple and straightforward waywhich is easy to follow by a study of consecutive stages.

At anaphase of the first maturation division, the maturation spindle is providedwith an aster at both ends. The outer aster is flattened against the surface of theanimal pole. The inner aster has a moderate to large, more or less sphericalcentrosphere. The latter is clear and rather homogeneous in appearance; itconsists of a tenuous reticulum of delicate fibrils, and contains a number ofdispersed mitochondria. There is, moreover, a dense cloud of mitochondria allaround the spindle and asters. The mitochondria penetrate in rows betweenthe astral rays. Part of them are further accumulated in dense layers beneath thecortex in the neighbourhood of the animal pole, where a distinct animal poleplasm has already been formed at this stage.

When the formation of the first polar body is initiated by the appearance of aconical projection at the animal pole, a sudden and very rapid transformation ofthe centrosphere of the deep maturation aster takes place. It enlarges con-siderably and elongates perpendicularly to the surface (Plate 1, Fig. A). At thesame time small vacuoles appear in it, which rapidly grow in size, so that thewhole centrosphere changes into a spongy mass of rather large vacuoles, separatedby delicate protoplasmic partitions in which lie the mitochondria (Plate 1, Fig. B).Narrow vacuolar clefts also become visible around the dyads; they seem to be inconnexion with the centrosphere. The outer end of the centrosphere, againstwhich the dyads are situated, meanwhile becomes more or less flat, while itsinner end remains rounded; therefore, the originally ovoid centrosphere nowbecomes the shape of a vase or an amphora. By continued swelling it maybecome so large that it extends almost to the centre of the egg (Plate 1, Fig. C).From the stages of extrusion of the polar body it may be estimated that the wholeswelling process, from the condition of Fig. A to that of Fig. C, takes less thanminute.

Meanwhile the conical bulge at the animal pole has become more pronounced;it becomes hemispherical, then its base begins to contract (Plate 1, Figs. A-C).The maturation spindle is taken along outwards; at the same time it shortensslightly. In its equatorial region the spindle fibres become thickened, and themitochondria begin to penetrate into the spindle substance here. When the

808 CHR. P. RAVEN

base of the polar body contracts, this equatorial part of the spindle is compressed;it will give rise to the 'mid-body', which connects for some time the polar bodywith the egg surface.

With beginning constriction of the base of the polar body, the centrosphereof the maturation aster enters upon a new phase. No further swelling takesplace. On the contrary, the fluid contents of the vacuoles seem to be extruded, sothat the centrosphere shrinks (Plate 1, Figs. D-E). It is not clear whether thefluid is extruded from the egg altogether or expressed into the surroundingcytoplasm. The whole aster, which had become greatly extended during theprevious phase, contracts. The clefts around the dyads remain clearly visiblefor some time, then they also disappear. Finally, the whole centrosphere hascollapsed to a small rudiment in the centre of the aster. The mitochondria,previously seen in the meshes of the centrosphere, are now all concentrated atthis place (Plate 1, Fig. F). The dyads are still situated in the outer part of theaster, at a short distance below the surface. This stage is reached, on an average,in about 15-20 min. The maturation aster may apparently remain in this statefor some time, but then its collapsed centre develops once again into a centro-sphere. This is more or less spherical or transversely oval, non-vacuolar, ratherdense in appearance, and contains a darker, vaguely delimited 'central body'(Plate 1, Figs. G-J). The mitochondria previously found in the centrospherenow arrange themselves in a ring at the boundary between the centrosphere andthe astral rays (Plate 1, Fig. G). The whole aster shifts slightly outwards, andflattens more or less against the surface, where a shallow indentation appears atthe animal pole (Plate 1, Figs. H-J).

Meanwhile the sperm aster has made its appearance. It first becomes visible,about 20 min. after the extrusion of the first polar body, as a very small astersomewhere in the cytoplasm, often far removed from the maturation aster.Soon it increases in size, and develops a clear centrosphere with rather unsharpboundary, and with a dark 'central body' in its centre. At the same time itapproaches the maturation aster, and comes to lie at a short distance beneath it(Plate 1, Figs. H-J). Like the maturation aster, the sperm aster is surrounded bya dense cloud of mitochondria, partially penetrating between its astral rays.

Two particulars of the sperm aster deserve special mention. The rays of thisaster belong more or less clearly to two categories: a large number of thin andrather short rays, forming the main substance of the aster, and a small number ofthicker and longer, spoke-like rays, which extend a large distance into thesurrounding cytoplasm in the meshes between the vacuoles formed aroundy-granules (Plate 1, Fig. H). A similar distinction cannot be made amongst therays of the maturation aster at this stage.

Secondly, for the first time these eggs permitted a clear view of the delicatestructure of the sperm centriole in pulmonates. In previous papers (Raven et ah,1958; Raven, 1959), I have designated by 'centriole' the dark, globular, oftenmore or less vaguely delimited bodies regularly occurring in the focal points of


centrospheres, assuming that they represent the best possible image of this cellorganelle obtainable with the light microscope in pulmonate eggs. It nowappears, however, that under favourable conditions the true centriole, probablythe light-microscopic counterpart of the structure described as such by electronmicroscopists, may be visible. We will now therefore designate the dark bodiesmentioned before (the ' centrioles' of our previous papers) by the unpretentiousterm 'central bodies'.

The sperm centriole of L. peregra has the shape of a slightly curved rod, about3-4 /A in length, surrounded by a clear space which often seems to be linedexternally by a fine membrane. The thicker end of the centriolar rod occupiesthe centre of the 'central body' of the sperm aster. From this point it tapersgradually towards the other end, which continues into an extremely delicatefibril that traverses the centrosphere, and runs radially outwards between theastral rays. This fibril, in its turn, is continuous with a sperm tail that can befollowed for some distance in the cytoplasm (Text-fig, la).

In the central body of the maturation aster no distinct centriole can be seen.When the sperm aster has approached the maturation aster, the formation

of the second maturation spindle begins. This occurs about 35-40 min. after theextrusion of the first polar body. At first a connexion between the two asters isestablished by a few thick and slightly curved fibres (Plate 1, Figs. K-L). Theyresemble the stout rays of the sperm aster. These connecting fibres graduallyseem to increase in number, and in this way form the central part of the secondmaturation spindle consisting of continuous fibres. In a second phase part ofthe astral radiations, both of the maturation aster and of the sperm aster, curvearound this central spindle providing it with a layer of mantle fibres (Plate 1,Fig. M).

By this same movement, the dyads are conveyed to the surface of the spindle.From their initial position against the centrosphere in the outer half of thematuration aster the dyads at a rather early stage begin to move centrifugallyalong the astral rays (Plate 1, Figs. G-J). At the time when the second maturationSpindle is being formed they have reached the tips of the rays, and are now takenalong by the latter bending inwards toward the equatorial part of the spindle(Plate 1, Fig. M). At first their arrangement is still rather irregular. This prometa-phase stage lasts till about 60 min. Then the dyads penetrate into the spindle,connect with its chromosomal fibres (Plate 2, Fig. A) and begin to arrangethemselves into a metaphase plate (Plate 2, Fig. B).

In some of the eggs, starting at the time when the formation of the secondmaturation spindle begins, a division of the central body of the sperm aster isadumbrated (Plate 1, Fig. L). At later stages this occurs in most, though (up to theend of anaphase) apparently not in all eggs. The division of the central body isaccompanied by a reduplication of the centriole. The two centrioles are notequivalent, however: one of the two has the original shape, and is still connectedwith a fibril running outward through the astral rays; the other one is much

810 CHR. P. RAVEN

smaller, often hardly visible, and its surrounding clear area is less distinct. In afew cases the impression was gained that this centriole arises as a lateral outgrowthof the original one, but, as these relationships are at the limit of visibility, nocertainty could be obtained in the matter.

In two of the eggs the sperm aster as a whole had divided into two astersconnected by an achromatic central spindle. Only one of the daughter astershad participated in the formation of the second maturation spindle and formedits deep aster. The other daughter aster lay beneath the former in one case,obliquely to one side in the other. This situation, which is the rule in Planorbis(cf. Raven, 1959), apparently occurs as a rare variation in L. peregra.

2. Limnaea ovata, Myxas glutinosaThese two species show a great deal of conformity as regards the development

of the second maturation spindle, so that it appears justified to treat them to-gether. The sections of their eggs are, at least with the histological techniqueemployed, much less clear than those of L. peregra, and give poor photographicpictures; therefore only a few photomicrographs will be given.

The deep aster of the anaphase spindle of first maturation in L. ovata has aclear spherical centrosphere of moderate size, with a reticulate structure. Thereis a broad belt of mitochondria along the boundary of the centrosphere; they liemainly between the inner parts of the astral rays. When the first polar body isextruded, the aster and its centrosphere become slightly flattened parallel to the

EXPLANATION OF PLATES1

PLATE 1

FIGS. A-M. Formation of second maturation spindle in Limnaea peregra. x 800.FIG. A. Twenty minutes. Telophase of first maturation division. Beginning formation of

first polar body. Elongation of deep centrosphere.FIG. B. Twenty minutes. Further swelling and vacuolization of centrosphere.FIG. C. Fifteen minutes. Beginning constriction of base of polar body. Maximum swelling

of centrosphere.FIG. D. Five minutes. First polar body extruded. Shrinking of centrosphere.FIG. E. Fifteen minutes. Further shrinkage of centrosphere.FIG. F. Twenty-five minutes. First polar body with mid-body. Centrosphere collapsed;

mitochondria concentrated in centre of maturation aster.FIG. G. Twenty minutes. First polar body with mid-body. In centre of maturation aster a

centrosphere has once more been formed. Beginning of centrifugal movement of dyads.FIG. H. Thirty-five minutes. Maturation aster with central body; centrifugal movement of

dyads. Sperm aster beneath maturation aster.FIG. J. Fifty minutes. Dyads have reached peripheral part of maturation aster. Sperm aster

approaches maturation aster.FIG. K. Twenty-five minutes. Beginning connexion between maturation aster and sperm

aster by cytoplasmic fibres.FIG. L. Thirty minutes. Connecting fibres between maturation aster and sperm aster form

rudiment of central spindle. Sperm aster with reduplicated central body.FIG. M. Forty minutes. Peripheral part of second maturation spindle formed by astral rays

of both asters bending around central spindle. Dyads taken along toward spindle equator.

1 The magnifications indicated by scale-lines in Plate 1, Fig. A and Plate 2, Figs. A and C are incorrect.Please read 7JJL instead of IOJJL.

J. Embryol. exp. Morph. Vol. 12, Part 4

PLATE 1

CHR. P. RAVEN (Facingpage SIO)

J. Einbryol. exp. Morph. Vol. 12, Part 4

:'i

PLATE 2

CHR. P. RAVEN {Facing page 811)


surface. In Myxas the deep centrosphere at anaphase is smaller, elongated atright angles to the surface, and its circumference is rather unsharp; the belt ofmitochondria is less dense than in L. ovata. In both species, but especially inMyxas, the accumulation of mitochondria around the spindle and asters isvery evident. In L. ovata there is, moreover, a distinct animal pole plasm con-taining many mitochondria. In Myxas this is not yet visible at this stage, but itappears during the formation of the second maturation spindle.

The characteristic transformations (swelling and shrinking) of the centrospherethat occur in L. peregra, are entirely lacking in these species. After the extrusionof the first polar body the maturation aster and centrosphere exhibit no greatchanges for some time. At most one can say that the boundary between centro-sphere and astral rays, which was rather vague in Myxas from the beginning,now begins to become more and more blurred inZ,. ovata too. The dyads, whichat first form a crowded group against the centrosphere, gradually move apartin a centrifugal direction along the astral rays (Plate 2, Figs. C-D, F).

PLATE 2

FIGS. A-B. Completion of second maturation spindle in Limnaea peregra. x 800.FIG. A. Sixty minutes. Prometaphase of second maturation division. Dyads have penetrated

into spindle and connected with chromosomal fibres. Sperm aster with divided centralbody. A single centriolar rod is faintly visible between the two central bodies.

FIG. B. Sixty minutes. Early metaphase of second maturation division. Central body ofsperm aster divided.

FIGS. C-E. Some stages in the formation of the second maturation spindle in Limnaea ovata.x900.

FIG. C. Twenty-five minutes. Dyads near centre of maturation aster (above). Sperm asterbeneath maturation aster, surrounded by very dense cloud of mitochondria (below).

FIG. D. Twenty-five minutes. Dyads are moving centrifugally along astral rays. Spermaster has approached maturation aster, but not yet connected with it.

FIG. E. Forty minutes. Maturation aster has folded together, taking dyads along, andfused with sperm aster. Zone of mitochondria in region of fusion of the two asters.

FIGS. F-H. Some stages in the formation of the second maturation spindle in Myxas glutinosa.x900.

FIG. F. Fifty-five minutes. Dyads are moving centrifugally along astral rays of maturationaster. The latter have begun to swing downwards. Sperm aster (below) beneath maturationaster.

FIG. G. Fifty-five minutes. Maturation aster has folded together, taking along dyads, buthas not yet fused with sperm aster.

FIG. H. Ninety minutes. Early metaphase of second maturation division.FIGS. J-M. Stages in the formation of the second maturation spindle in Limnaea palustris.

x900.FIG. J. Twenty-five minutes. Centrosphere of maturation aster dense, astral rays disappear-

ing. Dyads arranged along surface of centrosphere.FIG. K. Twenty-five minutes. Centrosphere with irregular outline capped by dyads (above).

Sperm aster (below) has approached maturation aster.FIG. L. Thirty minutes. Dyads have penetrated shrunken centrosphere (above). Sperm aster

has in this case divided to sperm amphiaster with achromatic central spindle (below).FIG. M. Thirty minutes. Centrosphere has given rise to second maturation spindle, which

has fused with sperm aster. Zone of mitochondria in region of fusion.

812 CHR. P. RAVEN

The sperm aster appears inL. ovata when the polar body has just been extruded,in M. glutinosa somewhat later. Like in L. peregra, it is at first very small, andshows no fixed topographic relationship to the maturation aster. Then it growsin size and approaches the latter. Especially in L. ovata it attracts a great mass ofmitochondria from the surrounding cytoplasm, which penetrate deeply betweenits rays, so that it is nearly obscured by them in the photographs (Plate 2, Figs.C, D). In L. ovata the sperm aster contains a centriolar rod exhibiting the samecharacteristics as that of L. peregra; in Myxas no clear centriole has been seen.

When the sperm aster has reached a position just beneath the maturationaster (Plate 2, Fig. C), the formation of the second maturation spindle begins.This occurs in a peculiar way, and quite differently from the process in L. peregra.No 'spinning' of central spindle fibres between the two asters can be seen.Instead, the rays of the maturation aster swing downwards in the direction ofthe sperm aster (Plate 2, Figs. D, F-G), taking the dyads along. The wholematuration aster is, so to speak, folded together like a shut-up umbrella. Thena fusion occurs between its lower end and the sperm aster, which up to thismoment did not visibly participate in the process. In the region of fusion forsome time a transverse band of mitochondria remains (Plate 2, Fig. E). Fromits localization, some distance below the spindle equator, one can deduce thatby far the greater part of the spindle derives from the maturation aster, whilethe sperm aster at most supplies its deeper part. Later the boundary between thetwo parts is no longer visible (Plate 2, Fig. H).

The dyads, after being conveyed to the equatorial region of the spindle by therays of the maturation aster bending downwards, for some time remain inmore or less irregular arrangement in the superficial part of the spindle. Then theypenetrate into it and arrange themselves into an equatorial plate.

In one Myxas egg at the anaphase of the second maturation division the deepaster (sperm aster) had a biscuit-shaped centrosphere with two central bodies.In two more eggs of this species division of the central body was adumbrated.No further indications of a reduplication of the sperm cytocentre were observed,neither in Myxas nor in L. ovata.

3. Limnaea palustris

Like in the other species, the deep aster of the anaphase spindle of the firstmaturation division has a big, clear spherical centrosphere. A belt of mito-chondria between the inner ends of the astral rays can also be observed in thisspecies. With the extrusion of the first polar body, the centrosphere becomestemporarily elongated perpendicularly to the surface. On its outer side it iscapped by the dyads, forming a densely crowded group. The accumulation ofmitochondria around the spindle and asters at this stage is somewhat lesspronounced than in the previous species. The animal pole plasm becomesvisible a short time after the first polar body has been extruded.


In the 20 min. following the extrusion of the first polar body, a gradual trans-formation of the maturation aster and its centrosphere occurs. The centro-sphere, which had at first a rather tenuous reticulate structure, gradually increasesin density and stainability. At the same time, the astral radiations become moreor less blurred. The boundary between centrosphere and astral rays becomesmore and more pronounced. The centrosphere becomes acorn- or pillow-shaped,with a more strongly curved outer and a flatter inner surface. The dyads moveapart, sliding along the surface of the centrosphere, until they have attained amore less even spacing along its convex outer side (Plate 2, Fig. J). This stage hasbeen reached in most eggs at 25 min.

The sperm aster makes its appearance at about 15-20 min. From a verysmall aster, lying near the centre of the egg, it soon grows to a moderate size anddevelops a centrosphere with rather unsharp boundary. In many cases itscentre appears more or less reduplicated. In three eggs the sperm aster has dividedas a whole, the two daughter asters remaining connected by an achromatic centralspindle (Plate 2, Fig. L). Soon it approaches the maturation aster and its centro-sphere (Plate 2, Fig. K).

The latter now shows a rapid change. While its astral radiations disappearaltogether, the centrosphere becomes irregular. Its originally smooth surfacedevelops fibrous spurs, so that the regular arrangement of the dyads becomesdisturbed (Plate 2, Fig. K). Then the centrosphere shrinks to a strongly erythro-sinophil lump of dense cytoplasm into which the dyads become incorporated(Plate 2, Fig. L). This erythrosinophil body now transforms very rapidly into thesecond maturation spindle, which becomes connected with the sperm aster(Plate 2, Fig. M). This occurs at about 30 min. A zone of mitochondria at theplace of junction of spindle and sperm aster remains visible for some time; itgives evidence of the fact that the spindle proper arises as a whole from theerythrosinophil substance of the egg centrosphere, whereas the sperm astersupplies the deep aster of the maturation spindle only. The dyads are at first moreor less irregularly distributed in the equatorial region of the spindle, but thisprometaphase stage is of short duration; soon the dyads become arranged intoan equatorial plate. In the deep aster of the spindle a rod-like sperm centriolecan be observed at this stage; it resembles that found in L. peregra and L. ovata,but no fibrillar connexion with a sperm tail has been observed in L. palustris.

4. Limnaea stagnalis

The development of the second maturation spindle in L. stagnalis has beendescribed in detail in an earlier paper (Raven et al, 1958).

DISCUSSION

1. The object of this study was to investigate whether the variations in develop-ment, as regards the second maturation spindle, are of a lower order of magnitude

814 CHR. P. RAVEN

within a same family than between families. Considering the results as a whole,this question must be answered in the negative. We have found that the secondmaturation spindle is formed, within the genus Limnaea, in at least three differentways, between which there are great and apparently rather fundamental differ-ences. The manner of formation of this spindle inL.peregra shows a much greaterresemblance to that in Planorbis planorbis than in the congeneric species L.stagnate. LikewiseL. ovata resembles Myxasglutinosa much more, in this respect,than either L. peregra or L. stagnalis. Apparently, therefore, the variability inthe course of the second maturation division in these snails does not respect theboundaries drawn up by the taxonomists, but obeys its own rules.

On the other hand, all Limnaeidae studied so far exhibit some commoncharacteristics. In all, the egg cytocentre apparently remains undivided after theextrusion of the first polar body. The centre and aster at the deep pole of thesecond maturation spindle are supplied by the sperm aster, which arises inde-pendently at some distance from the animal pole, but then moves toward this poleand participates secondarily in the formation of the maturation spindle. It mustbe added, however, that this description does not suffice to distinguish theLimnaeidae from other families, as it also holds for the planorbids hithertostudied.

2. When the sperm aster in Limnaea peregra has approached the maturationaster to within a certain distance, the central parts of the two asters becomeconnected by a few rather thick curved fibres, which show a great resemblanceto the thick rays of the sperm aster. Whether these connecting fibres arise byoutgrowth of astral rays from the sperm aster or from the maturation aster,by end-to-end fusion of astral rays of both asters, or, finally, whether theyrepresent a particular kind of fibre arising in some other way, could not bedecided with certainty, although the observations seem to favour the first possi-bility. By an increase in the number of these connecting fibres, a central spindleconsisting of continuous fibres is formed. Then part of the astral radiations ofboth maturation aster and sperm aster, curving around this central spindle,surround it with a layer of mantle fibres; in this process the thin fibres of thesperm aster also take part.

This mode of formation of the second maturation spindle shows a greatresemblance to that found in Planorbis planorbis and PL corneus (Raven, 1959),the main difference being that the sperm aster in the latter species at this time hasreduplicated as a rule, and formed an amphiaster, whereas this occurs onlyoccasionally in Limnaea peregra.

While the part played by the sperm aster in the development of the spindlein L.peregra is at least equivalent to, and perhaps even more important than, thatof the maturation aster, different relationships are found in L. ovata and Myxasglutinosa. Here the main mass of the second maturation spindle is formed byfolding together of the maturation aster, whereas the sperm aster supplies atmost a small part at the lower end of the spindle proper, and its deep aster.


No preliminary formation of a central spindle by fibres connecting the twocytocentres can be observed.

In L. palustris, the second maturation spindle proper is formed exclusively bythe maturation aster, notably its centrosphere, which grows in size, and becomesdistinctly marked off from its surroundings by a smooth outline, along which thedyads line up. It then shrinks down to an irregular body, which becomes trans-formed into the second maturation spindle. This fuses secondarily with thesperm aster.

The development of the second maturation spindle in L. palustris greatlyresembles that in L. stagnalis described previously (Raven et ah, 1958). Theshapes assumed by the centrospheres in the two species are slightly different,that of L. stagnalis becoming more elongated and egg-shaped. Moreover, inthe latter species the centrosphere is transformed directly into the spindle body,keeping substantially the same shape, whereas this transformation is precededby shrinkage and loss of shape in L. palustris. But these are minor differenceswhich do not detract from the general conformity between the two.

3. In all Limnaeidae the deep aster of the second maturation spindle issupplied by the sperm aster, which either takes part in the formation of the spindlefrom the beginning, or fuses secondarily with its deeper end. The same holds forPlanorbidae. In those cases where the sperm aster exhibits an early division(usually in planorbids, occasionally in limnaeids), as a rule one of its daughterasters functions as the deep aster of the maturation spindle, while the other oneremains connected with the former by means of an achromatic central spindle.

The origin of the superficial aster of the second maturation spindle is muchmore variable. In L. peregra the maturation aster (i.e. the original deep aster ofthe first maturation spindle, which remained in the egg at the extrusion of thefirst polar body) co-operates with the sperm aster in the formation of the secondspindle, and becomes its superficial aster without any further change. The sameholds for Planorbis (Raven, 1959).

In L. ovata and Myxas glutinosa, the maturation aster is folded together as awhole, and forms the main mass of the second spindle. The upper end of thisspindle is rather pointed at first, but soon new astral radiations develop aroundthis region. Apparently, therefore, the superficial aster is mainly a new formationin these species.

Finally, in L. palustris and L. stagnalis, the rays of the maturation asterbecome blurred and vanish, while its centrosphere develops to the second spindle.Only after the latter is more or less completely formed does a new superficialaster appear, apparently being entirely formed de novo.

4. With these differences in the development of the second maturationspindle and its asters a different behaviour of the dyads is correlated. In all speciesthe latter come together at the end of anaphase of the first maturation divisioninto a tightly packed group at the deep pole of the spindle, lying immediatelyagainst the outer side of the centrosphere of the maturation aster. In L. peregra,

816 CHR. P. RAVEN

L. ovata and Myxas glutinosa (and also in the species of Planorbis studiedpreviously) they then move centrifugally along the astral rays. When the latterbend inwards to participate in the formation of the second maturation spindle,the dyads are taken along. In this way they are carried to the outer surface ofthe equatorial region of the spindle. They subsequently penetrate into thespindle and arrange themselves into an equatorial plate.

In L. palustris and L. stagnalis, on the other hand, the dyads behave quitedifferently. No centrifugal movement along the astral rays takes place. Thelatter begin to disintegrate at an early stage and finally vanish altogether. Thedyads remain connected with the outer boundary of the centrosphere, movingalong its convex surface. In L. stagnalis they soon become arranged into a moreor less regular ring, which slides over the surface of the centrosphere (that mean-while develops into a spindle), until it has reached its equatorial region. Onlythen do the dyads begin to invade the spindle area. In L. palustris this annulararrangement of the dyads is less clear; they rather become distributed more orless evenly along the convex outer surface of the centrosphere. When the latterloses its regular outline and collapses, the dyads are incorporated into it. Sothey find themselves from the beginning within the developing spindle.

5. A special peculiarity of egg maturation in L. peregra is the rapid swellingand vacuolization of the centrosphere of the maturation aster during the extrusionof the first polar body, followed by its collapse and the return of the aster tonormal size after the polar body has been separated from the egg. In no otherpulmonate species studied so far has a similar phenomenon been observed.Owing to its conspicuousness one is inclined to conjecture about its functionalsignificance. It is obvious to assume that it has something to do with the expulsionof the polar body. One might think, for instance, that by this swelling a turgoris produced which helps in driving the polar body out by overcoming the physicalforces that offer resistance to its expulsion. But, of course, this is only an explana-tion ad hoc, and becomes rather doubtful by the fact that in none of the relatedspecies does a similar mechanism seem to be necessary. So we are left with theconclusion that this is perhaps only one of the many for the moment unaccount-able variations in the course of the maturation divisions in pulmonates.

6. In a previous paper (Raven et al, 1958) we have drawn the attention to theappearance, in the region of fusion of the maturation spindle and the spermaster in Limnaea stagnalis, of a zone of dark granules and small vacuoles, andsuggested that they are indicative of a certain cytochemical activity in thisregion. A similar zone of dark granules has also been observed in L. palustris,L. ovata and Myxas glutinosa in the zone of junction between the derivatives ofthe maturation aster and the sperm aster, respectively. It has now become clear,however, that these granules are mitochondria of the intervening cytoplasm,which become entrapped, so to speak, between the two fusing structures, andremain pinched between the spindle fibres, in this way clearly marking the regionof fusion. After some time they disappear. Whether they become lysed or


extruded from the spindle is not clear; the small vacuoles seen in L. stagnalismight perhaps argue for the first possibility, but they have not been observed inthe other species.

7. A newly-discovered feature of the cytology of these pulmonate eggs isprovided by the centriolar rod in the sperm aster described above (Text-fig, la).It was first observed in L. peregra, where it is clearly visible in the sperm aster ofnearly all eggs of the right stages. Subsequently it has also been found in L. ovataand L. palustris. A reinvestigation of the eggs of L. stagnalis has shown that thesperm aster in this species probably contains a similar rod. It appears thereforeto be present in all species ofLimnaea studied so far.

• V \:-vli***/•;'•''

TEXT-FIG. 1. Sperm aster, centriolar apparatus and sperm tail in (a) Limnaea peregra, (b)Planorbis planorbis.

In Planorbis planorbis, on the other hand, a different structure can be observed.The disintegrating sperm tail in the cytoplasm of these eggs has the appearance ofa bundle of about ten coarse irregularly twisted fibres staining heavily with ironhaematoxylin. Often the sperm aster is connected with the sperm tail. Then alltail fibres but one end about half-way along the rays of the sperm aster. One fibrecontinues in a radial direction through the astral radiations into the centrosphere,where it ends in a small ovoid vesicle, about 2 fx wide by 2 • 5 n long, near thecentre of the sperm aster. This vesicle bears one or, more often, two pairs ofdark granules, of which those lying nearer the central end appear to be somewhatsmaller than those of the other pair (Text-fig, lb). When the sperm aster hasdeveloped to an amphiaster, as is the rule in this species, such a structure is onlyfound in the half connected with the sperm tail; but occasionally a smaller vesicle,with a single central dark granule, can be observed in the other half.

It is difficult to assess the significance of the observed structures. The twistedcoarse fibres of the disintegrating sperm tail in Planorbis, whose number could

818 CHR. P. RAVEN

not be determined with absolute certainty, but most probably is about ten,might be supposed to represent the well-known nine peripheral and two centralfibrils of the flagella. The fact that these extremely thin fibrils, ordinarily onlyvisible by means of the electron microscope, here appear as rather coarse fibresin light-optical sections, might be due to the process of disintegration, wherebysubstances staining heavily with iron haematoxylin are accumulated around eachfibril. In Limnaea peregra, splitting up of the sperm tail into its componentfibrils in the egg cytoplasm apparently does not occur as a rule; in this species itgenerally appears as a single, rather thick dark strand, though occasionally onegets the impression that it consists of several sub-units.

If the above interpretation is correct, it is obvious to assume that the singlefibre continuing inwards towards the central area of the sperm aster (extremelydelicate in L. peregra, rather coarse in Planorbis planorbis) represents the doublecentral fibril of the flagella. Greater difficulties arise in the interpretation of thecentral structure with which it is continuous.

In light-microscopic studies of spermatogenesis in gastropods the centrioleof the spermatid, which is located next to the nuclear envelope, and becomes thebasal body of the flagellum, as a rule has been described as a small granule orglobule. Grasse, Carasso & Favard (1956), studying spermatogenesis in Helixpomatia by means of the electron microscope, still speak of an ovoid corpuscle,with its broader end turned backwards. It is true that more recent electron-microscopic studies (e.g., de Harven & Bernhard, 1956; Gall, 1961) have demon-strated that the centrioles, in representatives of various groups of animals, havethe shape of short hollow cylinders, with a wall consisting of nine complexsubcylinders or fibres running parallel to the long axis of the cylinder. But asthe length of these cylindrical centrioles is of the order of about 0-3 /x theycannot be identified with the rod-like structure observed in the sperm aster ofL. peregra, which measures about 3 to 4 /x in length.

The latter rather resembles the rod-shaped centrioles found by Costello (1961)at the ends of the cleavage spindles in Polychoerus, which are slightly curvedrods about 4- 5 to 5 /z in length. They lie transversely or slightly obliquely to thelong axis of the spindle, while the centrioles at the two ends of a given spindle areoriented at right angles to each other. Costello, in this paper, mentions severalother instances of rod-shaped centrioles in definite orientations, from which hedraws rather far-reaching conclusions on the possible significance of centrioleorientation and behaviour for the determination of spindle axes and cell arrange-ment during cleavage.

I have checked the position of the centriolar rod (or, in case of its beingreduplicated, of the larger one) in the sperm aster of L. peregra, after the latterhad become connected with the second maturation spindle. The observed rodswere, at a rough estimate, classed into three categories: transverse (i.e. at anangle of 90° ±22-5° to the long axis of the maturation spindle), oblique(45° ±22-5°) and longitudinal (less than 22-5°). Among twenty-three cases


where the position of the rod could be estimated with any degree of accuracy,thirteen were transverse, eight oblique and two longitudinal. At first sight, thisseems to argue for a preponderance of the transverse position. However, onsecond thoughts it appears that the three classes are not equivalent, and that theobserved distribution is compatible with an arbitrary direction of the rod.Anyhow, it is evident that nearly longitudinal positions do occur. Therefore, itis probable that there is in this case no fixed relationship between the orientationof the centriolar rod and the longitudinal axis of the spindle.

While the rod-like centrioles in Limnaea thus may perhaps be considered asenlarged counterparts of the structures observed by electron microscopists, itis not clear how we have to interpret the vesicular bodies found in a similar positionin Planorbis. A study of the sperm aster in Limnaea and Planorbis by means ofelectron microscopy might be rewarding.

The observation that the two centrioles found side by side in a single spermaster of L. peregra at first are not equivalent, one being much smaller than theother, is in agreement with modern views on the replication of centrioles (cf.Gall, 1961).

8. In Limnaea stagnalis an animal pole plasm becomes visible some time afterthe extrusion of the second polar body. It forms a layer of protoplasm imme-diately beneath the egg cortex in the animal hemisphere, staining dark violet bluewith iron haematoxylin. It is very rich in mitochondria (Raven, 1945).

Centrifugation experiments have shown that the formation of the animal poleplasm is probably due to specific attractive actions, exerted by the cortex in theneighbourhood of the animal pole upon certain components of the inner cyto-plasm. However, it is only the ground substance ('matrix') of the pole plasmwhich is accumulated by this means. The mitochondria are conveyed towardthe animal pole region by way of the maturation spindles and asters. The firstmaturation spindle and its asters are already surrounded by a dense cloud ofmitochondria, which partly penetrate in rows between the astral rays. A newsupply of mitochondria is carried toward the animal pole by the sperm aster,approaching the second maturation spindle and fusing with its inner end.Apparently the mitochondria are attracted towards the asters from a greatpart of the egg, perhaps by centripetally directed currents between the astralrays. After the extrusion of the second polar body and the disappearance of theremnant of the maturation spindle the mitochondria then accumulate in layersbeneath the cortex in the animal pole plasm (Raven & Van der Wai, 1964).

Similar relationships apparently hold in the other Limnaeidae. The accumula-tion of mitochondria around the asters and spindles is especially marked inLimnaea ovata (Plate 2, Fig. E) and Myxas glutinosa, but is also evident inL. peregra (e.g. Plate 2, Figs. A-B) and L. palustris (Plate 2, Fig. M). There is adifference among the species, however, with respect to the time of formation ofthe animal pole plasm. In L. peregra and L. ovata this plasm is already visible atthe time of formation of the first polar body (cf. Plate 1, Figs. A-C), in L.

53

820 CHR. P. RAVEN

palustris a short time afterwards (Plate 2, Figs. L-M), while it appears in Myxasglutinosa during the second maturation division. Though it is most conspicuousby the dense accumulation of mitochondria beneath the cortex in this region, astudy of the sections shows that these are embedded in a layer of 'matrix', sothat we apparently have to do with a pole plasm indeed.

9. The discovery of a great variability in the development of the secondmaturation spindle among species of a single genus raises the question as to itsimplications for taxonomy. As we have seen above, this variability does notconform to the classifications drawn up by taxonomists, but partly obeys otherrules. It is tempting to interpret the results in terms of affinity between species,and to conjecture, e.g., that L. stagnalis and L. palustris are closely allied species,thatZ,. ovata has affinities to the genus Myxas, and L. peregra to the Planorbidae.But it is evident that such speculations, based on a single character, though afavourite pastime of phylogenetic taxonomists of the past, have no real value.On the other hand, taxonomists cannot afford to disregard such striking similari-ties and dissimilarities established by developmental biology, if they aim at anatural system of classification.

Hubendick, in his monograph of the Limnaeidae (1951), considers L. peregra(Mull.) to be synonymous with L. ovata Drap. As we have seen, however, thetwo species (or, more specifically, the Scottish specimens of L. peregra and theDutch specimens of L. ovata) differ markedly, and probably rather fundamentally,in the course of the maturation divisions, and especially in the mode of develop-ment of the second maturation spindle. It is evident that these two populationsare different. Of course it must be left to the taxonomists to evaluate the weightof this difference, but they may be expected to take it into account. The sameholds for the other facts described in this paper.

SUMMARY

1. The formation of the second maturation spindle has been studied in theeggs of Limnaea peregra, L. ovata, L. palustris and Myxas glutinosa.

2. In all Limnaeidae studied so far the deep pole of the second maturationspindle is supplied by the sperm aster, which either takes part in the formation ofthis spindle from the beginning or fuses secondarily with its inner end. Occasion-ally the sperm aster divides and forms a sperm amphiaster; then one of itsdaughter asters functions as the deep aster of the second maturation spindle.

3. The second maturation spindle proper in the Limnaeidae is formed in atleast three different ways. In L. peregra, the maturation aster and sperm asterfirst become connected by thick fibres, forming a central spindle; then parts ofthe astral radiations of both asters curve around this central spindle and forma layer of mantle fibres. InZ,. ovata and M. glutinosa, the main mass of the secondmaturation spindle is formed by folding together of the maturation aster,whereas the sperm aster supplies at most a small part at the lower end of the


spindle. Finally, in L. palustris and L. stagnalis, the spindle forms as a wholefrom the centrosphere of the maturation aster; only afterwards does it fuse withthe sperm aster.

4. In L. peregra the original deep aster of the first maturation spindle, withoutmuch change, becomes the superficial aster of the second spindle. In L. ovataand M. glutinosa the superficial aster of the second maturation spindle is mainlyformed de novo. In L. palustris and L. stagnalis it is entirely formed de novo.

5. In L. peregra, L. ovata and M. glutinosa the dyads, after the extrusion of thefirst polar body, move centrifugally along the rays of the maturation aster.When these rays bend inwards to participate in the formation of the secondmaturation spindle, the dyads are taken along and carried to the equatorialregion of the spindle. In L. palustris and L. stagnalis, the radiations of thematuration aster soon disintegrate, and the dyads move along the surface of thecentrosphere. In L. stagnalis they penetrate into the latter only after it has beentransformed into the second maturation spindle; in L. palustris this occurssomewhat earlier when the centrosphere loses its regular shape.

6. In L. peregra the centrosphere of the deep aster of the first maturationspindle shows a rapid swelling and vacuolization during the extrusion of the firstpolar body. When the polar body becomes constricted off, the centrospherecollapses and the aster returns to normal size.

7. In all species of Limnaea the sperm aster contains a centriolar rod, about3-4 /x in length; it is, at least inL.peregra, connected through an extremely delicatefibril to the proximal end of the sperm tail in the cytoplasm. In older sperm astersit may be duplicated.

8. In all Limnaeidae the mitochondria tend to concentrate around thematuration spindles and asters. Later they become accumulated beneath theegg cortex in the region of the animal pole plasm. The time of formation of thispole plasm differs among the Limnaeidae. In L. peregra and L. ovata it is alreadyvisible at the time of formation of the first polar body; in L. palustris it appearsshortly afterwards, in M. glutinosa during the second maturation division, andin L. stagnalis some time after the extrusion of the second polar body.

9. Taxonomic implications of the observed similarities and dissimilarities inthe course of the second maturation division in Limnaeidae are discussed.

RESUME

La formation du deuxieme fuseau de maturation chez les oeufs de Limnaeidae divers

1. La formation du deuxieme fuseau de maturation a ete etudiee chez les oeufsde Limnaea peregra, L. ovata, L. palustris, et Myxas glutinosa.

2. Dans tous les Limnaeidae etudies jusqu'a present, le pole profond dudeuxieme fuseau de maturation est fourni par Taster spermique, qui peutparticiper a la formation de ce fuseau des le debut, ou qui fuse secondairementavec son extremite interieure. Parfois Taster spermique se divise, et forme un

822 CHR. P. RAVEN

amphiaster spermique; dans ce cas, un des asters filiaux sert comme asterprofond du deuxieme fuseau de maturation.

3. Le deuxieme fuseau de maturation, proprement dit, chez les Limnaeidae,peut se former de trois manieres differentes au moins. Chez L. peregra, Tasterde maturation et Taster spermique se lient d'abord par des fibres epaisses,formant un fuseau central; puis partant des deux asters, une partie de leur rayonsse courbe autour de ce fuseau central, Tentourant d'une couche de fibres. ChezL. ovata et M. glutinosa, la masse principale du deuxieme fuseau de maturation seforme en repliant Taster de maturation, tandis que Taster spermique fournittout au plus une petite partie de Textremite inferieure du fuseau. Enfin chezL. palustris etL. stagnalis, le fuseau se forme entierement a partir du centrospherede Taster de maturation; par apres seulement, il fusionne avec Taster spermique.

4. Chez L. peregra, Taster profond originel du premier fuseau de maturationdevient, sans grand changement, Taster superficiel du deuxieme fuseau. ChezL. ovata et M. glutinosa, Taster superficiel du deuxieme fuseau de maturation estpour la plupart, et chez L. palustris et L. stagnalis est entierement, forme de novo.

5. Chez L. peregra, L. ovata et M. glutinosa, les diades, apres Texpulsion dupremier globule polaire, centrifugent le long des rayons de Taster de maturation.Lorsque ces rayons se plient vers Tinterieur, pour participer a la formation dudeuxieme fuseau de maturation, les diades sont prises dans le mouvement etemigrent dans le plan equatorial du fuseau de maturation. Chez L. palustriset L. stagnalis, les rayons de Taster de maturation se desagregent rapidement etles diades avancent le long du centrosphere. Chez L. stagnalis, elles penetrentce dernier seulement apres sa transformation en deuxieme fuseau de maturation;chezL. palustris, ceci se fait un peu plus tot, quand le centrosphere perd sa formereguliere.

6. Chez L. peregra, le centrosphere de Taster profond du premier fuseau dematuration montre un enflement rapide avec formation de vacuoles, pendantTexpulsion du premier globule polaire. Apres cela, le centrosphere disparait, etTaster retrouve sa taille normale.

7. Chez toutes les especes de Limnaea, Taster spermique contient une baguettecentriolaire, d'environ 3-4 /x de longueur; elle s'attache, au moins chez L.peregra, par une fibrille extremement delicate a Textremite proximale de laqueue du sperme, dans le cytoplasme. Dans les asters spermique plus ages, ellepeut se doubler.

8. Chez tous les Limnaeidae, les mitochondries ont une tendence a s'accumulerautour des fuseaux et des asters de maturation. Plus tard, elles s'accumulentendessous du cortex de Toeuf, dans la region du plasme du pole animale. Ceplasme polaire est forme a des temps differents selon Tespece de Limnaea.Chez L. peregra et L. ovata, il est visible deja au moment de la formation dupremier globule polaire; chez L. palustris il apparait un peu plus tard, chezM. glutinosa pendant la deuxieme division de maturation, et chez L. stagnalisquelque temps apres Texpulsion du deuxieme globule polaire.


9. Les implications taxonomiques des ressemblances et des dissemblancesremarquees au cours de la deuxieme division de maturation chez les Limnaeidaesont discutees.

REFERENCES

COSTELLO, D. P. (1961). On the orientation of centrioles in dividing cells, and its significance:a new contribution to spindle mechanics. Biol. Bull. 120, 285-312.

GALL, J. G. (1961). Centriole replication. A study of spermatogenesis in the snail Viviparus.J. biophs. biochem. Cytol. 10,163-93.

GRASSE, P. P., CARASSO, N. & FAVARD, P. (1956). Les ultrastructures cellulaires au cours dela spermiogenese de l'escargot {Helix pomatia L.): evolution des chromosomes, duchondriome, de l'appareil de Golgi, etc. Ann. Sci. nat. 18, 339-80.

HARVEN, E. DE & BERNHARD, W. (1956). Etude au microscope electronique de l'ultrastructuredu centriole chez les Vertebres. Z. Zellforsch. 45, 378-97.

HUBENDICK, B. (1951). Recent Lymnaeidae. Their variation, morphology, taxonomy,nomenclature, and distribution. K. svenska VetenskAkad. Handl. Fj. Ser., 3, no. 1,Stockholm.

RAVEN, CHR. P. (1945). The development of the egg o£ Limnaea stagnalis L. from ovipositiontill first cleavage. Arch, neerl. Zool. 7, 91-121.

RAVEN, CHR. P. (1949). On maturation in the eggs of Limnaea stagnalis L. Bijdr. Dierk.28, 372-84.

RAVEN, CHR. P. (1959). The formation of the second maturation spindle in the eggs ofSuccinea,Physa, and Planorbis. J. Embryol. exp. Morph. 7, 344-60.

RAVEN, CHR. P., ESCHER, F. C. M., HERREBOUT, W. M. & LEUSSINK, J. A. (1958). The forma-tion of the second maturation spindle in the eggs of Limnaea, Limax, and Agriolimax.J. Embryol. exp. Morph. 6, 28-51.

RAVEN, CHR. P. & VAN DER WAL, U. P. (1964). Analysis of the formation of the animal poleplasm in the eggs of Limnaea stagnalis. J. Embryol. exp. Morph. 12, 123-39.

{Manuscript received 24th July 1964)

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