The Works of Francis Balfour 2-2
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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.
- The Ovum and Spermatozoon | The Maturation and Impregnation of the Ovum | The Segmentation of the Ovum | Dicyemae and Orthonectidae Dicyema | Porifera | Coelenterata | Platyhelminthes | Rotifera | Mollusca | Polyzoa | Brachiopoda | Chilopoda | Discophora | Gephyrea | Chaetognatha | Nemathelminthes | Tracheata | Crustacea | Pcecilopoda | Echinodermata | Enteropneusta | Bibliography
The Works of Francis Balfour Foster M. and Sedgwick A. The Works of Francis Balfour Vol. I. Separate Memoirs (1885) MacMillan and Co., London.
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 II. A Treatise on Comparative Embryology (1885)
Chapter II. The Maturation and Impregnation of the Ovum
Maturation of the ovum and formation of the polar bodies
IN the preceding chapter the changes in the ovum were described nearly up to the period when it became ripe, and ready to be impregnated. Preparatory to the act of impregnation there take place however a series of remarkable changes, which more especially concern the germinal vesicle.
The attention of a large number of investigators has recently been directed to these changes as well as to the phenomena of impregnation. The results of their investigations will be described in the present chapter ; but for an historical account of these investigations, as well as for a determination of the delicate questions of priority, the reader is referred to Fol's memoir (No. 87), and to a paper by the author (No. 81).
The nature of the changes which take place in the maturation of the ovum 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 (87), may be selected.
The ripe ovum (fig. 22), when detached from the ovary is formed of a granular vitellus enveloped in a mucilaginous coat, the zona radiata. It contains an eccentrically-situated germinal vesicle and a germinal spot. In the former is -present the usual protoplasmic reticulum. As soon as the ovum reaches the seawater the germinal vesicle commences to undergo a peculiar metamorphosis. It exhibits frequent changes of form, the reticulum vanishes, its membrane becomes gradually absorbed, its outline indented and indistinct, and finally its contents become to a certain extent confounded with the vitellus (fig. 23).
FIG. 11. RIPE OVUM OF ASTERIAS GLACIALIS ENVELOPED IN A MUCILAGINOUS ENVELOPE, AND CONTAINING AN ECCENTRIC GERMINAL VESICLE AND GERMINAL SPOT (copied from Fol).
FIG. 23. 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).
The germinal spot .at the same time loses its clearness of outline and gradually disappears from view.
At this stage, and between it and the stage represented in fig. 26, the action of reagents brings to light certain appearances the nature of which is not yet fully cleared up for Asterias, which have been described somewhat differently by Fol for Ast. glacialis and Hertwig for Asteracanthion.
Fol finds immediately after the stage just described that a star is visible between the remains of the germinal vesicle and the surface of the egg, which is connected with an imperfectlyformed nuclear spindle extending towards the germinal vesicle 1 . At the end of the nuclear spindle may be seen the broken up fragments of the germinal spot.
1 By the term 'nuclear spindle' I refer to the peculiar form of a double striated cone assumed by the nucleus just before division, which is no doubt familiar to all my readers. I use the term star for the peculiar stellate figure usually visible at the poles of the nuclear spindle. For a further description of these parts the reader is referred to Chapter IV.
FIG. 24. OVUM OF ASTERIAS GLACIALIS, SHEWING THE CLEAR SPACES IN THE PLACE OF THE GERMINAL VESICLE. FRESH PREPARATION (copied from Fol).
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. 24), one ovoid and nearer the surface, and the second more irregular in form and situated rather deeper in the vitellus. In the upper space parallel stride may be observed. By treatment with reagents the first clear space is found to be formed of a horizontally-placed spindle with two terminal stars, near which irregular remains of the germinal spot may be seen. Slightly later (fig. 25) there may be seen on the lower side of the spindle a somewhat irregular body, which may possibly be part of the remains of the germinal spot, though Fol holds that it is probably part of the membrane of the germinal vesicle. The lower clear space visible in the fresh ovum now contains a round body, fig. 25. Fol concludes that the spindle is formed out of part of the germinal vesicle and not from 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.
The following is Hertwig's (No. 92) account of the changes in the germinal vesicle in Asteracanthion. Shortly 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 prominence first mentioned as projecting inwards towards the germinal vesicle first one star, formed by radial striae of protoplasm, and then a second make their appearance ; while 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 its vacuole forms a rod-like body, the free end of which is situated between the two stars which occupy the prominence indenting the germinal vesicle. At a later period granules may be seen at the end of the rod and finally the rod itself vanishes. After these -changes by the aid of reagents there may be demonstrated a spindle between the two stars, which Hertwig believes to grow in size as the last remnants of the germinal spot gradually vanish, and he maintains that the spindle is formed at the expense of the germinal spot. The stage with this spindle corresponds with fig. 25.
Several of Hertwig's figures closely correspond with those of Fol, and considering how conflicting is the evidence before us, it seems necessary to leave open for Asterias the question as to what parts of the germinal vesicle are concerned in forming the first spindle.
FlG. 25. OVUM OF ASTERIAS GLACIALIS, AT THE SAME STAGE AS FIG. 24, TREATED WITH PICRIC ACID (copied from Fol).
A clearer view of the phenomena which take place at this stage has been obtained by Fol in the case of Heteropods (Pterotrachaea). In the ovum a few minutes after it has been laid the germinal vesicle becomes very pale, and two stars make their appearance round a clear substance near its poles. The nucleus itself is somewhat elongated, and commences to exhibit at its poles longitudinal striae, which gradually extend towards the centre at the expense of the nuclear reticulum, from a metamorphosis of which they are directly derived. When the striae of the two sides have nearly met, thickenings may be observed in the recticulum between them, which give rise, where the striae of the two sides unite, to the central thickenings of the fibres (nuclear plate). In this way a complete nuclear spindle is established 1 .
The important result of Fol's observations on Heteropods, which tallies also with what is found in Asterias, is that a spindle with two stars at its poles is formed from the metamorphosis of the germinal vesicle and surrounding protoplasm (fig. 25).
Polar cells. The spindle has up to this time been situated with its axis parallel to the surface of the egg, but in somewhat older specimens a vertical spindle is found, with one end projecting into a protoplasmic prominence which makes its appearance on the surface of the egg (fig. 26). Hertwig believes that the spindle simply travels towards the surface, and while doing so changes the direction of its axis. Fol asserts, however, that this is not the case, but that between the two phases 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 body with a dentated outline. He has not been able to arrive at a conclusion as to what meaning is to be attached to this occurrence, which does not appear to take place in Heteropods.
For the further details on the nuclear spindle vide the next Chapter.
Fig. 26. PORTION OF 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).
FIG. 27. PORTION OF THE OVUM OF ASTERIAS GLACIALIS, WITH THE FIRST POLAR CELL AS IT APPEARS WHEN LIVING (copied from Fol).
In any case the spindle which projects into the prominence on the surface of the egg divides into two parts, one in the prominence and one in the egg (fig. 26). The prominence itself with the enclosed portion of the spindle becomes constricted off from the egg to form a body, well known to embryologists as the polar body or cell (fig. 27). Since more than one polar cell is formed, that which is the earliest to appear may be called the first polar cell.
The part of the 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. 28), and the portion of the spindle remaining in the egg becomes converted into two or three clear vesicles (fig. 29), which soon unite to form a single nucleus (fig. 30). The new nucleus which is clearly derived from part of the original germinal vesicle is called the female pronucleus, for reasons which will appear in the sequel.
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 process of cell-division and have the value of cells.
FIG. 28. PORTION OF THE OVUM OF ASTERIAS GLACIALIS IMMEDIATELY AFTER THE FORMATION OF THE SECOND POLAR CELL. PICRIC ACID PREPARA
FIG. 29. PORTION OF THE OVUM OF ASTERIAS GLACIALIS AFTER THK FORMATION OF THE SF.COND POLAR CELL, SHEWING THE PART OF THE SPINDLE REMAINING IN THE OVUM BECOMING CONVERTED INTO TWO CLEAR VESICLES. PlCRIC ACID PREPARATION (copied from Fol).
A peculiar phenomenon makes its appearance in the eggs of Clepsine shortly after the formation of the polar cells, which has been spoken of by Whitman (No. 100) as the formation of the polar rings. The following is his description of the occurrence.
" Fifteen minutes after the elimination of the polar globules (i.e. cells) a ring-like depression or constriction appears in the yolk around the oral pole, and in this depression a transparent liquid substance (nuclear ?) is collected forming the first polar ring.... The same phenomena repeat themselves later at the aboral pole.... The rings concentrate to form two discs.... Before the first cleavage both discs plunge deep into the egg."
The nature of these rings is at present quite obscure.
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.
There is however 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 an 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, the disappearance of the reticulum, 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.
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 of the ovum 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.
FIG. 30. OVUM OF ASTERIAS GLACIALIS WITH THE TWO POLAR CELLS AND THE FEMALE PRONUCLEUS SURROUNDED BY RADIAL STRIDE, AS SEEN IN THE LIVING EGG (copied from Fol).
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, some of the Vermes [Turbellarians (Leptoplana], Nematodes, Hirudinea, Alciope, Sagitta], Ascidians, etc. It is very possible, not to say probable, that such changes are universal in the animal kingdom, but the present state of our knowledge does not justify us in saying so.
In the Craniata especially our knowledge of the formation of the polar bodies is very unsatisfactory. In Petromyzon Kupffer and Benecke have brought forward evidence to shew that one polar body is formed prior to the impregnation, and a second in connection with a peculiar prominence of protoplasm after impregnation. Part of the germinal vesicle remains in the egg as the female pronucleus. In the Sturgeon the germinal vesicle atrophies and breaks up before impregnation, and afterwards part is found as a granular mass on the surface of the egg, while part forms a female pronucleus.
In Amphibia the observations of Hertwig (90) and Bambeke (77) tend to shew that after the germinal vesicle has assumed a superficial situation at the pigmented pole of the ovum its contents become intermingled with the yolk, and are in part extruded from the ovum as a granular mass after impregnation. Part of them remains in the ovum and forms a female pronucleus. Whether there is a proper division of the germinal vesicle as in typical cases is not known.
Oellacher (95) by a series of careful observations upon the egg of the trout, and subsequently of the bird, demonstrated that in the ovum while still in the ovary, the germinal vesicle underwent a kind of degeneration and eventually became ejected, in part at any rate. My own observations on Elasmobranchs, which require enlargement and confirmation, tend to shew that this part may be the membrane. Ed. van Beneden (78) has contributed some important observations on the rabbit. His account is as follows. As the ovum approaches maturity the germinal vesicle assumes an eccentric position, and fuses with the peripheral layer of the egg to constitute the cicatricular 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 plasma of the nucleus 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 parts of the polar body are the two ejected products of the germinal vesicle. We may be perhaps permitted to hold that further observations on this difficult object will demonstrate that part of the germinal vesicle remains in the ovum to form the female pronucleus.
With reference to invertebrate forms attention may be called to the observations of Biitschli (80). Although in Cucullanus a normal formation of the polar bodies takes place, yet in the Nematodes generally, Biitschli has been unable to find the spindle modification of the germinal vesicle, but 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.
In addition to the types just quoted, which may very probably turn out to be normal in the mode of formation of the polar bodies, there is a large number of types, including the whole of the Rotifera and Arthropoda with a few doubtful exceptions 1 , in which the polar cells cannot as yet be said to have been satisfactorily observed.
The more important of the doubtful cases amongst the Rotifera and Arthropoda are the following.
Flemming (83) 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.
Hoek 2 believes that he has found a polar body in the ovum of Balanus balanoides, but his observations are not perfectly satisfactory.
1 The best instance of what appears like a polar cell in Arthropoda is a body recently found by Grobben (" Entwicklungsgeschichte d. Moina rectirostris." Claus' Arbeiten, Vol. II., Wien, 1879) near the surface of the protoplasm at the animal pole of the summer and parthenogenetic eggs of Moina rectirostris, one of the Cladocera. The body stains deeply with carmine, but differs from normal polar cells in not being separated from the ovum ; and its identification as a polar cell must remain doubtful till it has been shewn to originate from the germinal vesicle.
2 "Zur -Entwicklung d. Entomostraken." Niederlandischer Archiv. f. Zoologie, Vol. in. p. 62.
Biitschli, who has expressly searched for the polar bodies in the ova of Rotifera, was unable to find any trace of them, though he found that as the egg became ripe the germinal vesicle became half its original size. In the parthenogenetic eggs of Aphis he also failed to find a trace of polar bodies, though the germinal vesicle, after the germinal spot had broken up into fragments, approached the surface and disappeared.
Whatever may be the eventual result of more extended investigation, it is clear that the formation of polar cells according to the type described above is a very constant occurrence. Its importance is 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, etc., but, according to our present knowledge, far more usually after the ovum has been laid. In some instances the budding-off of the polar cells precedes, and in other instances follows impregnation ; but there is no evidence to shew that in the latter cases the process is influenced by the contact with the male element. In Asterias, as has been shewn by O. Hertwig and Fol, the 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 bodies 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 Butschli, takes place during conjugation in Infusoria as a preliminary to the formation of a fresh nucleus. This comparison is due to Butschli, and according to it the formation 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, is removed, to make room for the supply of the necessary parts to it again by the spermatic nucleus.
My view amounts to the following, viz. that after the formation of the polar cells the remainder of the germinal vesicle within the ovum (the female pronucleus) is incapable of further development without the addition of the nuclear part of the male element (spermatozoon), and that if polar cells were not formed parthenogenesis might normally occur. A strong support for this hypothesis would be afforded were it to be definitely established that a polar body is not formed in the Arthropoda and Rotifera ; since the normal occurrence of parthenogenesis is confined to these two groups. It is certainly a remarkable coincidence that they are the only two groups in which polar bodies have not so far been satisfactorily observed.
It is perhaps possible that the part removed in the formation of the polar cells is not absolutely essential ; and this seems at first sight to follow from the fact of parthenogenesis being possible in instances where impregnation is the normal occurrence. The genuineness of 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 such 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 impossible.
1 The instances quoted by Siebold, Parthenogenesis d. Arthropoden, are not quite satisfactory. In Hensen's case, p. 234, impregnation would have been possible if we can suppose the spermatozoa to be capable of passing into the body-cavity through the
Selenka found in the case of Purpura lapillus that no polar body was formed in the eggs which did not develop, but in the case of Neritina, Biitschli has found that this does not hold good.
The remarkable observations of Greeff (No. 88) on the parthenogenetic development of the eggs of Asterias rubens tell, however, very strongly against the above hypothesis. Greeff has found that under normal circumstances the eggs of this species of starfish will develop 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. GreefPs 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.
To the suggestion already made with reference to the function of the polar cells, I will venture to add the further one, that the function of forming polar cells has been acquired by the ovum for the express purpose of Preventing parthenogenesis.
The explanation given by Mr Darwin of the evil effects of self-fertilization, viz. the want of sufficient differentiation in the sexual elements 1 , would apply with far greater force to cases of parthenogenesis.
In the production of fresh individuals, two circumstances are obviously favourable to the species, (i) That the maximum number possible of fresh individuals should be produced, (2) That the individuals should be as vigorous as possible. Sexual differentiation (even in hermaphrodites) is clearly very inimical to the production of the maximum number of individuals. There can be little doubt that the ovum is potentially capable of developing by itself into a fresh individual, and therefore, unless the absence of sexual differentiation was very injurious to the vigour of the progeny, parthenogenesis would most certainly be a very constant occurrence ; and, on the analogy of the arrangements in plants to prevent selffertilization, we might expect to find some contrivance both in animals and in plants to prevent the ovum developing by itself without fertilization. If my view about the polar cells is correct, the formation of these bodies functions as such a contrivance.
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. fur Wiss. Zool., Bd. xxii., p. 202. A better instance is that of a sow given by Bischoff, Ann. Sci. Nat., series 3, Vol. n., 1844. The unimpregnated eggs were found divided into segments, but the segments did not contain the usual nucleus, and were perhaps nothing else than the parts of an ovum in a state of disruption.
1 Darwin, Cross- and Self- Fertilization of Plants, p. 443.
Reproduction by budding or fission has probably arisen as a means of increasing the number of individuals produced, so that the co-existence of asexual with sexual reproduction is to be looked on as a kind of compromise for the loss of the power of rapid reproduction due to the absence of parthenogenesis. In the Arthropoda and Rotifera the place of budding has been taken by parthenogenesis, which may be a frequent, though not always a necessary occurrence, as in various Branchiopoda (Apus, Limnadia, etc.) and Lepidoptera (Psyche helix:, etc.); or a regular occurrence for the production of one sex, as in Bees, Wasps, Nematus, etc. ; or an occurrence confined to a certain stage in the cycle of development in which all the individuals reproduce their kind parthenogenetically, as in Aphis, Cecidomyia, Gall Insects (Neuroterus, etc.), Daphnia 1 .
On my hypothesis the possibility of parthenogenesis, or at any rate its frequency, in Arthropoda and Rotifera is possibly due to the absence of polar cells. In the case of all animals, so far as is known to me, fertilization of the ovum occasionally occurs 2 , but there are instances in the vegetable kingdom where so-called parthenogenesis appears to be capable of recurring for an indefinite period. One of the best instances appears to be that of Ccelebogyne, an introduced exotic Euphorbiaceous plant which regularly produces fertile seeds although a male flower never appears. The recent researches of Strasburger have however shewn that in Ccelebogyne and other parthenogenetic flowering plants, embryos are formed by the budding and subsequent development of cells belonging to the ovule. This being the case, it is impossible to assert of these plants that they are really parthenogenetic, for the embryos contained in the seed of a flower which has certainly not been fertilized, may have been formed, not by the development of the ovum, but by budding from the surrounding tissue of the ovule.
The above view with reference to the nature of the polar bodies is not to be regarded as forming more than an hypothesis.
Impregnation of the Ovum
A far greater amount of certainty has 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.
1 Mr J. A. Osborne has recently shewn (Nature, Sept. 4, 1879), that the eggs of a Beetle (Gastrophysa raphani) may occasionally develop, up to a certain point at any rate, without the male influence.
2 Dicyema, which is an apparent exception, has not yet been certainly shewn to develop true ova. If its germs are true ova it forms an exception to the above rule.
FIG. 31. SMALL PORTIONS OF THE OVUM OF ASTERIAS GLACIALIS. THE SPERMATOZOA ARE SHEWN ENVELOPED IN THE MUCILAGINOUS COAT. IN A. A PROMINENCE IS RISING FROM THE SURFACE OF THE EGG TOWARDS THE NEAREST SPERMATOZOON; AND IN B. THE SPERMATOZOON AND PROMINENCE HAVE MET. (Copied from Fol.)
It will be convenient again to take Asterias glacialis as the type. 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. 29), which aggregate themselves into a single clear nucleus, which toward the centre of the egg and around which, as a centre, the protoplasm becomes radiately striated (fig. 30). This nucleus is known as the female pronucleus. By the action of reagents a nucleolus may be shewn in it. 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 (fig. 31, A and B). Under normal circumstances the spermatozoon which meets the prominence is the only one concerned in the fertilization, and it makes its way into the egg by passing through the prominence. The tail of the spermatozoon, 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. 32). It eventually becomes absorbed into the body of the ovum.
FIG. 32. 1'OKTION OF THE OVUM OF ASTERIAS GLACIALIS AFTER THE ENTRANCE OF A SPERMATOZOON INTO THE OVUM, IT SHEWS THE PROMINENCE OF THE OVUM THROUGH WHICH THE SPERMATOZOON HAS ENTERED. A VITELLINEMEMBRANEW1TH A CRATER-LIKE OPENING HAS BECOME DISTINCTLY FORMED (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 a distinct membrane, which separates from the egg and prevents the entrance of other spermatozoa. At the point where the spermatozoon entered a crater-like opening is left in the membrane, through which the metamorphosed tail of the spermatozoon may at first be seen projecting (fig. 32).
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, probably by assimilating material from the ovum, and around it is formed a clear space free from yolk-spherules. Shortly after its formation the protoplasm in its neighbourhood assumes a radiate arrangement (fig. 33). At whatever point of the egg the spermatozoon may have entered, it gradually travels towards the female pronucleus. The latter, around which the protoplasm no longer has a radiate arrangement, remains motionless till the rays of the male pronucleus come in contact with it, after which its condition of repose is exchanged for one of activity, and it rapidly approaches the male pronucleus, apparently by means of its inherent amoeboid contractions, and eventually fuses with it (figs. 3436).
FlG . 33 QVUM OF ASTERIAS GLACIALIS, WITH MALE AND FEMALE PRONUCLEUS AND A RADIAL STRIATION OF THE PROTOPLASM AROUND THE FORMER. (Copied from FoL)
As the male pronucleus approaches the female the latter, according to Selenka, sends out protoplasmic processes which embrace the former. The actual fusion does not take place till after the pronuclei have been in contact for some time. While the two pronuclei are approaching one another the protoplasm of the egg exhibits amoeboid movements.
The product of the fusion of the two pronuclei forms the first segmentation nucleus (fig. 37), which soon, however, divides into the two nuclei of the two first segmentation spheres.
The phenomenon which has just been described consists essentially in the fusion of the male cell and the female cell. In this act the protoplasm of the two cells as well as their nuclei coalesce, since the whole spermatozoon which has been absorbed into the ovum is a cell of which the head is the nucleus.
It is clear that the ovum after fertilization is an entirely different body to the ovum prior to that act, and unless the use of the same term for the two conditions of the ovum had become very familiar, a special term, such as oosperm, for the ovum after its fusion with the spermatozoon, would be very convenient.
FIGS. 34, 35, AND 36. THREE SUCCESSIVE STAGES IN THE COALESCENCE OF THE MALE AND FEMALE PRONUCLEI IN ASTERIAS GLACIALIS. FROM THE LIVING OVUM. (Copied from Fol.)
Of the earlier observations on this subject there need perhaps only be cited one of E. van Beneden, on the rabbit's ovum, shewing the presence of two nuclei before the commencement of segmentation. Butschli 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 (76). Neither of these authors gave at the first the correct interpretation of their results. At a later period Butschli (80) arrived at the conclusion that in a large number of instances (Lymnaus, Nephelis, &c.), the nucleus in question was formed Cucullanus, by the fusion of two or more nuclei, and Strasburger at first made a similar statement for Phallusia, though he has since withdrawn it. Though Biitschli'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 (78) described in the rabbit the formation of the original segmentation nucleus from two nuclei, one peripheral and the other central, and deduced from his observations that the 'peripheral nucleus was derived from the spermatic element. It was reserved for Oscar Hertwig (89) to describe in Echinus lividus the entrance of a spermatozoon into the egg and the formation from it of the male pronucleus.
FIG. 37 OVUM OF ASTERIAS GLACIALIS, AFTER THE COALESCENCE OF THE MALE AND FEMALE PRONUCLEI . (Copied from Fol.)
The general fact that impregnation consists in the fusion of the spermatozoon and ovum has now been established for some forms in the majority of invertebrate groups (Arthropoda and Rotifera excepted). Amongst Vertebrata also it has been shewn by E. van Beneden that the first segmentation nucleus is formed by the coalescence of the male and female pronucleus. Calberla, and Kupffer and Benecke have demonstrated that a single spermatozoon enters at first the ovum of Petromyzon.
The contact of the spermatozoon with the egg-membrane causes in Petromyzon active movements of the protoplasm of the ovum, and a retreat of the protoplasm from the membrane.
In Amphibia the appearance of a peculiar pigmented streak extending inwards from the surface of the pigmented pole of the ovum,, and containing in a clear space at its inner extremity a nucleus, has been demonstrated as the result of impregnation by Bambeke (77) and Hertwig (90). There can be little doubt that this nucleus is the male pronucleus, and that the pigmented streak indicates its path inwards. Close to it Hertwig has shewn that another nucleus is to be found, the female pronucleus, and that eventually the two join together. In Amphibia the phenomena accompanying impregnation are clearly of the same nature as in the Invertebrata. A precisely similar series of phenomena to those in Amphibia has been shewn by Salensky to take place in the Sturgeon.
Although 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 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 the 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 Nematoidea 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 cells are 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.
One important point has been so far only indirectly alluded to, viz. the number of spermatozoa required to effect impregnation.
The concurrent testimony of almost all observers tends to shew that one only is required for this purpose. But the number of cases tested is too small to admit of satisfactory generalization.
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 star ; 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 up to a certain point 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 several may be concerned in the act. The development continues, however, to be normal up to the gastrula stage, at any rate, if three or even four spermatozoa enter the egg almost simultaneously. Under such circumstances each spermatozoon forms a separate pronucleus and star. Selenka is of opinion (apparently rather on a priori grounds than as a result of direct observation) that normal development cannot occur when more than one male pronucleus fuses with the female pronucleus ; and holds that, where he has observed such normal development after the entrance of more than one spermatozoon, the majority of male pronuclei become absorbed.
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.
Kupffer and Benecke have further shewn that although only one spermatozoon enters the ovum directly in Petromyzon yet other spermatozoa pass through the vitelline membrane, and are taken into a peculiar protoplasmic protuberance of the ovum which appears after impregnation.
The act of impregnation may be described as the fusion of the ovum and spermatozoon, and the most important feature in this act appears to be 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, as stated above, contains part of the nucleus of the primitive spermatic cell. The spermatic cells originate from cells indistinguishable from the primitive ova, so that the fusion which takes place is the fusion of morphologically similar parts in the two sexes.
These conclusions tally very satisfactorily with the view adopted in the Introduction, that impregnation amongst the Metazoa was derived from the process of conjugation amongst the Protozoa.
In what may probably be regarded as a normal case the following series of events accompanies the maturation and impregnation of an ovum :
- Transportation of the germinal vesicle to the surface of the egg.
- Absorption of the membrane of the germinal vesicle and metamorphosis of the germinal spot and nuclear reticulum.
- Assumption of a spindle character by the remains of the germinal vesicle, these remains being probably in part formed from the germinal spot.
- Entrance of one end of the spindle into a protoplasmic prominence at the surface of the egg.
- Division of the spindle into two halves, one remaining in the egg, the other in the prominence ; the prominence becoming at the same time nearly constricted off from the egg as a polar cell.
- Formation of a second polar cell in the same manner as the first, part of the spindle still remaining in the egg.
- Conversion of the part of the spindle remaining in the egg into a nucleus the female pronucleus.
- Transportation of the female pronucleus towards the centre of the egg.
- Entrance of one spermatozoon into the egg.
- Conversion of the head of the spermatozoon into a nucleus the male pronucleus.
- Appearance of radial striae round the male pronucleus, which gradually travels towards the female pronucleus.
- Fusion of male and female pronuclei to form the first segmentation nucleus.
(76) Auerbach. Organologische Stiidien, Heft 2. Breslau, 1874.
(77) Bambeke. " Recherchess. Embryologie des Batraciens." Bull.detAcad. royale de Belgique, sme Ser., T. LXI., 1876.
(78) E. van Beneden. "La Maturation de 1'OZuf des Mammiferes." Bull.de fAcad. royale de Belgique, 2me Sen, T. XL. No. 12, 1875.
(79) Idem. " Contributions a 1'Histoire de la Vesicule Germinative, &c." Bull. de TAcad. royale de Belgique, sme Se"r., T. XLI. No. i, 1876.
(80) O. Biitschli. Eizelle, Zelltheilung, und Conjugation der Infusorien. Frankfurt, 1876.
(81) F. M. Balfour. "On the Phenomena accompanying the Maturation and Impregnation of the Ovum." Quart. J. of Micros. Science, Vol. xvni., 1878.
(82) Calberla. " Befruchtungsvorgang beim Ei von Petromyzon Planeri." Zeit. f. wiss. Zool., Vol. XXX.
(83) W. Flemming. " Studien in d. Entwickelungsgeschichte der Najaden." Sitz. d. k. Akad. Wien, B. LXXL, 1875.
(84) H. Fol. "Die erste Entwickelung des Geryonideneies." Jenaische Zeitschrift, Vol. vii., 1873.
(85) Idem. " Sur le Developpement des Pteropodes." Archives de Zoologie Experimentale et Generale, Vol. IV. and V., 1875 6.
(86) Idem. "Sur le Commencement de 1'Henogenie." Archives des Sciences Physiques et Naturelles. Geneve, 1877.
(87) Idem. Recherches s. /. Fecondation et I. cornmen. d. FHenogenie. Geneve, 1879.
(88) R. Greeff. " Ueb. d. Bau u. d. Entwickelung d. Echinodermen. " Sitzun. der Gesellschaft z. Beforderung d. gesammten Naturiviss. z. Marburg, No. 5, 1876.
(89) Oscar Hertwig. "Beit. z. Kenntniss d. Bildung, &c., d. thier. Eies." Morphologisches Jahrbuch, Vol. I., 1876.
(90) Idem. Ibid. Morphologisches Jahrbuch, Vol. in. Heft i, 1877.
(91) Idem. " Weitere Beitrage, &c." Morphologisches Jahrbuch, Vol. ill., 1877, Heft 3.
(92) Idem. "Beit. z. Kenntniss, &c." Morphologisches Jahrbuch, Vol. IV. Heft i and 2, 1878.
(93) N. Kleinenberg. Hydra. Leipzig, 1872.
(94) C. Kupffer u. B. Benecke. Der Vorgang d. Befruchtung am Eie d. Neunaugen. Konigsberg, 1878.
(95) J. Oellacher. "Beitrage zur Geschichte des Keimblaschens im Wirbelthiereie." Archivf. micr. Anat., Bd. viil., 1872.
(96) W. Salensky. " Befruchtung u. Furchung d. Sterlets-Eies." Zoologischer Anzeiger, No. u, 1878.
(97) E. Selenka. Befruchtung des Eies von Toxopneustesvariegatus. Leipzig, 1878.
(98) Strasburger. Ueber Zellbildung u. Zelltheilung. Jena, 1876.
(99) Idem. Ueber Befruchtung u. Zelltheilung. Jena, 1878.
(100) C. O. Whitman. "The Embryology of Clepsine." Quart. J. of Micr. Science, Vol. xvm., 1878.