The Works of Francis Balfour 1-11
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Foster M. and Sedgwick A. The Works of Francis Balfour Vol. I. Separate Memoirs (1885) MacMillan and Co., London.
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Foster M. and Sedgwick A. The Works of Francis Balfour Vol. II. A Treatise on Comparative Embryology 1. (1885) MacMillan and Co., London. Foster M. and Sedgwick A. The Works of Francis Balfour Vol. III. A Treatise on Comparative Embryology 2 (1885) MacMillan and Co., London. Foster M. and Sedgwick A. The Works of Francis Balfour Vol. IV. Plates (1885) MacMillan and Co., London.
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Vol I. Separate Memoirs (1885)
XI. On the Phenomena Accompanying the Maturation and Impregnation of the Ovum
The brilliant discoveries of Strasburger and Auerbach have caused the attention of a large number of biologists to be turned to the phenomena accompanying the division of nuclei and the maturation and impregnation of the ovum. The results of the recent investigations on the first of these points formed the subject of an article by Mr Priestley in the sixteenth volume of this Journal, and the object of the present article is to give some account of what has so far been made out with reference to the second of them. The matters to be treated of naturally fall under two heads : (i) the changes attending the ripening of the ovum, which are independent of impregnation ; (2) the changes which are directly due to impregnation.
Every ovum as it approaches maturity is found to be composed (Fig. i) of (i) a protoplasmic body or vitellus usually containing yolk-spherules in suspension ; (2) of a germinal vesicle or nucleus,
FIG. i. Unripe ovum of Toxopneustes lividus (copied from Hertwig).
1 From the Quarterly Journal of Microscopical Science, April, 1878.
containing (3) one or more germinal spots or nucleoli. It is
with the germinal vesicle and its contents that we are especially
concerned. This body at its full development has a more
or less spherical shape, and is enveloped by a distinct membrane.
Its contents are for the most part fluid, but may be more or
less granular. Their most characteristic component is, however, a
protoplasmic network which stretches from the germinal spot to
the investing membrane, but is especially concentrated round
the former (Fig. i). The germinal spot forms a nearly homogeneous body, with frequently one or more vacuoles. It occupies
an often excentric position within the germinal vesicle, and is
usually rendered very conspicuous by its high refrangibility. In
many instances it has been shewn to be capable of amoeboid
movements (Auerbach, and Os. Hertwig), and is moreover more
solid and more strongly tinged by colouring reagents than the
remaining constituents of the germinal vesicle. These peculiarities have caused the matter of which it is composed to be
distinguished by Auerbach and Hertwig as nuclear substance.
In many instances there is only one germinal spot, or one main spot, and two or three accessory smaller spots. In other cases, e.g. Osseous Fish, there are a large number of nearly equal germinal spots. The eggs which have been most investigated with reference to the changes of germinal vesicle are those with a single germinal spot, and it is with these that I shall have more especially to deal in the sequel.
The germinal vesicle occupies in the first instance a central position in the ovum, but at maturity is almost always found in close proximity to the surface. Its change of position in a large number of instances is accomplished during the growth of the ovum in the ovary, but in other cases does not take place till the ovum has been laid.
The questions which many investigators have recently set themselves to answer are the two following: (i) What becomes of the germinal vesicle when the ovum is ready to be impregnated ? (2) Is any part of it present in the ovum at the commencement of segmentation ? According to their answers to these questions the older embryologists roughly fall into two groups: (i) By one set the germinal vesicle is stated to completely disappear and not to be genetically connected with the subsequent nuclei of the embryo. (2) According to the other set it remains in the ovum and by successive divisions forms the parent nucleus of all the nuclei in the body of the embryo. Though the second of these views has been supported by several very distinguished names the first view was without doubt the one most generally entertained, and Haeckel (though from his own observations he was originally a supporter of the second view) has even enunciated the theory that there exists an anuclear stage, after the disappearance of the germinal vesicle, which he regards as an embryonic repetition of the monad condition of the Protozoa.
While the supporters of the first view agree as to the disappearance of the germinal vesicle they differ considerably as to the manner of this occurrence. Some are of opinion that the vesicle simply vanishes, its contents being absorbed in the ovum ; others that it is ejected from the ovum and appears as the polar cell or body, or Ricktungskb'rper of the Germans a small body which is often found situated in the space between the ovum and its membrane, and derives its name from retaining a constant position in relation to the ovum, and thus serving as a guide in determining the similar parts of the embryo through the different stages. The researches of Oellacher (I5) 1 in this direction deserve special mention, as having in a sense formed the foundation of the modern views upon this subject. By a series of careful observations upon the egg of the trout and subsequently of the bird, he demonstrated that the germinal vesicle of the ovum, while still in the ovary, underwent partial degeneration and eventually became ejected. His observations were made to a great extent by means of sections, and the general accuracy of his results is fairly certain, but the nature of the eggs he worked on, as well as other causes, prevented his obtaining so deep an insight into the phenomena accompanying the ejection of the germinal vesicle as has since been possible. Loven, Flemming (6), and others have been led by their investigations to adopt views similar in the main to Oellacher's. As a rule, however, it is held by believers in the disappearance of the germinal vesicle that it becomes simply absorbed, and many very accurate
1 The numbers appended to authors' names refer to the list of publications at the end of the paper.
accounts, so far as they go, have been given of the gradual
atrophy of the germinal vesicle. The description of Kleinenberg
(14) for Hydra, and Gbtte for Bombinator, may perhaps be
selected as especially complete in this respect ; in both instances
the germinal vesicle commences to atrophy at a relatively early
period.
Coming to the more modern period the researches of five workers, viz. Biitschli, E. van Beneden, Fol, Hertwig, and Strasburger have especially thrown light upon this difficult subject. It is now hardly open to doubt that while part of the germinal vesicle is concerned in the formation of the polar cell or cells, when such are present, and is therefore ejected from the ovum, part also remains in the ovum and forms a nuclear body which will be spoken of as the female pronucletis, the fate of which is recorded in the second part of this paper. The researches of Biitschli and van Beneden have been especially instrumental in demonstrating the relation between the polar bodies and the germinal vesicle, and those of Hertwig and Fol, in shewing that part of the germinal vesicle remained in the ovum. It must not, however, be supposed that the results of these authors are fully substantiated, or that all the questions connected with these phenomena are settled. The statements we have are in many points opposed and contradictory, and there is much that is still very obscure.
In the sequel an account is first given of the researches of the above-named authors, followed by a statement of those results which appear to me the most probable.
The researches of van Beneden (3 and 4) were made on the ovum of the rabbit and of Asterias, and from his observations on both these widely separated forms he has been led to conclude that the germinal vesicle is either ejected or absorbed, but that it has in no case a genetic connection with the first segmentation sphere. He gives the following description of the changes in the rabbit's ovum. The germinal vesicle is enclosed by a membrane, and contains one main germinal spot, and a few accessory ones, together with a granular material which he calls nucleoplasma, which affects, as is usual in nuclei, a reticular arrangement. The remaining space in the vesicle is filled by a clear fluid. As the ovum approaches maturity the germinal
MATURATION AND IMPREGNATION OF THE OVUM. 525
vesicle assumes an excentric position, and fuses with the peripheral layer of the egg to constitute the cicatriciilar lens. The germinal spot next travels to the surface of the cicatricular lens and forms the nuclear disc: at the same time the membrane of the germinal vesicle vanishes though it probably unites with the nuclear disc. The nucleoplasma then collects into a definite mass and forms the nucleoplasmic body. Finally the nuclear disc assumes an ellipsoidal form and becomes the nuclear body. Nothing is now left of the original germinal vesicle but the nuclear body and the nucleoplasmic body both still situated within the ovum. In the next stage no trace of the germinal vesicle can be detected in the ovum, but outside it, close to the point where the modified remnants of the vesicle were previously situated, there is present a polar body which is composed of two parts, one of which stains deeply and resembles the nuclear body, and the other does not stain but is similar to the nucleoplasmic body. Van Beneden concludes that the polar bodies are the two ejected products of the germinal vesicle. In the case of Asterias, van Beneden has not observed the mode of formation of the polar bodies, and mainly gives an account of the atrophy of the germinal vesicle, but adds very little to what was already known to us from Kleinenberg's (14) earlier observations. He describes with precision the breaking up of the germinal spot into fragments and its eventual disappearance.
Though there are reasons for doubting the accuracy of all the above details on the ovum of the rabbit, nevertheless, the observations of van Beneden taken as a whole afford strong grounds for concluding that the formation of the polar cells is connected with the disappearance, partial or otherwise, of the germinal vesicle. A very similar account of the apparent disappearance of the germinal vesicle is given by Greeff (19) who states that the apparent disappearance of the germinal spot precedes that of the vesicle.
The observations of Biatschli are of still greater importance in this direction. He has studied with a view to elucidating the fate of the germinal vesicle, the eggs of Nephelis, Lymnaeus, Cucullanus, and other Nematodes; and Rotifers. In all of these, with the exception of Rotifers, he finds polar bodies, and in this
526 MATURATION AND IMPREGNATION OF THE OVUM.
respect his observations are of value as tending to shew the wide-spread existence of these structures. Negative results with reference to the presence of the polar bodies have, it may be remarked, only a very secondary value. Biitschli has made the very important discovery that in perfectly ripe eggs of Nephelis, Lymnaeus and Cucullanus and allied genera a spindle, similar to that of ordinary nuclei in the act of division, appears close to the surface of the egg. This spindle he regards as the metamorphosed germinal vesicle, and has demonstrated that it takes part in the formation of the polar cells. He states that the whole spindle is ejected from the egg, and that after swelling up and forming a somewhat spherical mass it divides into three parts.
In the Nematodes generally. Biitschli has been unable to find the spindle modification of the germinal vesicle, but he states that the germinal vesicle undergoes degeneration, its outline becoming indistinct and the germinal spot vanishing. The position of the germinal vesicle continues to be marked by a clear space which gradually approaches the surface of the egg. When it is in contact with the surface a small spherical body, the remnant of the germinal vesicle, comes into view, and eventually becomes ejected. The clear space subsequently disappears. This description of Biitschli resembles in some respects that given by van Beneden of the changes in the rabbit's ovum, and not impossibly refers to a nearly identical series of phenomena. The discovery by Biitschli of the spindle and its relation to the polar body has been of very great value.
The publications of van Beneden, and more especially those of Biitschli, taken by themselves lead to the conclusion that the whole germinal vesicle is either ejected or absorbed. Nearly simultaneously with their publications there appeared, however, a paper by Oscar Hertwig (11) on the eggs of one of the common sea urchins ( Toxopneustes lividus), in which he attempted to shew that part of the germinal vesicle, at any rate, was concerned in the formation of the first segmentation nucleus. He believed (though he has himself now recognised that he was in error on the point) that no polar cell was formed in Toxopneustes, and that the whole germinal vesicle was absorbed, with the exception of the germinal spot which remained in the egg as the female pronucleus.
MATURATION AND IMPREGNATION OF THE OVUM. 527
The following is the summary which he gives of his results, PP- 3578.
" At the time when the egg is mature the germinal vesicle undergoes a retrogressive metamorphosis and becomes carried towards the surface of the egg by the contraction of the protoplasm. Its membrane becomes dissolved and its contents disintegrated and finally absorbed by the yolk. The germinal spot appears, however, to remain unaltered and to continue in the yolk and to become the permanent nucleus of the ripe ovum capable of impregnation."
After the publication of Butschli's monograph, O. Hertwig (12) continued his researches on the ova of Leeches (Hcemopis and Nephelis], and not only added very largely to our knowledge of the history of the germinal vesicle, but was able to make a very important rectification in Butschli's conclusions. The following is a summary of his results : The germinal vesicle, as in other cases, undergoes a form of degeneration, though retaining its central position ; and the germinal spot breaks up into fragments. The stages in which this occurs are followed by one when, on a superficial examination, the ovum appears to be absolutely without a nucleus ; but there can be demonstrated by means of reagents in the position previously occupied by the germinal vesicle a spindle nucleus with the usual suns at its poles, which Hertwig believes to be a product of the fragments of the germinal spot. This spindle travels towards the periphery of the ovum and then forms the spindle observed by Butschli. At the point where one of the apices of the spindle lies close to the surface a small protuberance arises which is destined to form the first polar cell. As the protuberance becomes more prominent one half of the spindle passes into it. The spindle then divides in the normal manner for nuclei, one half remaining in the protuberance, the other in the ovum, and finally the protuberance becomes a rounded body united to the egg by a narrow stalk. It is clear that if, as there is -every reason to think, the above description is correct, the polar cell is formed by a simple process of cell-division and not, as Butschli believed, by the forcible ejection of the spindle.
The portion of the spindle in the polar cell becomes a mass of granules, and that in the ovum becomes converted without
528 MATURATION AND IMPREGNATION OF THE OVUM.
the occurrence of the usual nuclear stage into a fresh spindle. A second polar cell is formed in the same manner as the first one, and the first one subsequently divides into two. The portion of the spindle which remains in the egg after the formation of the second polar cell reconstitutes itself into a nucleus the female pronucleus and travelling towards the centre of the egg undergoes a fate which will be spoken of in the second part of this paper.
The most obscure part of Hertwig's work is that which concerns the formation of the spindle on the atrophy of the germinal vesicle, and his latest paper, though it gives further details on this head, does not appear to me to clear up the mystery. Though Hertwig demonstrates clearly enough that this spindle is a product of the metamorphoses of the germinal vesicle, he does not appear to prove the thesis which he maintains, that it is the metamorphosed germinal spot.
Fol, to whom we are indebted in his paper on the development of Geryonia (7) for the best of the earlier descriptions of the phenomena which attend the maturation of the egg, and later for valuable contributions somewhat similar to those of Biitschli with reference to the development of the Pteropod egg (8), has recently given us a very interesting account of what takes place in the ripe egg of Asterias glacialis (9). In reference to the formation of the polar cells, his results accord closely with those of Hertwig, but he differs considerably from this author with reference to the preceding changes in the germinal vesicle. He believes that the germinal spot atrophies more or less completely, but that in any case its constituents remain behind in the egg, though he will not definitely assert that it takes no share in the formation of the spindle at the expense of which both the polar cells and the female pronucleus are formed. The spindle with its terminal suns arises, according to him, from the contents of the germinal vesicle, loses its spindle character, travels to the surface, and reacquiring a spindle character is concerned in the formation of the polar cells in the way described by Hertwig.
Giard (10) gives a somewhat different account of the behaviour of the germinal vesicle in Psammechinus miliaris. At maturity the contents of the germinal vesicle and spot mix
MATURATION AND IMPREGNATION OF THE OVUM. 529
together and form an amoeboid mass, which, assuming a spindle form, divides into two parts, one of which travels towards the centre of the egg and forms the female pronucleus, the other remains at the surface and gives origin to two polar cells, both of which are formed after the egg is laid. What Giard regards as the female pronucleus is perhaps the lower of the two bodies which take the place of the original germinal vesicle as described by Fol. Vide the account of Fol's observations on p. 531.
Strasburger, from observations on Phallusia, accepts in the main Hertwig's conclusion with reference to the formation of the polar bodies, but does not share Hertwig's view that either the polar bodies or female pronucleus are formed at the expense of the germinal spot alone. He has further shewn that the socalled canal-cell of conifers is formed in the same manner as the polar cells, and states his belief that an equivalent of the polar cells is widely distributed in the vegetable subkingdom.
This sketch of the results of recent researches will, it is hoped, suffice to bring into prominence the more important steps by which the problems of this department of embryology have been solved. The present aspects of the question may perhaps be most conveniently displayed by following the history of a single ovum. For this purpose the eggs of Asterias glacialis, which have recently formed the subject of a series of beautiful researches by Fol (9), may conveniently be selected.
The ripe ovum (fig. 2), when detached from the ovary, is formed of a granular vitellus without a vitelline membrane, but enveloped in a mucilaginous coat. It contains an excentrically situated germinal vesicle and germinal spot. In the former is present the usual protoplasmic reticulum. As soon as the ovum reaches the sea water the germinal vesicle commences to undergo a peculiar metamorphosis. It exhibits frequent changes of form, its membrane becomes gradually absorbed and its outline indented and indistinct, and finally its contents become to a certain extent confounded with the vitellus (Fig. 3).
The germinal spot at the same time loses its clearness of outline and gradually disappears from view.
At a slightly later stage in the place of the original germinal vesicle there may be observed in the fresh ovum two clear spaces (fig. 4), one ovoid and nearer the surface, and the second
530 MATURATION AND IMPREGNATION OF THE OVUM.
FIG. 2. Ripe ovum of Asterias glacialis enveloped in a mucilaginous envelope, and
containing an excentric germinal vesicle and germinal spot (copied from Fol).
FlG. 3. Two successive stages in the gradual metamorphosis of the germinal vesicle
and spot of the ovum of Asterias glacialis immediately after it is laid (copied
from Fol).
FIG. 4. Ovum of Asterias glacialis, shewing, the clear spaces in the place of the germinal vesicle. Fresh preparation (copied from Fol).
more irregular in form and situated rather deeper in the vitellus. By treatment with reagents the first clear space is found to be formed of a spindle with two terminal suns on the lower side of which is a somewhat irregular body (Fig. 5). The second clear space by the same treatment is she\vn to contain a round body.
MATURATION AND IMPREGNATION OF THE OVUM. 531
FIG. 5. Ovum of Asterias glacialis, at the same stage as Fig. 4, treated with picric
acid (copied from Fol).
Fol concludes that the spindle is formed out of part of the germinal vesicle and not of the germinal spot, while he sees in the round body present in the lower of the two clear spaces the metamorphosed germinal spot. He will not, however, assert that no fragment of the germinal spot enters into the formation of the spindle. It may be observed that Fol is here obliged to fill up (so far at least as his present preliminary account enables me to determine) a lacuna in his obseivations in a hypothetical manner, and O. Hertwig's (13) most recent observations on the ovum of the same or an allied species of Asterias tend to throw some doubt upon Fol's interpretations.
The following is Hertwig's account of the changes in the germinal vesicle. A quarter of an hour after the egg is laid the protoplasm on the side of the germinal vesicle towards the surface of the egg develops a prominence which presses inwards the wall of the vesicle. At the same time the germinal spot develops a large vacuole, in the interior of which is a body consisting of nuclear substance, and formed of a firmer and more refractive material than the remainder of the germinal spot. In the above-mentioned prominence towards the germinal vesicle, first one sun is formed by radial striae of protoplasm, and then a second makes its appearance, while in the living ovum the germinal spot appears to have vanished, the outline of the germinal vesicle to have become indistinct, and its contents to have mingled with the surrounding protoplasm. Treatment with reagents demonstrates that in the process of disappearance of the germinal spot the nuclear mass in the vacuole forms a
532 MATURATION AND IMPREGNATION OF THE OVUM.
rod-like body, the free end of which is situated between the two suns which occupy the prominence of the germinal vesicle. At a slightly later period granules may be seen at the end of the rod and finally the rod itself vanishes. After these changes there may be demonstrated by the aid of reagents a spindle between the two suns, which Hertwig believes to grow in size as the last remnants of the germinal spot gradually vanish, and he maintains, as before mentioned, that the spindle is formed at the expense of the germinal spot. Without following Hertwig so far as this 1 it may be permitted to suggest that his observations tend to shew that the body noticed by Fol in the median line, on the inner side of his spindle, is in reality a remnant of the germinal spot and not, as Fol supposes, part of the germinal vesicle. Considering how conflicting is the evidence before us it seems necessary to leave open for the present the question as to what parts of the germinal vesicle are concerned in forming the first spindle.
The spindle, however it be formed, has up to this time been situated with its axis parallel to the surface of the egg, but not long after the stage last described a spindle is found with one end projecting into a protoplasmic prominence which makes its appearance on the surface of the egg (Fig. 6). Hertwig believes
FIG. 6. Portion of the ovum of Asterias glacialis, shewing the spindle formed from
the metamorphosed germinal vesicle projecting into a protoplasmic prominence
of the surface of the egg. Picric acid preparation (copied from Fol).
that the spindle simply travels towards the surface, and while doing so changes the direction of its axis. Fol finds, however, that this is not the case, but that between the two conditions
1 Hertwig's full account of his observations, with figures, in the 4th vol. of the Morphologische Jahrbuch, has appeared since the above was written. The figures given strongly support Hertwig's views.
MATURATION AND IMPREGNATION OF THE OVUM. 533
of the spindle an intermediate one is found in which a spindle can no longer be seen in the egg, but its place is taken by a compact rounded body. He has not been able to arrive at a conclusion as to what meaning is to be attached to this occurrence. In any case the spindle which projects into the prominence on the surface of the egg divides it into two parts, one in the prominence and one in the egg (Fig. 7). The prominence itself with the
FIG. 7. Portion of the ovum of Asterias glacialis at the moment of the detachment
of the first polar body and the withdrawal of the remaining part of the spindle
within the ovum. Picric acid preparation (copied from Fol).
enclosed portion of the spindle becomes partially constricted off from the egg as the first polar body (Fig. 8). The part of the
FIG. 8. Portion of the ovum of Asterias glacialis, with the first polar body as it
appears when living (copied from Fol).
spindle which remains in the egg becomes directly converted into a second spindle by the elongation of its fibres without passing through a typical nuclear condition. A second polar cell next becomes formed in the same manner as the first (Fig. 9), and
FIG. 9. Portion of the ovum of Asterias glacialis immediately after the formation of
the second polar body. Picric acid preparation (copied from Fol).
534 MATURATION AND IMPREGNATION OF THE OVUM.
the portion of the spindle remaining in the egg becomes converted into two or three clear vesicles (Fig. 10) which soon unite to form a single nucleus, the female pronucleus (Fig. 11).
fC^ap^ 6 -^
FIG. 10. Portion of the ovum of Asterias glacialis after the formation of the second polar cell, shewing the part of the spindle remaining in the ovum becoming converted into two clear vesicles. Picric acid preparation (copied from Fol).
FIG. n. Ovum of Asterias glacialis with the two polar bodies and the female
pronucleus surrounded by radial strife, as seen in the living egg (copied from Fol).
The two polar cells appear to be situated between two membranes, the outer of which is very, delicate and only distinct where it covers the polar cells, while the inner one is thicker and becomes, after impregnation, more distinct and then forms what Fol speaks of as the vitelline membrane. It is clear, as Hertwig has pointed out, that the polar bodies originate by a regular cell division and have the value of cells.
MATURATION AND IMPREGNATION OF THE OVUM. 535
General conclusions.
Considering how few ova have been adequately investigated with reference to the behaviour of the germinal vesicle any general conclusions which may at present be formed are to be regarded as provisional, and I trust that this will be borne in mind by the reader in perusing the following paragraphs.
There is abundant evidence that at the time of maturation of the egg the germinal vesicle undergoes peculiar changes, which are, in part at least, of a retrogressive character. These changes may begin considerably before the egg has reached the period of maturity, or may not take place till after it has been laid. They consist in appearance of irregularity and obscurity in the outline of the germinal vesicle, the absorption of its membrane, the partial absorption of its contents in the yolk, and the breaking up and disappearance of the germinal spot. The exact fate of the single germinal spot, or the numerous spots where they are present, is still obscure; and the observations of Oellacher on the trout, and to a certain extent my own on the skate, tend to shew that the membrane of the germinal vesicle may in some cases be ejected from the egg, but this conclusion cannot be accepted without further confirmation.
The retrogressive metamorphosis of the germinal vesicle is followed in a large number of instances by the conversion of what remains into a striated spindle similar in character to a nucleus previous to division. This spindle travels to the surface and undergoes division to form the polar cell or cells in the manner above described. The part which remains in the egg forms eventually the female pronucleus.
The germinal vesicle has up to the present time only been observed to undergo the above series of changes in a certain number of instances, which, however, include examples from several divisions of the Ccelenterata, the Echinodermata, and the Mollusca, and also some of the Vermes (Nematodes, Hirudinea, Sagitta). It is very possible, not to say probable, that it is universal in the animal kingdom, but the present state of our knowledge does not justify us in saying so. -It maybe that in the case of the rabbit, and many Nematodes as described by van Beneden
536 MATURATION AND IMPREGNATION OF THE OVUM.
and by Butschli, we have instances of a different mode of formation of the polar cells.
The case of Amphibians, as described by Bambeke (2) and Hertwig (12) cannot so far be brought into conformity with our type, though observations are so difficult to make with such opaque eggs that not much reliance can be placed upon the existing statements. In both of these types of possible exceptions it is fairly clear that, whatever may be the case with reference to the formation of the polar cells, part of the germinal vesicle remains behind as the female pronucleus.
There are a large number of types, including the whole of the Rotifera J and Arthropoda, with a few doubtful exceptions, in which the polar cells cannot as yet be said to have been satisfactorily observed.
Whatever may be the eventual result of more extended investigation, it is clear that the formation of polar cells according to our type is a very constant occurrence. Its importance is also very greatly increased by the discovery by Strasburger of the existence of an analogous process amongst plants. Two questions about it obviously present themselves for solution : (i) What are the conditions of its occurrence with reference to impregnation ? (2) What meaning has it in the development of the ovum or the embryo ?
The answer to the first of these questions is not difficult to find. The formation of the polar bodies is independent of impregnation, and is the final act of the normal growth of the ovum. In a few types the polar cells are formed while the ovum is still in the ovary, as, for instance, in some species of Echini, Hydra, &c., but, according to our present knowledge, far more usually after the ovum has been laid. In some of the instances the budding off of the polar cells precedes, and in others follows impregnation ; but there is no evidence to shew that in the later cases the process is influenced by the contact with the male element. In Asterias, as has been shewn by O. Hertwig, the
1 Flemming (6) finds that, in the summer and probably parthenogenetic eggs of Lacinularia socialis, the germinal vesicle approaches the surface and becomes invisible, and that subsequently a slight indentation in the outline of the egg marks the point of its disappearance. In the hollow of the indentation Flemming believes a polar cell to be situated, though he has not definitely seen one,
MATURATION AND IMPREGNATION OF THE OVUM. 537
formation of the polar cells may indifferently either precede or follow impregnation a fact which affords a clear demonstration of the independence of the two occurrences.
To the second of the two questions it does not unfortunately seem possible at present to give an answer which can be regarded as satisfactory.
The retrogressive changes in the membrane of the germinal vesicle which usher in the formation of the polar bodies may very probably be viewed as a prelude to a renewed activity of the contents of the vesicle ; and are perhaps rendered the more necessary from the thickness of the membrane which results from a protracted period of passive growth. This suggestion does not, however, help us to explain the formation of polar cells by a process identical with cell division. The ejection of part of the germinal vesicle in the formation of the polar cells may probably be paralleled by the ejection of part or the whole of the original nucleus which, if we may trust the beautiful researches of Biitschli, takes place during conjugation in Infusoria as a preliminary to the formation of a fresh nucleus. This comparison is due to Biitschli, and according to it the forma'tion of the polar bodies would have to be regarded as assisting, in some as yet unknown way, the process of regeneration of the germinal vesicle. Views analogous to this are held by Strasburger and Hertwig, who regard the formation of the polar bodies in the light of a process of excretion or removal of useless material. Such hypotheses do not unfortunately carry us very far.
I would suggest that in the formation of the polar cells part of the constituents of the germinal vesicle which are requisite for its functions as a complete and independent nucleus are removed to make room for the supply of the necessary parts to it again by the spermatic nucleus (vide p. 541). More light on this, as on other points, may probably be thrown by further investigations on parthenogenesis and the presence or absence of a polar cell in eggs which develope parthenogenetically. Curiously enough the two groups in which parthenogenesis most frequently occurs in the ordinary course of development (Arthropoda and Rotifera) are also those in which polar cells, with the possible exception mentioned above, of the parthenogenetic eggs of Lacenularia, are stated to be absent. This curious coincidence,
B. 35
538 MATURATION AND IMPREGNATION OF THE OVUM.
should it be confirmed, may perhaps be explained on the hypothesis, I have just suggested, viz. that a more or less essential part of the nucleus is removed in the formation of the polar cells ; so that in cases, .e.g. A rthropoda and Rotifera, where polar cells are not formed, and an essential part of the nucleus not therefore removed, parthenogenesis can much more easily occur than when polar cells are formed.
That the part removed in the formation of the polar cells is not absolutely essential, seems at first sight to follow from the fact of parthenogenesis being possible in instances where impregnation is the normal occurrence. The genuineness of all the observations on this head is too long a subject to enter into here 1 , but after admitting, as we probably must, that there are genuine cases of parthenogenesis, it cannot be taken for granted without more extended observation that the occurrence of development in these rare instances may not be due to the polar cells not having been formed as usual, and that when the polar cells are formed the development without impregnation is less possible.
The remarkable observations of Professor Greeff (19) on the parthenogenetic development of the eggs of Asterias rubens tell, however, very strongly against this explanation. Greeff has found that under normal circumstances the eggs of this species of starfish will develope without impregnation in simple sea water. The development is quite regular and normal though much slower than in the case of impregnated eggs. It is not definitely stated that polar cells are formed, but there can be no doubt that this is implied. Professor Greeffs account is so precise and circumstantial that it is not easy to believe that any error can have crept in ; but neither Hertwig nor Fol have been able to repeat his experiments, and we may be permitted to wait for further confirmation before absolutely accepting them.
1 The instances quoted by Siebold from Hensen and Oellacher are not quite satisfactory. In Hensen's case impregnation would have been possible if we can suppose the spermatozoa to be capable of passing into the body-cavity through the open end of the uninjured oviduct; and though Oellacher's instances are more valuable, yet sufficient care seems hardly to have been taken, especially when it is not certain for what length of time spermatozoa may be able to live in the oviduct. For Oellacher's precautions, vide Zeit. fiir -iviss. Zool. Bd. xxil. p. 202.
MATURATION AND IMPREGNATION OF THE OVUM. 539
It is possible that the removal of part of the protoplasm of the egg in the formation of the polar cells may be a secondary process due to an attractive influence of the nucleus on the cell protoplasm, such as is ordinarily observed in cell division.
Impregnation of the Ovum.
A far greater amount of certainty appears- to me to have been attained as to the effects of impregnation than as to the changes of the germinal vesicle which precede this, and there appears, moreover, to be a greater uniformity in the series of resulting phenomena. For convenience I propose to reverse the order hitherto adopted and to reserve the history of the literature and my discussion of disputed points till after my general account. Fol's paper on Asterias glacialis, is again my source of information. The part of the germinal vesicle which remains in the egg, after the formation of the second polar cell, becomes converted into a number of small vesicles (Fig. 10), which aggregate themselves into a single clear nucleus which gradually travels toward the centre of the egg and around which as a centre the protoplasm becomes radiately striated (Fig. n). This nucleus is known as \.\\Q female pronnclcus 1 . In Asterias glacialis the most favourable period for fecundation is about an hour after the formation of the female pronucleus. If at this time the spermatozoa are allowed to come in contact with the egg, their heads soon become enveloped in the investing mucilaginous coat. A prominence, pointing towards the nearest spermatozoon, now arises from the superficial layer of protoplasm of the egg and grows till it comes in contact with the spermatozoon (Figs. 12 and 13). Under normal circumstances the spermatozoon, which meets the prominence, is the only one concerned in the fertilisation, and it makes its way into the egg by passing through the prominence. The tail of the spermatozoa, no longer motile, remains visible for some time after the head has bored its way in, but its place is soon taken by a pale conical body which is, however, probably in part a product of the metamorphosis of the tail itself (Fig. 14). This body vanishes in its turn.
1 According to Hertwig's most recent statement a nucleolus is present in this nucleus.
352
540 MATURATION AND IMPREGNATION OF THE OVUM.
FIG. 12.
FIG. 13.
FIGS. 12 and 13. Small portion of the ovum of Asterias glacialis. The spermatozoa are shewn enveloped in the mucilaginous coat. In Fig. 12 a prominence is rising from the surface of the egg towards the nearest spermatozoon ; and in Fig. 13 the spermatozoon and prominence have met. From living ovum (copied from Fol).
At the moment of contact between the spermatozoon and the egg the outermost layer of the protoplasm of the latter raises itself as distinct membrane, which separates from the egg and prevents the entrance of any more spermatozoa. At the point where the spermatozoon entered a crater-like opening is left in the membrane (Fig. 14).
FIG. 14. Portion of the ovum of Asterias glacialis after the entrance of a spermatozoon into the ovum. It shows the prominence of the ovum through which the spermatozoon has entered. A vitelline membrane with a crater-like opening has become distinctly formed. From living ovum (copied from Fol).
The head of the spermatozoon when in the egg forms a nucleus for which the name male pronucleus may be conveniently adopted. It grows in size by absorbing, it is said, material from the ovum, though this may be doubted, and around it is formed a clear space free from yolk-spherules. Shortly after its forma
MATURATION AND IMPREGNATION OF THE OVUM. 54!
tion the protoplasm in its neighbourhood assumes a radiate arrangement (Fig. 15). At whatever point of the egg the
FIG. 15. Ovum of Asterias glacialis, with male and female pronucleus and a radial
striation of the protoplasm around the former. From living ovum (copied from
Fol).
spermatozoon may have entered, it gradually travels towards the female pronucleus. This latter, around which the protoplasm no longer has a radial arrangement, icmains motionless till it comes in contact with the rays of the male pronucleus, after which its condition of repose is exchanged for one of activity, and it rapidly approaches the male pronucleus, and eventually fuses with it (Fig. 16).
FIG. 16. Three successive stages in the coalescence of the male and female pronucleus in Asterias glacialis. From the living ovum (copied from Fol).
The product of this fusion forms the first segmentation nucleus (Fig. 17), which soon, however, divides into the two nuclei of the two first segmentation spheres. While the two pronuclei are approaching one another the protoplasm of the egg exhibits amoeboid movements.
Of the earlier observations on this subject there need perhaps only be cited one of E. van Beneden, on the rabbit's ovum,
542 MATURATION AND IMPREGNATION OF THE OVUM.
FIG. 17. Ovum of Asterias glacialis, after the coalescence of the male and female
pronucleus (copied from Fol).
shewing the presence of two nuclei before the commencement of segmentation. Btitschli was the earliest to state from observations on Rhabditis dolichura that the first segmentation nucleus arose from the fusion of two nuclei, and this was subsequently shewn with greater detail for Ascaris nigrovenosa, by Auerbach (i). Neither of these authors gave at first the correct interpretation of their results. At- a later period Biitschli (5) arrived at the conclusion that in a large number of instances (Lymntzus, Nephelis, Cucullanus, &c.), the nucleus in question was formed by the fusion of two or more nuclei, and Strasburger at first made a similar statement for PJiallusia, though he has since withdrawn it. Though Biatschli's statements depend, as it seems, upon a false interpretation of appearances, he nevertheless arrived at a correct view with reference to what occurs in impregnation. Van Beneden (3) described in the rabbit the formation of the original segmentation nucleus from two nuclei, one peripheral and the other central, and he gave it as his hypothetical view that the peripheral nucleus was derived from the spermatic element. It was reserved for Oscar Hertwig (n) to describe in Echinus lividus the entrance of a spermatozoon into the egg and the formation from it of the male pronucleus.
Though there is a general agreement between the most recent observers, Hertwig, Fol, Selenka, Strasburger, &c., as to the main facts connected with the entrance of one spermatozoon into
MATURATION AND IMPREGNATION OF THE OVUM. 543
the egg, the formation of the male pronucleus, and its fusion with the female pronucleus, there still exist differences of detail in the different descriptions which partly, no doubt, depend upon the difficulties of observation, but partly also upon the observations not having all been made upon the same species. Hertwig does not enter into details with reference to the actual entrance of the spermatozoon into the egg, but in his latest paper points out that considerable differences may be observed in occurrences which succeed impregnation, according to the relative period at which this takes place. When, in Asterias, the impregnation is effected about an hour after the egg is laid and previously to the formation of the polar cells, the male pronucleus appears at first to exert but little influence on the protoplasm, but after the formation of the second polar cell, the 'radial striae around it become very marked, and the pronucleus rapidly grows in size. When it finally unites with the female pronucleus it is equal in size to the latter. In the case when the impregnation is deferred for four hours the male pronucleus never becomes so large as the female pronucleus. With reference to the effect of the time at which impregnation takes place, Asterias would seem to serve as a type. Thus in Hirudinea, Mollusca, and Nematodes impregnation normally takes place before the formation of the polar bodies is completed, and the male pronucleus is accordingly as large as the female. In Echinus, on the other hand, where the polar bodies are formed in the ovary, the male pronucleus is always small.
Selenka, who has investigated the formation of the male pronucleus in Toxopneustes variegatus, differs in certain points from Fol. He finds that usually, though not always, a single spermatozoon enters the egg, and that though the entrance may be effected at any part of the surface, it generally occurs at the point marked by a small prominence where the polar cell was formed. The spermatozoon first makes its way through the mucous envelope of the egg, within which it swims about, and then bores with its head into the polar prominence. The head of the spermatozoon on entering the egg becomes enveloped by the superficial protoplasm, and travels inward with its envelope, while the tail remains outside. As Fol has described, a delicate membrane becomes formed shortly after the entrance of the
544 MATURATION AND IMPREGNATION OF THE OVUM.
spermatozoon. The head continues to make its way by means of rapid oscillations, till it has traversed about one eighth of the diameter of the egg, and then suddenly becomes still. The tail in the meantime vanishes, while the neck swells up and forms the male pronucleus. The junction of the male and female pronucleus is described by Fol and Selenka in nearly the same manner.
Giard gives an account of impregnation which is not easily brought into harmony with that of the other investigators. His observations were made on Psammechinus miliaris. At one point is situated a polar body and usually at the pole opposite to it a corresponding prominence. The spermatozoa on gaining access to the egg attach themselves to it and give it a rotatory movement, but according to Giard none of them penetrate the vitelline membrane which, though formed at an earlier period, now retires from the surface of the egg.
Giard believes that the prominence opposite the polar cells serves for the entrance of the spermatic material, which probably passes in by a process of diffusion. Thus, though he regards the male pronucleus as a product of impregnation, he does not believe it to be the head of a spermatozoon.
Both Hertwig and Fol have made observations on the result of the entrance into the egg of several spermatozoa. Fol finds that when the impregnation has been too long delayed the vitelline membrane is formed with comparative slowness and several spermatozoa are thus enabled to penetrate. Each spermatozoon forms a separate pronucleus with a surrounding sun ; and several male pronuclei usually fuse with the female pronucleus. Each male pronucleus appears to exercise a repulsive influence on other male pronuclei, but to be attracted by the female pronucleus. When there are several male pronuclei the segmentation is irregular and the resulting larva a monstrosity. These statements of Fol and Hertwig are at first sight in contradiction with the more recent results of Selenka. In Toxopneustes variegatus Selenka finds that though impregnation is usually effected by a single spermatozoon yet that several may be concerned in the act. The development continues, however, to be normal if three or even four spermatozoa enter the egg almost simultaneously. Under such circumstances each spermatozoon forms a separate pronucleus and sun.
MATURATION AND IMPREGNATION, OF THE OVUM. 545
It may be noticed that, while the observations of Fol and Hertwig were admittedly made upon eggs in which the impregnation was delayed till they no longer displayed their pristine activity, Selenka's were made upon quite fresh eggs ; and it seems not impossible that the pathological symptoms in the embryos reared by the two former authors may have been due to the imperfection of the egg and not to the entrance of more than one spermatozoon. This, of course, is merely a suggestion which requires to be tested by fresh observations. We have not as yet a sufficient body of observations to enable us to decide whether impregnation is usually effected by a single spermatozoon, though in spite of certain conflicting evidence the balance would seem to incline towards the side of a single spermatozoon 1 .
The discovery of Hertwig as to the formation of the male pronucleus throws a flood of light upon impregnation.
The act of impregnation is seen essentially to consist in the fusion of a male and female nucleus ; not only does this appear in the actual fusion of the two pronuclei, but it is brought into still greater prominence by the fact that the female pronucleus is a product of the nucleus of a primitive ovum, and the male pronucleus is the metamorphosed head of the spermatozoon which is itself developed from the nucleus of a spermatic cell 2 . The spermatic cells originate from cells (in the case of Vertebrates at least) identical with the primitive ova, so that the fusion which takes place is the fusion of morphologically similar parts in the two sexes.
It must not, however, be forgotten, as Strasburger has pointed out, that part of the protoplasm of the generative cells of the two sexes also fuse, viz. the tail of the spermatozoon with the protoplasm of the egg. But there is no evidence that the former is of importance for the act of impregnation. The fact that impregnation mainly consists in the union of two nuclei gives an importance to the nucleus which would probably not have been accorded to it on other grounds.
1 The recent researches of Calberla on the impregnation of the ovum of Petromyzon Planeri support this conclusion.
2 This seems the most probable view with reference to the nature of the head of the spermatozoon, though the point is not perhaps yet definitely decided.
546 MATURATION AND IMPREGNATION OF THE OVUM.
Hertwig's discovery is in no way opposed to Mr Darwin's theory of pangenesis and other similar theories, but does not afford any definite proof of their accuracy, nor does it in the meantime supply any explanation of the origin of two sexes or of the reasons for an embryo becoming male or female.
Summary.
In what may probably be regarded as a normal case the following series of events accompanies the maturation and impregnation of an egg :
(1) Transportation of the germinal vesicle to the surface of the egg.
(2) Absorption of the membrane of the germinal vesicle and metamorphosis of the germinal spot.
(3) Assumption of a spindle character by the remains of germinal vesicle, these remains being probably largely formed from the germinal spot.
(4) Entrance of one end of the spindle into a protoplasmic prominence at the surface of the egg.
(5) Division of the spindle into two halves, one remaining in the egg, the other in the prominence. The prominence becomes at the same time nearly constricted off from the egg as a polar cell.
(6) Formation of a second polar cell in same manner as first, part of the spindle still remaining in the egg.
(7) Conversion of the part of the spindle remaining in the egg after the formation of the second polar cell into a nucleus the female pronucleus.
(8) Transportation of the female pronucleus towards the centre of the egg.
(9) Entrance of one spermatozoon into the egg.
(10) Conversion of the head of the spermatozoon into a nucleus the male pronucleus.
(11) Appearance of radial striae round the male pronucleus which gradually travels towards the female pronucleus.
(12) Fusion of male and female pronuclei to form the first segmentation nucleus.
MATURATION AND IMPREGNATION OF THE OVUM. 547
List of important recent Publications on the Maturation and Impregnation of the Ovum.
1. Auerbach. Organologische Studien, Heft 2.
2. Bambeke. "Recherches s. Embryologie des Batraciens." Bull, de I'Acad. royale de Belgique, 2me ser., t. LXI, 1876.
3. E. Van Beneden. "La Maturation de 1'CEuf des Mammiferes." Bull, de PAcad. royale de Belgique, 2me se*r., t. XL, no. 12, 1875.
4. E. Van Beneden. "Contributions a 1'Histoire de la Ve"sicule Germinative, &c." Bull, de PAcad. royale de Belgiqtte, 2me se"r., t. XLI, no. i, 1876.
5. Biitschli. Eizelle, Zelltheilung, und Conjugation der Infusorien.
6. Flemming. "Studien in d. Entwickelungsgeschichte der Najaden." Sitz. d. k. Akad. Wien, B. LXXI, 1875.
7. Fol. "Die erste Entwickelung des Geryonideneies." Jenaische Zeitschrift, Vol. VII.
8. Fol. " Sur le DeVeloppement des Pteropodes." Archives de Zoologie Experimental et Generate, Vols. IV and V.
9. Fol. "Sur le Commencement de 1'He'noge'nie." Archives des Sciences Physiques et Naturelles. Geneve, 1877.
10. Giard. Note sur les premiers phe"nomenes du developpement de I'Oursin. 1877.
11. Hertwig, Oscar. "Beit. z. Kenntniss d. Bildung, &c., d. thier. Eies." Morphologisches Jahrbuch, Bd. I.
12. Hertwig, Oscar. Ibid. Morphologisches Jahrbuch, Bd. in, Heft. J.
13. Hertwig, Oscar. "Weitere Beitrage, &c." Morphologisches Jahrbuch, Bd. in, Heft 3.
14. Kleinenberg. Hydra. Leipzig, 1872.
15. Oellacher, J. "Beitrage zur Geschichte des Keimblaschens im Wirbelthiereie." Archiv f. micr. Anat., Bd. vin.
1 6. Selenka. Befruchtung u. Theilung des Eies von Toxopneustes variegatus (Vorlaufige Mittheilung). Erlangen, 1877.
17. Strasburger. tleber Zellbildung u. Zelltheilung. Jena, 1876.
18. Strasburger. Ueber Befruchtung u. Zelltheilung. Jena, 1878.
19. R. Greeff. " Ueb. d. Bau u. d. Entwickelung d. Echinodermen." Sitzun. der Gesellschaft z. Beforderung d. gesammten Naturiviss. z. Marburg, No. 5. 1876.
548 MATURATION AND IMPREGNATION OF THE OVUM.
Postscript. Two important memoirs have appeared since this paper was in type. One of these by Hertwig, Morphologisches Jahrbuch, Bd. iv, contains a full account with illustrations of what was briefly narrated in his previous paper (13); the other by Calberla, "Der Befruchtungsvorgang beim Ei von Petromyzon Planeri" Zeit. fiir wiss. Zool., Bd. xxx, shews that the superficial layer of the egg is formed by a coating of protoplasm free from yolk- spheres, which at one part is continued inwards as a column, and contains the germinal vesicle. The surface of this column is in contact with a micropyle in the egg-membrane. Impregnation is effected by the entrance of the head of a single spermatozoon (the tail remaining outside) through the micropyle, and then along the column of clear protoplasm to the female pronucleus.
