Notes on the Fecundation of the Egg of Sphærechinus granularia, and on the Maturation and Fertilisation of the Egg of Phallusia mammillata

The form I chose to work upon was Sphærechinus granularis, which is exceedingly abundant at Naples, and could always be had in any number. Mr. Wilson studied Toxopneustes variegatus, while Mr. Matthews divided his attention between Asterias Forbesii andArbacia punctulata. With regard to Wilson’s interesting observations on the polarity of the egg and the axial relations of the two pronuclei, I am not in a position either to corroborate or refute his statements, my attention having been almost entirely directed to the behaviour of the centrosomes. I will therefore pass over the first part of his paper, which has to do with the living egg, without further comment, The result of his and Mr. Matthews’ study of series of sections of the eggs of the different forms they worked upon are summed up in the following words :—” After the formation of the second polar body the egg archoplasm soon disappears, and no egg centrum, or egg archoplasm (‘ovocentre ‘as opposed to ‘spermcentre’) can be discovered at any subsequent period. There is nothing like a quadrille to be seen save in doubly fertilised eggs (Toxopneustes). The archoplasm of the first cleavage-amphiaster is developed entirely from, or under the influence of the sperm archoplasm (‘spermocentre ‘of Fol), and this is derived not from the apex of the spermatozoon, but from its base, undoubtedly from the middle piece (Toxopneustes Arbacia)…. There is no centrum save as an artefact.”

With regard to all but the last sentence, my results are practically the same as Wilson’s. In as far, therefore, as my work agrees with his, I shall deal very shortly with the facts, dwelling in greater detail on those points where our results differ.

Method.—At once after the fertilisation of a great number of ova certain quantities were preserved, in a mixture of corrosive sublimate and acetic acid, at intervals of about five minutes, until the first cleavage-plane made its appearance. This usually took place about one and a half hours after fertilisation, but the time varied greatly with the temperature of the surroundings. After being hardened in alcohol, the eggs were embedded in paraffin, cut into sections, and stained with Heidenhain’s iron-hæmatoxylin.

Fig. 1 is a drawing of a section of an unfertilised ovum, killed immediately after leaving the parent’s body. The polar bodies are given off before the egg leaves the mother, but the nucleus has not yet taken up a central position. At this stage there is no sign of an astrosphere or of a centrosome. The next stage is taken (fig. 2) shortly after the entrance of the spermatozoon, which may be at any point on the surface, and is not affected by the position of the female pronucleus. The tail of the spermatozoon has dropped off, and the head and middle piece have turned completely round, so that the latter is nearest the centre of the egg. That this rotation takes place, as Wilson is the first to point out for Echinoderm eggs, I had Convinced myself long before seeing his paper. The process is exactly parallel to that described by Fick (3) as occurring in the egg of the axolotl. For a very short time the middle piece persists as a small faintly-stained body attached to the sperm head. It soon becomes separated, and is converted into the astrosphere. At first the rays are very short, and all start from a central point, hut gradually they lengthen out, and a finely granulated central mass makes its appearance, in the midst of which lies a single centrosome. As the astrosphere grows the granular central mass at first becomes reticulate, and in this condition the division of the astrosphere takes place, Fig. 5, but finally the network disappears, leaving a clear homogeneous central mass. The centrosome is of extreme minuteness. Figs. 2, 3, and 4 show the above stages.

The sperm head consists of a mass of chromatin enclosed in a loose membrane. At first cone-shaped, it gradually becomes more irregular in contour, till it appears as a roundish or oval lump, and the membrane so closely approximated as to become indistinguishable. As regards the so-called “fusion “I have nothing to add to Wilson’s account. The astrosphere divides into two about the same time as the sperm head comes into contact with the egg-nucleus, which has by this time taken up a central position. The two products of division both resemble the original astrosphere at the stage drawn in fig. 3. There is a finely reticular central mass in each, but in no single instance was I able to see a centrosome. Although I find a similar absence at this stage in Phallusia, still I believe that further examination is all that is necessary to prove that the centrosome exists at this stage also. The two astrospheres gradually travel to opposite poles of the ovum, as Wilson has already described. Shortly after taking up their positions there, they exhibit each a clear inside space¹ (the granular central mass having disappeared), in the middle of which are two clearly distinct centrosomes (fig. 6). It is probably this stage that Fol interpreted as being the one in which the two halves of the egg- and sperm-centrosomes respectively are about to fuse, one sperm half with one egg half, to form two single composite centrosomes (cf. Fol, fig. 9). From his drawings, however, even when it is taken into consideration that they are but rough woodcuts, one is tempted to doubt whether Fol ever really saw a centrosome at all. It is quite certain, as Wilson points out, that the clear area round the nucleus in which the “quadrille “is drawn as taking place is an artefact. I have obtained like results after using certain reagents, especially with the eggs of sea-urchins which had been kept some time in the tanks previously. With regard to the decrease in the length of the rays (mentioned by Wilson) during the Amphiaster (two-starred) stage, I cannot confirm his results. On the contrary, in some instances they are larger than at any other time, reaching nearly to the periphery of the ovum, and the “stratum corneum” is extremely’well marked.

Further than the Amphiaster (two-starred) stage of the first segmentation nucleus I have not investigated. The conclusions which I think may be drawn from these facts I shall reserve till later, in the general summary at the end of the paper.

I studied the maturation and fertilisation of the ova of this Ascidian, partly with a view to comparing the origin of and the rôle played by the centrosomes with what I had found to be the case in the egg of Sphærechinus, but more especially to compare the case of Phallusia with that of Styelopsis described fairly recently by Julin (5). The method employed was precisely similar to the one already given for Sphærechinus. A mixture of 90 to 95 per cent, saturated solution of corrosive sublimate with 10 to 5 per cent, glacial acetic was found in both cases to be the best preservative when followed by Heidenhain’s iron hæmatoxylin stain.

Here again I took no account of the changes to be observed in the living egg. These were sufficiently described by Strasburger (7) so long ago as 1875.

The ova were examined by means of sections in the ovary, oviduct, unfertilised after leaving the parent’s body, and after fecundation. Unfortunately with regard to the development of the ova I can give no details. I never succeeded in getting satisfactory preparations of the nuclear figures of the ovogones while in the germinative zone. (I use Boveri’s (1) nomenclature in his well-known diagram of the sexual cells of Ascaris.)

I can therefore give no such details as described by Julin for Styelopsis. Although I obtained preparations showing karyokinetic division in the ovaries of very young Ascidians, yet the cells themselves were so small that counting the chromosomes was impossible. I found precisely the same difficulty with the testes.

Transverse sections of the ovary show ovogones in various stages in the zone of growth. The nucleus is relatively of enormous size, is vesicular, and contains a large circular “nucleolus” of chromatin (fig. 8). As the egg passes into the branches of the oviduct, the nucleus begins to get smaller, and lessens so quickly in size that it becomes hardly one sixth of its original diameter. The nucleolus disappears, the chromatin is more regularly dispersed in the form of a long thread, and then is split into eight chromosomes. The details I have not been able to follow (fig. 9).

The polar bodies are given off shortly after the ova are shed into the sea water, irrespective of whether they are fertilised or not. The nucleus loses its membrane, a spindle is formed, and the eight chromosomes are arranged in the equatorial plane. As to what follows I do not wish to lay down any absolute facts. The polar spindles are so excessively small, and the chromosomes lie so close to one another, that accurate observation is a matter of extreme difficulty. From the study of a large seriesof sections, however, I am convinced that the rôle played by the chromosomes is very different from what has been described for Ascaris, and on which so many theoretical speculations have been based. The eight chromosomes of the first polar spindle lengthen out, become dumbbell-shaped, and finally divide in the middle (fig. 10). Eight chromosomes pass into the first polar body, which also divides karyokinetically into two, each having eight chromosomes. (I have never counted more than six or seven chromosomes in the products of division of the first polar body, but I think it may be taken for granted that there must be eight [fig. 11].) The chromosomes left in the first polar spindle again divide in the same manner (?), and about eight—certainly more than four—chromosomes pass into the second polar body (I have counted six distinctly), and eight (?) remain. To exactly determine the number of chromosomes left in the female pronucleus after the formation of the second polar body, and before it passes into the resting condition, is a matter of great difficulty, as the time between the two phases is very short (fig. 12). I have counted four, six, seven, eight, and nine in different instances. This discrepancy is partly due to the great tendency the chromosomes have to lump themselves together into one mass, so that the female pronucleus resembles the nucleus of the ovogone in having a large “nucleolus “of chromatin. This is broken up into a network as soon as the nucleus develops a membrane and passes into the resting condition. It then withdraws somewhat from the periphery of the egg, though still maintaining an excentric position. It should here be remarked that throughout the whole process of maturation there is no sign of a centrosome or archoplasmic sphere.

As is shown, however, in fig. 9 c, at either end of the spindle is a deeply-stained body, which may be called a pseudocentrosome. This is nothing more than the point where the slightly stained spindle-threads meet. Although there is no astrosphere, the protoplasm at the ends of the spindle is distinctly modified, the reticular structure giving way to a more granulated condition.

The spermatozoa consist of the usual three pieces—head, middle piece, and tail. When ripe they are very active, piercing through the two layers of test and follicle cells in a very short space of time. The egg puts out a “cone d’attraction,” which embraces the head of the spermatozoa. The tail drops off, and the head rotates very rapidly. In fig. 13 the stage is drawn where the head has rotated 90°. At the end of the middle piece is a deeply-staining body, which may be the centrosome. Already the rays of the astrosphere are apparent. The “cone d’attraction “subsides as soon as the sperm-head has made its way into the ovum. The head itself broadens and grows rapidly, until it reaches about twice its original size. It then suddenly splits into two. At first regular, these pieces gradually take on a beaded irregular shape, and subsequently break up into small irregularly-shaped chromosomes, usually about eight or nine in number (figs. 14,15, and 16). These chromosomes are only transitory structures, at least as far as outward appearances go, for very shortly afterwards the male pronucleus passes completely into the resting condition. Meanwhile the astrosphere has grown considerably, and passed through the same phases of formation as described in the case of Sphærechinus. The rays are remarkable for their length and thickness, the whole structure being much coarser than the astrosphere of Sphærechinus. The centrosome is also very large and distinct, and soon after the centre of the astrosphere has become finely granular, divides into two, although the corresponding division of the astrosphere itself does not take place till somewhat later. As Boveri (2) has described and figured for Ciona intestinalis, the astrosphere lengthens out,—the rays contractingsomewhat,—becomesdumbbell-shaped, and finally constricted off in the middle into two separate spheres. In Phallusia, however, the pronuclei fuse somewhat differently, as will be seen by comparing fig. 18 with fig. 29 in Boveri’s paper (2). The two astrospheres and also the two nuclei are at this stage very difficult to stain, and this may account for the fact that from this until the Amphiaster stage there was no trace of a centrosome to be found. The interior of the astropheres show a reticular structure, which is maintained till the Amphiaster stage, when the centrosomes become visible again, and the network gives place to a clear “space,” or at most a slightly reticular central mass (figs. 17 and 18).

In the first cleavage-spindle I have counted from thirteen to sixteen chromosomes, and on theoretical grounds it is necessary to assume that the latter is the correct number, which would give eight derived from the female, and eight from the male pronucleus respectively. I do not wish, however, to lay too much stress on the exactitude of these numbers, though I believe they are approximately correct (fig. 19).

With regard to the conditions of the chromatin in the development of the spermatozoa, it was only possible to substantiate the fact that one spermatocyte I gives rise to two spermatocytes II, and these again each divide into two, forming in all four spermatids. Further, the two nuclear divisions take place without any intermediate resting phase. Beyond this, however, nothing could be definitely ascertained as to the number of the chromosomes in any stage of development, owing to the extreme minuteness of the cells themselves.

It may be interesting to note that the ova of certain specimens, which had been kept in the aquarium tanks for a long time, were found to be infested with long rod-like bodies seemingly of a bacillic nature. Although these ova were mixed with ripe sperm, no single fertilisation ever took place. That they were not, however, quite destitute of vitality is shown by the fact that polar-spindles were formed, although of an apparently pathological nature (fig. 20).

For the structures needing high powers of the microscope, Zeiss’s apochromatic 2·0 mm., apert. 1·30, homogen. immers. was used with compensating ocular No. 8, and an Abbé condenser.

1. The above results may be shortly summarised as follows :

2. In Sphærechinus granularis and Phallusia mammillata there is no egg astrosphere or egg centrosome. Both these structures are brought into the ovum by the spermatozoon, and they give rise by division to all the subsequent astrospheres and centrosomes throughout ontogeny. There is, consequently, no such thing as a “quadrille.”

3. In both forms the sperm head rotates through 180°, and the astrophere and centrosome are elaborated out of or under the influence of the middle piece.

4. In Phallusia the nucleus of the ovocyte I contains eight chromosomes irregularly dispersed throughout its substance.

5. In the two succeeding nuclear divisions these eight chromosomes divide into sixteen each time, eight passing out into the first and eight into the second polar body. There is consequently no equalling or “reducing division” at this period.

6. The sperm head breaks off into eight chromosomes, and sixteen are found in the first segmentation spindle.

The origin and fate both of astrospheres and centrosomes in Sphærechinus and Phallusia certainly tend to support Boveri’s generalisation, that the ovum when ready for fertilisation possesses two out of the three essentials for cell division, viz. cytoplasm and nucleoplasm, but is without the third, or centrosome; while, on the other hand, the ripe [spermatozoon possess nucleoplasm and centrosome, but little or no cytoplasm. It is therefore only when the union of the male and female cells takes place that the requisite conditions for development are fulfilled. Where careful observations have been made this supposition has almost always been found to hold good, e.g. in the case of Rhynchelmis (Vejdoffsky, 8), Ascaris (Boveri, 2), Axolotl (Fick, 3), and Styelopsis (Julin, 5); while only in one instance has it been incontestably denied, viz. by Wheeler (9) for Myzostomum. When, however, it is borne in mind that nuclear and cell-division can take place without the presence of an astrosphere or centrosome, the supposed importance of the latter asan organ of division is greatly lessened. It is, moreover, extremely difficult to offer any explanation as to why the first cleavage spindle should have two astrospheres and two or four centrosomes, while the polar spindles (Phallusia, Ascaris, Sagitta, Ciona, &c.) may have none. Again, the relation between centrosome and astrosphere is very obscure; are the rays produced by the action of the centrosome, or vice versâ? The former alternative seems to the most probable in the case of Phallusia, where, as is shown in fig. 13, while the spermatozoon is quite at the surface of the ovum the posterior half of the middle piece has become a centrosome, and already acted on the cytoplasm of the egg to produce a radial appearance. In spermatozoa simply killed and stained there is no sign of a centrosome in the middle piece, which points to a direct metamorphosis of part of the middle piece into a centrosome as soon as the spermatozoon penetrates into the ovum.

Neither in Sphærechinus nor Phallusia is there any evidence to point to the centrosome as being artefact. In the former it appears to be all that remains of the middle-piece, and is brought to light by the latter’s disintegration into granules and final disappearance.

Finally, with regard to a more important matter, viz. the relations of the chromatin substance during the maturation of the ovum, Phallusia agrees more with the vertebrates than the invertebrates. I cannot go into the subject, however, in any great detail, because I have been unable to trace the history of the eight chromosomes in the nucleus of ovocyte I. Still, there are one or two points worth noting. In the first place, Phallusia agrees with all the other forms in the ovocyte I containing half the number of chromosomes typical for the given species. Rückert (6) puts this first and foremost among the few ascertained facts that we have at present in this complicated subject. He writes:—”Alie genauereUntersuchungen der letzen Jahre stimmen darin ueberein, dass schon vor der ersten Reifungstheilung Chromatin-portionem auftreten deren Zahl die Hälfte betragt von der Normalzahl der Chromosomen der betreffenden Species…. Am klarsten liegt der Reifungsvorgatig, wenn sie aus vier deutlich geschiedenen “Unterabtheilungen, Stäbchen oder Kugeln,bestehen (Vierergruppen). In diesem typischen Fall geht die Reifung bekanntlich folgendermassen vor sich : Durch zwei ohne Ruhephase auf einander folgende mitotische Reifungstheilungen werden die Vierergruppen in der Weise gevierteilt dass in jede Enkelzelle (Spermatiden, Reifes Ei) von jeder Gruppe ein einziges Stabchen als Chromosoma gelangt, womit die schon durch die Zahl der Vierergruppen vorbereitete Reduktion definitiv vollzogen wird.”

It is obvious that if these “Vierergruppen “are to be looked for in Phallusia, it is not in the nucleus of the ovocyte I that they are contained, but in the three polar bodies and the female pronucleus. That is to say, there are eight “Vierergruppen,” as each of the eight chromosomes divides twice, making thirtytwo pieces in all. Julin, on the other hand, looks upon the eight chromosomes in Styelopsis as themselves forming two “Vierergruppen;” and the subsequent formation of the polar bodies, albeit on these he confesses to have made few observations, bears out this view. At the first polar division four chromosomes pass out, and at the second two, leaving two quarters of the two original chromosomes, which had divided twice precociously to give rise to two “Vierergruppen.” The spermatozoon brings into the ovum two quarters likewise, so that the first segmentation nucleus possesses four chromosomes, or double the number contained in the nucleus of the ovocyte I. In fact, as regards the number of chromosomes, Styelopsis exactly resembles Ascaris bivaleus. In Phallusia each of the original eight chromosomes is a single complete structure; and if the process were like what occurs in Styelopsis, the nucleus of the ovocyte I would contain thirty-two chromosomes. Owing to .its extremely small size this is a mechanical impossibility, and hence there is no such precocious splitting to form eight “Vierergruppen.”

Hence it seems to follow that those who would see in this precocious splitting a process especially brought about in order to ensure a more varied combination of ancestral units, are obliged to recognise that it may take place in one species of the same group and not in another. In Phallusia at no one stage of maturation there are thirty-two chromosomes “to choose from,” so to speak, but the division takes place exactly as in normal cell-division, except that the chromosomes divide transversely and not longitudinally (? in second polar body). On the contrary, the two cases of Phallusia and Styelopsis seem to point towards the phenomena of maturation being nothing more than normal cell-division. Whereas in the former case the large number of chromosomes prevents precocious division, in the latter the small number allows it, and it is possible that some good to the organism may be gained thereby. The point to be found out is at what stage does the reduction from sixteen to eight chromosomes in the development of the sexual cells of Phallusia take place, which is the only real “reducing division “of the kind.

The fact that the process of maturation of the egg of Phallusia resembles what has been described for the egg of certain vertebrates, may be an additional point of evidence for a phylogenetic connection between them and the Ascidians.

NAPLES ; March, 1805.

SHORTLY after the above notes were sent in to await publication an interesting and suggestive paper by Boveri¹ appeared, which it is necessary for me here to briefly notice.

If I understand Boveri aright, his results and mine agree up to the stage I have figured in fig. 3, but after that they differ considerably. In the stage I have drawn in fig. 6 Boveri finds the centrosome, not as one, or two, very minute intensively staining granules, but as a hollow vesicle swollen to such a size as to be separated from the radial striations by a very narrow “heller Hof.” He finds no trace of any deeply-staining central body. In going carefully over my preparations for a second time, I cannot discover the slightest reason for Boveri’s interpretation. The one, or two, central bodies are so clearly defined that I am surprised Boveri has not found them. Of course it is possible that, as he suggests, this body, or bodies, may be merely the “Centralkbrner “of the centrosome ; but if this is so I am at a loss to know where the real centrosome is. I must, however, state that in one or two preparations I have distinctly seen a ring round the centrosomes, e.g. fig. 6, which according to Boveri should be the centrosome in an earlier stage of swelling up than he has figured. I consider, however, that this appearance is unimportant and may be an artefact, as in many cases where the centrosomes were very plain,— e.g. in figs. 3, 4, and 11,—there was no trace of it. It is, I think, a matter of some regret that Boveri has as yet only published a single figure illustrating his work. I hope, however, I may not be construed as in any way trying to depreciate the accurate and valuable work of such a distinguished observer as Professor Boveri, who has had such a far wider experience in cellular morphology than myself, but in this particular case I cannot help thinking that his interpretation is erroneous.

Finally, I may mention that the process I have described, viz. the homogeneous middle-piece of the spermatozoon becoming first the granular, then the reticular, and finally again the homogeneous central mass of the astrosphere (figs. 3, 4, 5, and 6 for Echinus, and figs. 14, 15, 17, 18, and 19 for Phallusia) is almost exactly the same as Vejdoffsky described for Rhynchelmis. From these results he believes that protoplasm is at first quite homogeneous and structureless ; that then very small granules appear which group themselves together to form a reticulum, which may become once more homogeneous. I do not pretend, however, to use this word “homogeneous” other than as before explained, in a purely relative sense. With higher powers of the microscope I see no reason to suppose that the middle-piece would not itself present a “Wabenstructur,” and that the network is anything more than a coarse protoplasmic reticulum. It is right, moreover, to mention that I was not biased by Vejdoffsky’s work when noting the above process, for my attention was not drawn to this particular point until reading his paper again after my own had been finished and sent in for publication.

A point worthy of notice is that, at least in one particular instance (fig. 14), the homogeneity of the central mass has been arrived at before the division of the spermastrosphere.

M. D. HILL.

June, 1895.

Illustrating Mr. M. D. Hill’s “Notes on the Fecundation of the Egg of Sphærechinus granularis, and on the Maturation and Fertilisation of the Egg of Phallusia mammillata.”

cent. Centrosome, chr. Chromosomes, f. pron. Female pronucleus. m. pron. Male pronucleus. 1st p. b. First polar body. 2nd p. b. Second polar body. Sp. ast. Sperm astrosphere. Sp. h. Sperm head. Sp. cent. Sperm centrosome. 1st sep. sp. First segmentation. Ist sep. n. First segmentation nucleus, m. p. Middle piece. Ist p. s. First polar spindle. 2nd p. s. Second polar spindle.

FIGS. 1—6.—Cross sections of eggs of Sphærechinus granularis.

Fig. 1. Section of unfertilised egg.

Fig. 2. Section of egg ten minutes after fertilisation.

Fig. 3. Section of egg fifteen minutes after fertilisation. Sperm astrosphere has grown in size, and the centrosome is apparent.

Fig. 4. Section of egg twenty minutes after fertilisation. Sperm astrosphere is in contact with female pronucleus.

Fig. 5. Section of egg twenty-five minutes after fecundation. Male pronucleus has “fused” with female, and astrosphere has divided. No trace of sperm centrosome.

Fig. 6. Section of egg forty-five minutes after fertilisation. Centrosome has appeared again and divided into two in each astrosphere. Central mass has become entirely homogeneous.

FIGS. 7—20.—Phallusia mammillata.

Fig. 7. Cross-section through ovary of Phallusia mammillata, with ovarian ova.

Fig. 8. Cross-section of an unfertilised ovum directly after leaving oviduct. Nucleus is enormously reduced in size, and chromosomes are eight in number.

Fig. 9. Shows changes in nucleus in forming first polar spindle.

Fig. 10. Shows extension of first polar body.

Fig. 11. The first polar body has divided into two, and the second polar body has also been formed. (Slightly less magnification than Fig. 10.)

Fig. 12. Shows the changes through which remaining chromosomes from second polar spindle pass during formation of female pronucleus.

Fig. 13. Cross-section of egg a few minutes after fertilisation. Sperm head has rotated through 90°.

Fig. 13a. Sperm head enlarged.

Fig. 14. Cross-section of egg ten minutes after fertilisation. Sperm head and astrosphere have increased in size, and centrosome is evident in the granular central mass.

Fig. 15. Cross-section of egg fifteen minutes after fertilisation. Sperm head has split into two.

Fig. 16. Cross-section of egg twenty minutes after fertilisation. Sperm head has broken up into eight chromosomes, and central mass has (unusually early) become homogeneous.

Fig. 17. Sperm head has passed into the resting stage of the male pronucleus. The astrosphere is in act of division, and central mass reticular.

Fig. 18. Sperm astrosphere has divided into two, and two pronuclei are in contact.

Fig. 19. First segmentation spindle with sixteen chromosomes. Central masses of atrospheres have become nearly homogeneous, and centrosomes divided into two.

Fig. 20. Cross-section of an unfertilised ovum, taken from oviduct of a specimen living in aquarium.

FIG. 21. Shows transformation of middle piece of spermatozoon into central mass of astrosphere. Slightly diagrammatic.