Chapter XII: The Organs of Excretion (3)

After the publication of Bütschli's monograph, O. Hertwig (12) continued his researches on the ova of Leeches (_Hæmopis_ 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 Bütschli'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 Bütschli. 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 Bütschli 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 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 Bütschli 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 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 so-called 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 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 shewn to contain a round body. 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 observations 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 striæ 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 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[364] 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.

Footnote 364: 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.

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 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 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 enclosed portion of the spindle becomes partially constricted off from the egg as the first polar body (Fig. 8). 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. 9), and 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). 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.

_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 Coelenterata, 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 may be that in the case of the rabbit, and many Nematodes as described by van Beneden and by Bütschli, 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[365] and Arthropoda, with a few doubtful exceptions, in which the polar cells cannot as yet be said to have been satisfactorily observed.

Footnote 365: 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.

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: (1) 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 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 Bütschli, takes place during conjugation in Infusoria as a preliminary to the formation of a fresh nucleus. This comparison is due to Bütschli, 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 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 develop 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, 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. Arthropoda 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[366], 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.

Footnote 366: 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. für
wiss. Zool._ Bd. XXII. p. 202.

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 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. Professor Greeff's 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.

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. 11). This nucleus is known as the _female pronucleus_[367]. 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.

Footnote 367: According to Hertwig's most recent statement a
nucleolus is present in this nucleus.

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).

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 formation the protoplasm in its neighbourhood assumes a radiate arrangement (Fig. 15). At whatever point of the egg the spermatozoon may have entered, it gradually travels towards the female pronucleus. This latter, around which the protoplasm no longer has a radial arrangement, remains 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).

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, shewing the presence of two nuclei before the commencement of segmentation. Bütschli 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 (1). Neither of these authors gave at first the correct interpretation of their results. At a later period Bütschli (5) arrived at the conclusion that in a large number of instances (_Lymnæus_, _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 _Phallusia_, though he has since withdrawn it. Though Bütschli'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 (11) 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 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 striæ 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 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.

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[368].

Footnote 368: The recent researches of Calberla on the
impregnation of the ovum of _Petromyzon Planeri_ support this
conclusion.

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[369]. 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.

Footnote 369: 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.

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.

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 striæ round the male pronucleus which gradually travels towards the female pronucleus.

(12) Fusion of male and female pronuclei to form the first segmentation nucleus.

_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 l'Acad. royale de Belgique_, 2me sér., t. LXI. 1876.

3. E. Van Beneden. "La Maturation de l'OEuf des Mammifères." _Bull. de l'Acad. royale de Belgique_, 2me sér., t. XL, no. 12, 1875.

4. E. Van Beneden. "Contributions à l'Histoire de la Vésicule Germinative, &c." _Bull. de l'Acad. royale de Belgique_, 2me sér., t. XLI, no. 1, 1876.

5. Bütschli. _Eizelle, Zelltheilung, und Conjugation der Infusorien._

6. Flemming. "Studien in d. Entwicklungsgeschichte der Najaden." _Sitz. d. k. Akad. Wien_, B. LXXI. 1875.

7. Fol. "Die erste Entwicklung des Geryonideneies." _Jenaische Zeitschrift_, Vol. VII.

8. Fol. "Sur le Développement des Pteropodes." _Archives de Zoologie Expérimentale et Générale_, Vols. IV and V.

9. Fol. "Sur le Commencement de l'Hénogénie." _Archives des Sciences Physiques et Naturelles_. Genève, 1877.

10. Giard. _Note sur les premiers phénomènes du développement de l'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. III, Heft. 1.

13. Hertwig, Oscar. "Weitere Beiträge, &c." _Morphologisches Jahrbuch_, Bd. III, Heft 3.

14. Kleinenberg. _Hydra_. Leipzig, 1872.

15. Oellacher, J. "Beiträge zur Geschichte des Keimbläschens im Wirbelthiereie." _Archiv f. micr. Anat._, Bd. VIII.

16. Selenka. _Befruchtung u. Theilung des Eies von Toxopneustes variegatus_ (Vorläufige Mittheilung). Erlangen, 1877.

17. Strasburger. _Ueber Zellbildung u. Zelltheilung._ Jena, 1876.

18. Strasburger. _Ueber Befruchtung u. Zelltheilung._ Jena, 1878.

19. R. Greeff. "Ueb. d. Bau u. d. Entwicklung d. Echinodermen." _Sitzun. der Gesellschaft z. Beförderung d. gesammten Naturwiss. z. Marburg_, No. 5. 1876.

_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. für 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.

XII. ON THE STRUCTURE AND DEVELOPMENT OF THE VERTEBRATE OVARY[370].

Footnote 370: From the _Quarterly Journal of Microscopical
Science_, Vol. 18, 1878.

(With Plates 24, 25, 26.)

The present paper records observations on the ovaries of but two types, viz., Mammalia and Elasmobranchii. The main points dealt with are three:--1. The relation of the germinal epithelium to the stroma. 2. The connection between _primitive ova_ in Waldeyer's sense and the permanent ova. 3. The homologies of the egg membranes.

The second of these points seems to call for special attention after Semper's discovery that the primitive ova ought really to be regarded as _primitive sexual cells_, in that they give rise to the generative elements of both sexes.

THE DEVELOPMENT OF THE ELASMOBRANCH OVARY.

The development of the Elasmobranch ovary has recently formed the subject of three investigations. The earliest of them, by H. Ludwig, is contained in his important work, on the 'Formation of the Ovum in the Animal Kingdom[371].' Ludwig arrives at the conclusion that the ovum and the follicular epithelium are both derived from the germinal epithelium, and enters into some detail as to their formation. Schultz[372], without apparently being acquainted with Ludwig's observations, has come to very similar results for Torpedo.

Footnote 371: _Arbeiten a. d. zool.-zoot. Institut Würzburg_,
Bd. I.

Footnote 372: _Archiv f. micr. Anat._ Vol. XI.

Semper[373], in his elaborate memoir on the urogenital system of Elasmobranchii, has added very greatly to our knowledge on this subject. In a general way he confirms Ludwig's statements, though he shews that the formation of the ova is somewhat more complicated than Ludwig had imagined. He more especially lays stress on the existence of nests of ova (Ureiernester), derived from the division of a single primitive ovum, and of certain peculiarly modified nuclei, which he compares to spindle nuclei in the act of division.

Footnote 373: _Arbeiten a. d. zool.-zoot. Institut Würzburg_,
Bd. II.

My own results agree with those of previous investigators, in attributing to the germinal epithelium the origin both of the follicular epithelium and ova, but include a number of points which I believe to be new, and, perhaps, of some little interest; they differ, moreover, in many important particulars, both as to the structure and development of the ovary, from the accounts of my predecessors.

The history of the female generative organs may conveniently be treated under two heads, viz. (1) the history of the ovarian ridge itself, and (2) the history of the ova situated in it. I propose dealing in the first place with the ovarian ridge.

_The Ovarian ridge in Scyllium._--At the stage spoken of in my monograph on Elasmobranch Fishes as stage L, the ovarian ridge has a very small development, and its maximum height is about 0.1 mm. It exhibits in section a somewhat rounded form, and is slightly constricted along the line of attachment. It presents two surfaces, which are respectively outer and inner, and is formed of a layer of somewhat thickened germinal epithelium separated by a basement membrane from a central core of stroma. The epithelium is far thicker on the outer surface than on the inner, and the primitive ova are entirely confined to the former. The cells of the germinal epithelium are irregularly scattered around the primitive ova, and have not the definite arrangement usually characteristic of epithelial cells. Each of them has a large nucleus, with a deeply staining small nucleolus, and a very scanty protoplasm. In stage N the ovarian ridge has a pointed edge and narrower attachment than in stage L. Its greatest height is about 0.17 mm. There is more stroma, and the basement membrane is more distinct than before; in other respects no changes worth recording have taken place. By stage P a distinction is observable between the right and left ovarian ridges; the right one has, in fact, grown more rapidly than the left, and the difference in size between the two ridges becomes more and more conspicuous during the succeeding stages, till the left one ceases to grow any larger, though it remains for a great part of life as a small rudiment.

The right ovarian ridge, which will henceforth alone engage our attention, has grown very considerably. Its height is now about 0.4 mm. It has in section (vide Pl. 24, fig. 1) a triangular form with constricted base, and is covered by a flat epithelium, except for an area on the outer surface, in length co-extensive with the ovarian ridge, and with a maximum breadth of about 0.25 mm. This area will be spoken of as the ovarian area or region, since the primitive ova are confined to it. The epithelium covering it has a maximum thickness of about 0.05 mm., and thins off rather rapidly on both borders, to become continuous with the general epithelium of the ovarian ridge. Its cells have the same character as before, and are several layers deep. Scattered irregularly amongst them are the primitive ova. The germinal epithelium in the ovarian region is separated by a basement membrane from the adjacent stroma.

In succeeding stages, till the embryo reaches a length of 7 centimetres, no very important changes take place. The ovarian region grows somewhat in breadth, though in this respect different embryos vary considerably. In two embryos of nearly the same age, the breadth of the ovarian epithelium was 0.3 mm. in the one and 0.35 mm. in the other. In the former of these embryos, the thickness of the epithelium was slightly greater than in the latter, viz. 0.09 mm. as compared with 0.08. In both the epithelium was sharply separated from the subjacent stroma. There were relatively more epithelial cells in proportion to primitive ova than at the earlier date, and the individual cells exhibited great variations in shape, some being oval, some angular, others very elongated, and many of them applied to part of an ovum and accommodating themselves to its shape. In some of the more elongated cells very deeply stained nuclei were present, which (in a favourable light and with high powers) exhibited the spindle modification of Strasburger with great clearness, and must therefore be regarded as undergoing division. The ovarian region is at this stage bounded on each side by a groove.

In an embryo of seven centimetres (Pl. 24, fig. 2) the breadth of the ovarian epithelium was 0.5, but its height only 0.06 mm. It was still sharply separated from the subjacent stroma, though a membrane could only be demonstrated in certain parts. The amount of stroma in the ovarian ridge varies greatly in different individuals, and no reliance can be placed on its amount as a test of the age of the embryo. In the base of the ovarian ridge the cells were closely packed, elsewhere they were still embryonic.

My next stage (Pl. 24, fig. 3, and fig. 4), shortly before the time of the hatching of the embryo, exhibits in many respects an advance on the previous one. It is the stage during which a follicular covering derived from the germinal epithelium is first distinctly formed round the ova, in a manner which will be more particularly spoken of in the section devoted to the development of the ovum itself. The breadth of the ovarian region is 0.56 mm., and its greatest height close to the central border, 0.12 mm.--a great advance on the previous stage, mainly, however, due to the larger size of the ova.

The ovarian epithelium is still in part separated from the subjacent stroma by a membrane close to its dorsal and ventral borders, but elsewhere the separation is not so distinct, it being occasionally difficult within a cell or so to be sure of the boundary of the epithelium. The want of a clear line between the stroma and the epithelium is rendered more obvious by the fact that the surface of the latter is somewhat irregular, owing to projections formed by specially large ova, into the bays between which are processes of the stroma. In an ovary about this stage, hardened in osmic acid, the epithelium stains very differently from the subjacent stroma, and the line of separation between the two is quite sharp. A figure of the whole ovarian ridge, shewing the relation between the two parts, is represented on Pl. 24, fig. 5.

The layer of stroma in immediate contact with the epithelium is very different from the remainder, and appears to be destined to accompany the vascular growths into the epithelium, which will appear in the next stage. The protoplasm of the cells composing it forms a loose reticulum with a fair number of oval or rounded nuclei, with their long axis for the most part parallel to the lower surface of the epithelium. It contains, even at this stage, fully developed vascular channels.

The remainder of the stroma of the ovarian ridge has now acquired a definite structure, which remains constant through life, and is eminently characteristic of the genital ridge of both sexes. The bulk of it (Pl. 24, fig. 3, _str_) consists of closely packed polygonal cells, of about 0.014 mm. with large nuclei of about 0.009. These cells appear to be supported by a delicate reticulum. The whole tissue is highly vascular, with the numerous capillaries; the nuclei in the walls of which stand out in some preparations with great clearness.

In the next oldest ovary, of which I have sections, the breadth of the ovarian epithelium is 0.7 mm. and its thickness 0.096. The ovary of this age was preserved in osmic acid, which is the most favourable reagent, so far as I have seen, for observing the relation of the stroma and epithelium. On Pl. 24, fig. 6, is represented a transverse section through the whole breadth of the ovary, slightly magnified to shew the general relations of the parts, and on Pl. 24, fig. 7, a small portion of a section more highly magnified. The inner surface of the ovarian epithelium is more irregular than in the previous stage, and it may be observed that the subjacent stroma is growing in amongst the ova. From the relation of the two tissues it is fairly clear that the growth which is taking place is a definite growth of the stroma into the epithelium, and not a mutual intergrowth of the two tissues. The ingrowths of the stroma are, moreover, directed towards individual ova, around which, outside the follicular epithelium, they form a special vascular investment in the succeeding stages. They are formed of a reticular tissue with comparatively few nuclei.

By the next stage, in my series of ovaries of _Scy. canicula_, important changes have taken place in the constitution of ovarian epithelium. Fig. 8, Pl. 24, represents a portion of the ovarian epithelium, on the same scale as figs. 1, 2, 3, &c., and fig. 9 a section through the whole ovarian ridge slightly magnified. Its breadth is now 1.3 mm., and its thickness 0.3 mm. The ova have grown very greatly, and it appears to me to be mainly owing to their growth that the greater thickness of the epithelium is due, as well as the irregularity of its inner surface (vide fig. 9).

The general relation of the epithelium to the surrounding parts is much the same as in the earlier stage, but two new features have appeared--(1) The outermost cells of the ovarian region have more or less clearly arranged themselves as a kind of epithelial covering for the organ; and (2) the stroma ingrowths of the previous stage have become definitely vascular, and have penetrated through all parts of the epithelium.

The external layer of epithelium is by no means a very marked structure, the character of its cells varies greatly in different regions, and it is very imperfectly separated from the subjacent layer. I shall speak of it for convenience as _pseudo-epithelium_.

The greater part of the germinal epithelium forms anastomosing columns, separated by very thin tracts of stroma. The columns are, in the majority of instances, continuous with the pseudo-epithelium at the surface, and contain ova in all stages of development. Many of the cells composing them naturally form the follicular epithelium for the separate ova; but the majority have no such relation. They have in many instances assumed an appearance somewhat different from that which they presented in the last stage, mainly owing to the individual nuclei being more widely separated. A careful examination with a high power shews that this is owing to an increase in the amount of protoplasm of the individual cells, and it may be noted that a similar increase in the size of the bodies of the cells has taken place in the pseudo-epithelium and in the follicular epithelium of the individual ova.

The stroma ingrowths form the most important feature of the stage. In most instances they are very thin and delicate, and might easily be overlooked, especially as many of the cells in them are hardly to be distinguished, taken separately, from those of the germinal epithelium. These features render the investigation of the exact relation of the stroma and epithelium a matter of some difficulty. I have, however, been greatly assisted by the investigation of the ovary of a young example of _Scyllium stellare_, 16-1/2 centimètres in length, a section of which is represented in Pl. 25, fig. 26. In this ovary, although no other abnormalities were observable, the stroma ingrowths were exceptionally wide; indeed, quite without a parallel in my series of ovaries in this respect. The stroma most clearly divides up the epithelium of the ovary into separate masses, or more probably anastomosing columns, the equivalents of the egg-tubes of Pflüger. These columns are formed of normal cells of the germinal epithelium, which enclose ovarian nests and ova in all stages of development. A comparison of the section I have represented, with those from previous stages, appears to me to demonstrate that the relation of the epithelium and stroma has been caused by an ingrowth or penetration of the stroma into the epithelium, and not by a mutual intergrowth of the two tissues. Although the ovary, of which fig. 26 represents a section was from _Scy. stellare_, and the previous ovaries have been from _Scy. canicula_, yet the thickness of the epithelium may still be appealed to in confirmation of this view. In the previous stage the thickness was about 0.096 mm., in the present one it is about 0.16 mm., a difference of thickness which can be easily accounted for by the growth of the individual ova and the additional tracts of stroma. A pseudo-epithelium is more or less clearly formed, but it is continuous with the columns of epithelium. In the stroma many isolated cells are present, which appear to me, from a careful comparison of a series of sections, to belong to the germinal epithelium.

The thickness of the follicular epithelium on the inner side of the larger ova deserves to be noted. Its meaning is discussed on p. 567.

Quite a different interpretation to that which I have given has been put by Ludwig and Semper upon the parts of the ovary at this stage. My _pseudo-epithelium_ is regarded by them as forming, together with the _follicular epithelium_ of the ova, the sole remnant of the original germinal epithelium; and the masses of cells below the pseudo-epithelium, which I have attempted to shew are derived from the original germinal epithelium, are regarded as parts of the ingrowths of the adjacent stroma.

Ludwig has assumed this interpretation without having had an opportunity of working out the development of the parts, but Semper attempts to bring forward embryological proofs in support of this position.