Chapter III: The Segmentation of the Ovum (1)

The immediate result of the fusion of the male and female pronucleus is the segmentation or division of the ovum usually into two, four, eight, etc. successive parts. The segmentation may be dealt with from two points of view, viz. (1) the nature of the vital phenomena which take place in the ovum during its occurrence, which may be described as the internal phenomena of segmentation. (2) The external characters of the segmentation.

_Internal Phenomena of Segmentation._

Numerous descriptions have been given during the last few years of the internal phenomena of segmentation. The most recent contribution on this head is that of Fol (No. 87). He appears to have been more successful than other observers in obtaining a complete history of the changes which take place, and it will therefore be convenient to take as type the ovum of _Toxopneustes (Echinus) lividus_, on which he made his most complete series of observations. The changes which take place may be divided into a series of stages. The ovum immediately after the fusion of the male and female pronucleus contains a central segmentation nucleus.

In the first stage a clear protoplasmic layer derived from the plasma of the cell is formed round the nucleus, from which there start outwards a number of radial striæ, which are rendered conspicuous by the radial arrangement of the yolk granules between them. The nucleus during this process remains perfectly passive.

In the second stage the nucleus becomes less distinct and somewhat elongated, and around it the protoplasmic layer of the earlier stage is arranged in the form of a disc-shaped ring, compared by Fol to Saturn's ring. The protoplasmic rays still take their origin from the perinuclear protoplasm. This stage has a considerable duration (20 minutes).

In the third stage the protoplasm around the nucleus becomes transported to the two nuclear poles, at each of which it forms a clear mass surrounded by a star-shaped figure formed by radial striæ. The nucleus is hardly visible in the fresh condition, but when brought into view by reagents is found to contain many highly refractive particles, and to be still enveloped in a membrane.

In the fourth stage the nucleus when treated by reagents has assumed the well-known spindle form. The striæ of which it is composed are continuous from one end of the spindle to the other and are thickened at the centre. The central thickenings constitute the so-called nuclear plate. The clear protoplasmic masses and stars are present as before at the apices of the nucleus, and the rays of the latter converge as if they would meet at the centre of the clear masses, but stop short at their periphery. There is no trace of a membrane round either the nuclear spindle or the clear masses; and in the centre of the latter is a collection of granules. The striæ of the polar stars are very fine but distinct.

Between the stage with a completely formed spindle and the previous one the intermediate steps have not been made out for Toxopneustes; but for Heteropods Fol has been able to demonstrate that the striæ of the spindle and their central thickenings are formed, as in the case of the spindle derived from the germinal vesicle, _from the metamorphosis of the nuclear reticulum_. They commence to be formed at the two poles, and are then (in Heteropods) in immediate contiguity with the striæ of the stars. The striæ gradually grow towards the centre of the nucleus and there meet.

In the fifth stage the central thickenings of the spindle separate into two sets, which travel symmetrically outwards towards the clear masses, growing in size during the process. They remain however united for a short time by delicate filaments--named by Fol connective filaments--which very soon disappear. The clear masses also increase in size. During this stage the protoplasm of the ovum exhibits active amoeboid movements preparatory to division.

In the sixth stage, which commences when the central thickenings of the spindle have reached the clear polar masses, the division of the ovum into two parts is effected by an equatorial constriction at right angles to the long axis of the nucleus. The inner vitelline membrane follows the furrow for a certain distance, but does not divide with the ovum. All connection between the two parts of the spindle becomes lost during this stage, and the thickenings of the fibres of the spindle give rise to a number of spherical vesicular bodies, which pass into the clear masses and become intermingled with the granules which are placed there. The radii of the stars now extend round the whole circumference of each of the clear masses.

In the seventh stage the two clear masses become elongated and travel towards the outer sides of their segments; while the radii connected with them become somewhat bent, as if a certain amount of traction had been exercised on them in the movement of the clear masses. Shortly afterwards the spherical vesicles, each of which appears like a small nucleus and contains a central nucleolus, begin to unite amongst themselves, and to coalesce with the neighbouring granules. Those in each segment finally unite to form a nucleus which absorbs the substance of the clear mass. _The new nucleus is therefore partly derived from the division of the old one and partly from the plasma of the cell._ The two segments formed by division are at first spherical, but soon become flattened against each other. In each subsequent division of these cells the whole of the above changes are repeated.

The phenomena which have just been described would appear to occur in the segmentation of ova with remarkable constancy and without any very considerable variations.

The division of the ovum constitutes a special case of cell division, and it is important to determine to what extent the phenomena of ordinary cell division are related to those which take place in the division of the ovum. Without attempting a full discussion of the subject I will confine myself to a few remarks suggested by the observations of Flemming, Peremeschko and Klein. The observations of these authors shew that in the course of the division of nuclei in the salamander, newt, etc. the nuclear reticulum undergoes a series of peculiar changes of form, and after the membrane of the nucleus has vanished divides into two masses. The masses form the basis for the new nuclei, and become reconverted into an ordinary nuclear reticulum after repeating, in the reverse order, the changes of form undergone by the reticulum previous to its division.

It is clear without further explanation that the conversion of the nuclear reticulum of the segmentation nucleus into the striæ of the spindle is a special case of the same phenomenon as that first described by Flemming in the salamander. There are however some considerable differences. In the first place the fibres in the salamander do not, according to Flemming, unite in the middle line, though they appear to do so in the newt. This clearly cannot be regarded as a fact of great importance; nor can the existence of the central thickenings of the striæ (nuclear plate), constant as it is for the division of the nucleus of the ovum, be considered as constituting a fundamental difference between the two cases. More important is the fact that the striæ in the case of the ovum do not appear, at any rate have not been shewn, to form themselves again into a nuclear network.

With reference to the last point it is however to be borne in mind (1) that the gradual travelling outwards of the two halves of the nuclear plate is up to a certain point a repetition, in the reverse order, of the mode of formation of the striæ of the spindle, since the striæ first appeared at the poles and gradually grew towards the middle of the spindle; (2) that there is still considerable doubt as to how the vesicular bodies formed out of the nuclear plate reconstitute themselves into a nucleus.

The layer of clear protoplasm around the nucleus during its division has its homologue in the case of the division of the nuclei of the salamander, and the rays starting from this are also found. Klein has suggested that the extra-nuclear rays of the stars around the poles of the nucleus are derived from a metamorphosis of the extra-nuclear reticulum, which he believes to be continuous with the intra-nuclear reticulum.

The delicate connective filaments usually visible between the two halves of the nuclear plate would seem from Strasburger's latest observations (No. 104) to be derived from the nuclear substance between the striæ of the spindle, and to become eventually reabsorbed into the newly-formed nuclei.

We are it appears to me still in complete ignorance as to the physical causes of segmentation. The view that the nucleus is a single centre of attraction, and that by its division the centre of attraction becomes double and thereby causes division, appears to be quite untenable. The description already given of the phenomena of segmentation is in itself sufficient to refute this view. Nor is it in the least proved by the fact (shewn by Hallez) that the plane of division of the cell always bears a definite relation to the direction of the axis of the nucleus.

The arguments by which Kleinenberg (93) attempted to demonstrate that cell division was a phenomenon caused by alterations in the molecular cohesion of the protoplasm of the ovum still in my opinion hold good, but recent discoveries as to the changes which take place in the nucleus during division probably indicate that the molecular changes which take place in the cohesion of the protoplasm are closely related to, and possibly caused by, those in the nucleus. These alterations of cohesion are produced by a series of molecular changes, the external indications of which are to be found in the visible alterations in the constitution of the body of the cell and of the nucleus prior to division.

BIBLIOGRAPHY.

In addition to the papers cited in the last Chapter, _vide_

(101) W. Flemming. "Beiträge z. Kenntniss d. Zelle u. ihrer Lebenserscheinungen." _Archiv f. mikr. Anat._, Vol. XVI., 1878.

(102) E. Klein. "Observations on the glandular epithelium and division of nuclei in the skin of the Newt." _Quart. J. of Micr. Science_, Vol. XIX., 1879.

(103) Peremeschko. "Ueber d. Theilung d. thierischen Zellen." _Archiv f. mikr. Anat._, Vol. XVI., 1878.

(104) E. Strasburger. "Ueber ein z. Demonstration geeignetes Zelltheilungs-Object." _Sitz. d. Jenaischen Gesell. f. Med. u. Naturwiss._, July 18, 1879.

_External Features of Segmentation._

[FIG. 38. VARIOUS STAGES IN PROCESS OF SEGMENTATION. (After
Gegenbaur.)]

In the simplest known type of segmentation the ovum first of all divides into two, then four, eight, sixteen, thirty-two, sixty-four, etc. cells (fig. 38). These cells so long as they are fairly large are usually known as segments or spheres. At the close of such a simple segmentation the ovum becomes converted into a sphere composed of segments of a uniform size. These segments usually form a wall (fig. 39, E), one row of cells thick, round a central cavity, which is known as the segmentation cavity or cavity of Von Baer. Such a sphere is known as a blastosphere. The central cavity usually appears very early in the segmentation, in many cases when only four segments are present (fig. 39, B).

[FIG. 39. THE SEGMENTATION OF AMPHIOXUX. (Copied from Kowalevsky.)

_sg._ segmentation cavity. A. Stage with two equal segments. B.
Stage with four equal segments. C. Stage after the four segments
have become divided by an equatorial furrow into eight equal
segments. D. Stage in which a single layer of cells encloses a
central segmentation cavity. E. Somewhat older stage in optical
section.]

In other instances, which however are rarer than those in which a segmentation cavity is present, there is no trace of a central cavity, and the sphere at the close of segmentation is quite solid. In such instances the solid sphere is known as a morula. It is found in some Sponges, many Coelenterata, some Nemertines, etc., and in Mammals; in which group the segmentation is not however quite regular. All intermediate conditions between a large segmentation cavity, and a very minute central cavity which may be surrounded by more than a single row of cells have been described.

The segmentation cavity has occasionally, as in Sycandra, the Ctenophora and Amphioxus, the form of an axial perforation of the ovum open at both extremities.

When the process of regular segmentation is examined somewhat more in detail it is found to follow as a rule a rather definite rhythm. The ovum is first divided in a plane which may be called vertical, into two equal parts (fig. 39, A). This division is followed by a second, also in a vertical plane, but at right angles to the first plane, and by it each of the previous segments is halved (fig. 39, B.) In the third segmentation the plane of division is horizontal or equatorial and divides each of the four segments into two halves, making eight segments in all (fig. 39, C). In the fourth period the segmentation takes place in two vertical planes each at an angle of 45° with one of the previous vertical planes. All the segments are thus again divided into two equal parts. In the fifth period there are two equatorial planes one on each side of the original equatorial plane, and thirty-two spheres are present at the close of this period. Sixty-four segments are formed at the sixth period, but beyond the fourth and fifth periods the original regularity is not usually preserved.

In many instances the type of segmentation just described cannot be distinctly recognized. All that can be noticed is that at each fresh segmentation every segment becomes divided into two equal parts. It is not absolutely certain that there is not always some slight inequality in the segments formed, by which, what are known as the animal and vegetative poles of the ovum, can very early be distinguished. A regular segmentation is found in species in most groups of the animal kingdom. It is very common in Sponges and Coelenterates. Though less common so far as is known amongst the Vermes, it is yet found in many of the lower types, viz. Nematoidea, Gordiacea, Trematoda, Nemertea (apparently as a rule), _Sagitta_, _Chætonotus_, some Gephyrea (_Phoronis_); though not usual it occurs amongst Chætopoda, E.G. SERPULA. It is the usual type of segmentation amongst the Echinodermata. Amongst the Crustacea it appears (for the earlier phases of segmentation at any rate) not infrequently amongst the lower forms, and even occurs amongst the Amphipoda (_Phronima_). It is however very rare amongst the Tracheata, _Podura_ affording the one example of it known to me. It is almost as rare amongst Mollusca as amongst the Tracheata, but occurs in _Chiton_ and is nearly approached in some Nudibranchiata. In Vertebrata it is most nearly approached in _Amphioxus_[36].

[36] In the Rabbit and probably other Monodelphous Mammalia the
segmentation is nearly though not quite regular.

Most of the eggs which have a perfectly regular segmentation are of a very insignificant size and rarely contain much food-yolk: in the vast majority of eggs there is present however a considerable bulk of food material usually in the form of highly refracting yolk-spherules. These yolk-spherules lie embedded in the protoplasm of the ovum, but are in most instances not distributed uniformly, being less closely packed and smaller at one pole of the ovum than elsewhere. Where the yolk-spherules are fewest the active protoplasm is necessarily most concentrated, and we can lay down as a general law[37] that the velocity of segmentation in any part of the ovum is roughly speaking proportional to the concentration of the protoplasm there; and that the size of the segments is inversely proportional to the concentration of the protoplasm. Thus the segments produced from that part of an egg where the yolk-spherules are most bulky, and where therefore the protoplasm is least concentrated, are larger than the remaining segments, and their formation proceeds more slowly.

[37] _Vide_ F. M. Balfour, "Comparison of the early stages
of development in Vertebrates." _Quart. Jour. of Micr.
Science_, July, 1875.

Though where much food-yolk is present it is generally distributed unequally, yet there are many cases in which it is not possible to notice this very distinctly. In most of these cases the segmentation is all the same unequal, and it is probable that they form apparent rather than real exceptions to the law laid down above. Although before segmentation the protoplasm may be uniformly distributed, yet in many instances, _e.g._ Mollusca, Vermes, etc., during or at the commencement of segmentation the protoplasm becomes aggregated at one pole, and one of the segments formed consists of clear protoplasm, all the food-yolk being contained in the other and larger segment.

Unequal Segmentation. The type of segmentation I now proceed to describe has been called by Haeckel (No. 105) 'unequal segmentation', a term which may conveniently be adopted. I commence by describing it as it occurs in the well-known and typical instance of the Frog[38].

[38] _Vide_ Remak, _Entwicklung d. Wirbelthiere_; and Götte,
_Entwicklung d. Unke_.

The ripe ovum of the common Frog and of most other tailless Amphibians presents the following structure. One half appears black and the other white. The former I shall call the upper pole, the latter the lower. The ovum is composed of protoplasm containing in suspension numerous yolk-spherules. The largest of these are situated at the lower pole, the smaller ones at the upper pole, and the smallest of all in the peripheral layer of the upper pole, in which also pigment is scattered and causes the black colour visible from the surface.

[FIG. 40. SEGMENTATION OF COMMON FROG. RANA TEMPORARIA. (Copied from
Ecker.)

The numbers above the figures refer to the number of segments at the
stage figured.]

The first formed furrow is a vertical furrow. It commences in the upper half of the ovum, through which it extends rapidly, and then more slowly through the lower. As soon as the first furrow has extended through the egg, and the two halves have become separated from each other, a second vertical furrow appears at right angles to the first and behaves in the same way (fig. 40, 4).

The next furrow is equatorial or horizontal (fig. 40, 8). It does not arise _at the true equator of the egg_, but much nearer to its upper pole. It extends rapidly round the egg and divides each of the four previous segments into two parts, one larger and one smaller. Thus at the end of this stage there are present four small and four large segments. At the meeting point of these a small cavity appears, which is the segmentation cavity, already described for uniformly segmenting eggs. It increases in size in subsequent stages, its roof being formed of the smaller cells and its floor of the larger. The appearance of the equatorial furrow is followed by a period of repose, after which two rapidly succeeding vertical furrows are formed in the upper pole, dividing each of the four segments of which this is composed into two. After a short period these furrows extend to the lower pole, and when completed 16 segments are present--eight larger and eight smaller (fig. 40, 16). A pause now ensues, after which the eight upper segments become divided by an equatorial furrow, and somewhat later a similar furrow divides the eight lower segments. At the end of this stage there are therefore present 16 smaller and 16 larger segments (fig. 40, 32). After 64 segments have been formed by vertical furrows which arise symmetrically in the two poles (fig. 40, 64), two equatorial furrows appear in the upper pole before a fresh furrow arises in the lower; so that there are 128 segments in the upper half, and only 32 in the lower. The regularity is quite lost in subsequent stages, but the upper pole continues to undergo a more rapid segmentation than the lower. While the segments have been increasing in number the segmentation cavity has been rapidly growing in size; and at the close of segmentation the egg forms a sphere, containing an excentric cavity, and composed of two unequal parts (fig. 41). The upper part, which forms the roof of the segmentation cavity, is formed of smaller cells: the lower of larger yolk-containing cells.

[FIG. 41. SECTION THROUGH FROG'S OVUM AT THE CLOSE OF SEGMENTATION.

_sg._ segmentation cavity. _ll._ large yolk-containing cells. _ep._
small cells at formative pole (epiblast).]

The mode of segmentation of the Frog's ovum is typical for unequally segmenting ova, and it deserves to be noticed that as regards the first three or more furrows the segmentation occurs with the same rhythm in the unequally segmenting ova as in those which have an uniform segmentation. There appear two vertical furrows followed by an equatorial furrow. The general laws which were stated with reference to the _velocity_ of segmentation and the size of the resulting segments are well exemplified in the case of the Frog's ovum.

The majority of the smaller segments in the segmented Frog's ovum are destined to form into the epiblast, and the larger segments become hypoblast and mesoblast.

With a few exceptions (the Rabbit, Lymnæus, etc.) the majority of the smaller segments always become epiblast and of the larger segments hypoblast.

The Frog's ovum serves as a good medium type for unequally segmenting ova. There are many cases however in which a regular segmentation is far more closely approached, and others in which it is less so.

One familiar instance in which a regular segmentation is nearly approached is afforded by the Rabbit's ovum, which has indeed usually been regarded as offering an example of a regular segmentation.

The ovum of the Rabbit[39] becomes first divided into two sub-equal spheres. The larger and more transparent of the two may, from its eventual fate, be called the epiblastic sphere, and the other the hypoblastic. The two spheres are divided into four, and then by an equatorial furrow into eight--four epiblastic and four hypoblastic. One of the latter assumes a central position. The four epiblastic spheres now divide before the four hypoblastic. There is thus introduced a stage with twelve spheres. It is followed by one with sixteen, and that by one with twenty-four. During the stages with sixteen spheres and onwards the epiblastic spheres gradually envelop the hypoblastic, which remain exposed on the surface at one point only. There is no segmentation cavity.

[39] Van Beneden, "Développement embryonnaire des Mammifères."
_Bull. de l'Acad. Belgique_, 1874.

In Pedicellina, one of the entoproctous Polyzoa, there is a sub-regular segmentation, where however the two primary spheres can be distinguished much in the same way as in the case of the Rabbit.

A very characteristic type of unequal segmentation is that presented by the majority of Gasteropods and Pteropods and probably also of some Lamellibranchiata. It is also found in some Turbellarians, in Bonellia, some Annelids, etc. In many instances it offers a good example of the type where in the course of segmentation the protoplasm becomes aggregated at one pole of the ovum, or of its segments, to become separated off as a clear sphere.

The first four segments formed by two vertical furrows at right angles are equal, but from these there are budded off four smaller segments, which in subsequent stages divide rapidly, receiving however, a continual accession of segments budded off from the larger spheres. The four larger spheres remain conspicuous till near the close of the segmentation. The process of budding, by which the smaller spheres become separated from the larger, consists in a larger sphere throwing out a prominence, which then becomes constricted off from it.

In the extreme forms of this unequal segmentation we find at the end of the second cleavage two larger spheres filled with yolk material and two smaller clear spheres; and in the later stages, though the large spheres continue to bud off small spheres, only the two smaller ones undergo a regular segmentation, and eventually completely envelop the former. Such a case as this has been described in Aplysia by Lankester[40].

[40] _Phil. Trans._ 1875.

The types I have described serve to exemplify unequal segmentation. The Rabbit's ovum stands at one end of the series, that of Aplysia at the other; and the Frog's ovum between the two.

Great variations are presented by the ova with unequal segmentation as to the presence of a segmentation cavity. In some instances, _e.g._ the Frog, such a cavity is well developed. In other cases it is small, _e.g._ most Mollusca, while not unfrequently it is altogether absent.

Before leaving this important type of segmentation, it will be well to enter with slightly greater detail into some of the more typical as well as some of the special forms which it presents.

As an example of the typical Molluscan type the normal Heteropod segmentation, accurately described by Fol[41], may be selected.

[41] Fol, _Archives de Zoologie Expérimentale_, Vol. IV.
1875.

The ovum divides into two and then four equal segments in the usual vertical planes. Each segment has a protoplasmic and a vitelline pole. The protoplasmic pole is turned towards the polar bodies. In the third segmentation, which takes place along an equatorial plane, four small protoplasmic cells or segments are segmented or rather budded off from the four large segments, so that there are four small segments in one plane and four large below these. In the fourth segmentation the four large segments alone are active and give rise to four small and four large cells; so that there are formed in all eight small and four large cells. The four small cells of the third generation next divide, forming in all twelve small cells and four large. The small cells of the fourth generation then divide, and subsequently the four large cells give rise to four new small ones, so that there are twenty small cells and four large. The small cells form a cap embracing the upper pole of the large segments. It may be noted that from the third stage onwards the cells increase in arithmetical progression--a characteristic feature of the typical gasteropod segmentation.

In the later stages of segmentation the large cells cease to give rise to smaller ones in the same manner as before. One of them divides first into two unequal parts, of which the smaller becomes pushed in towards the centre of the egg. The larger cell then divides again into two, and the three cells so formed occupy the centre of a shallow depression. The remaining larger cells divide in the same way, and give rise to smaller cells which line a pit which becomes formed on one side of the ovum. The original smaller cells continue in the meantime to divide and so form a layer enclosing the larger, leaving exposed however the opening of the pit lined by the latest products of the larger cells.

The eggs of Anodon and Unio serve as excellent examples of the type in which the ovum has a uniform structure before the commencement of segmentation, but in which a separation into a protoplasmic and a nutritive portion becomes obvious during segmentation.

In Anodon[42] the egg is at first uniformly granular, but after impregnation it throws out on one side a protuberance nearly free from granules (fig. 42, 1)

[42] Flemming, "Entwick. der Najaden," _Sitz. d. Akad. Wiss.
Wien_, Bd. 4, 1875.

[FIG. 42. SEGMENTATION OF ANODON PISCINALIS. (Copied from Flemming.)
_r._ polar cells. _v._ vitelline sphere. 1. Commencing division into
two segments; one mainly formed of protoplasm, the other of yolk. 2.
Stage with four segments. 3. Formation of blastosphere, and
segmentation cavity. 4. Definite segmentation of the yolk sphere.]

In the case of this clear protuberance and of the similar protuberances which follow it, the protoplasm is not at first quite free from food-yolk, but only becomes so on being separated from the yolk-containing part of the ovum. We must therefore suppose that the production of the clear segments is in part at least due to the yolk-spherules becoming used up to form protoplasm. Such a formation of protoplasm from yolk-spherules has been clearly shewn to occur in other types by Bobretzky and Fol.

The protuberance soon becomes separated off from the larger part of the egg as a small segment composed of clear protoplasm. From the larger segment filled with food-yolk, a second small clear segment is next budded off, and simultaneously (fig. 42, 2) the original small segment divides into two. Thus there are formed four segments, one large and three small; the large segment as before being filled with food-yolk. The continuation of a similar process of budding off and segmentation eventually results in the formation of a considerable number of small and of one large segment (fig. 42, 3). Between this large and the small segments is a segmentation cavity.

Eventually the large yolk segment, which has hitherto merely budded off a series of small segments free from yolk, itself divides into two similar parts. This process is then repeated (fig. 42, 4) and there is at last formed a number of yolk segments filled with yolk spheres, which occupy the place of the original large yolk segment. Between these yolk segments and the small segments is placed the segmentation cavity.

The segmentation of the ovum of Euaxes[43] resembles that of Unio in the budding off of clear segments from those filled with yolk, but presents many interesting individualities.

[43] Kowalevsky, _Mem. Akad. Petersburg_, Series VII, 1871.

A very peculiar modification of the ordinary Gasteropod segmentation is that described by Bobretzky for Nassa mutabilis[44].

[44] _Archiv. f. mikr. Anat._ Vol. XIII. 1877.

The ovum contains a large amount of food-yolk, and the protoplasm is aggregated at the formative pole, adjoining which are placed the polar bodies. An equatorial and a vertical furrow (fig. 43 A), the former near the upper pole, appear simultaneously, and divide the ovum into three segments, two small, each with a protoplasmic pole, and one large entirely formed of yolk material. One of the two small segments next completely fuses with the large segment (fig. 43 B), and after the fusion is complete, a triple segmentation of the large segment takes place as at the first division, and at the same time the single small segment divides into two. In this way four partially protoplasmic segments and one yolk segment are formed (fig. 43 C). One of the small segments again fuses with the large segment, so that the number of segments becomes again reduced to four, three small and one large. The protoplasmic ends of these segments are turned towards each other, and where they meet four very small cells become budded off, one from each segment (fig. 43 D). Four small cells are again budded off twice in succession, while the original small cells remain passive, so that there come to be twelve small and four large cells. In later stages the four first-formed small cells give rise to still smaller cells and then the next-formed do the same. The large cells continue also to give rise to small ones, and finally, by a continuous process of division, and fresh budding of small cells from large cells, a cap of small cells becomes formed covering the four large cells which have in the meantime pressed themselves together (fig. 43 E). A segmentation cavity of not inconsiderable dimensions becomes established between this cap of small cells and the large cells.

[FIG. 43. SEGMENTATION OF NASSA MUTABILIS (from Bobretzky). A. Upper
half divided into two segments. B. One of these has fused with the
large lower segment. C. Four small and one large segment, one of the
former fusing with the large segment. D. Each of the four segments
has given rise to a small segment. E. Small segments have increased
to thirty-six.]

Many eggs, such as those of the Myriapods[45], present an irregular segmentation; but the segmentation is hardly unequal in the sense in which I have been using the term. Such cases should perhaps be placed in the first rather than in the present category.

[45] Metschnikoff, _Zeitschrift f. wiss. Zoologie_, 1874.

The type of unequal segmentation is on the whole the most widely distributed in the animal kingdom. There is hardly a group without examples of it.

It occurs in Porifera, Hydrozoa, Actinozoa and Ctenophora. Amongst the Ctenophora this segmentation is of the most typical kind. Four equal segments are first formed in the two first periods. In the third period a circumferential furrow separates four smaller from four larger segments.

This type is also widely distributed amongst the unsegmented (Gephyrea, Turbellaria), as well as the segmented Vermes, and is typical for the Rotifera. It appears to be very rare in Echinoderms (_Echinaster Sarsii_). It is not uncommon in early stages of the segmentation of the lower Crustacea.

For Mollusca (except Cephalopoda) it is typical. Amongst the Ascidia it occurs in several forms (_Salpa_, _Molgula_) and amongst the Craniata it is typical in the Cyclostomata, Amphibia, and some Ganoids, _e.g. Accipenser_.

Partial segmentation. The next type of segmentation we have to deal with has long been recognized as partial segmentation. It is a type in which only part of the ovum, called the germinal disc, undergoes segmentation, the remainder usually forming an appendage of the embryo known as the yolk-sack. Ova belonging to the two groups already dealt with are frequently classed together as holoblastic ova, in opposition to ova of the present group in which the segmentation is only partial, and which are therefore called meroblastic. For embryological purposes this is in many ways a very convenient classification, but ova belonging to the present group are in reality separated by no sharp line from those belonging to the group just described.

[FIG. 44. SURFACE VIEWS OF THE EARLY STAGES OF THE SEGMENTATION IN A
FOWL'S EGG. (After Coste.)

_a._ edge of germinal disc. _b._ vertical furrow. _c._ small central
segment. _d._ larger peripheral segment.]

The origin and nature of meroblastic ova will best be understood by taking an ovum with an unequal segmentation, such as that of the frog, and considering what would take place in accordance with the laws already laid down, supposing the amount of food-yolk at the vitelline pole to be enormously increased. What would happen may be conveniently illustrated by fig. 44, representing the segmentation of a fowl's egg. There would first obviously appear a vertical furrow at the formative or protoplasmic pole. (Fig. 44 A, _b_.) This would gradually advance round the ovum and commence to divide it into two halves. Before the furrow had however proceeded very far it would come to the vitelline part of the ovum; here, according to the law previously enunciated, it would travel very slowly, and if the amount of the food-yolk was practically infinite as compared with the protoplasm, it would absolutely cease to advance. A second vertical furrow would soon be formed, crossing the first at right angles, and like it not advancing beyond the edge of the germinal disc. (Fig. 44 B.)

The next furrow should be an equatorial one (as a matter of fact in the fowl's ovum an equatorial furrow is not formed till after two more vertical furrows have appeared). The equatorial furrow would however, in accordance with the analogy of the frog, _not be formed at the equator, but very close to the formative pole_. It would therefore separate off as a distinct segment (fig. 44 C, _c_), a small central, _i.e._ polar, portion of each of the imperfect segments formed by the previous vertical furrows. By a continuation of the process of segmentation, with the same alternation of vertical and equatorial furrows as in the frog, a cap or disc of small segments would obviously be formed at the protoplasmic pole of the ovum, outside which would be a number of deep radiating grooves (fig. 45), formed by the vertical furrows, the advance of which round the ovum has come to an end owing to the too great proportion of yolk spheres at the vitelline pole.

[FIG. 45. SURFACE VIEW OF THE GERMINAL DISC OF FOWL'S EGG DURING A
LATE STAGE OF THE SEGMENTATION.

_c._ small central segmentation spheres; _b._ larger segments
outside these; _a._ large, imperfectly circumscribed, marginal
segments; _e._ margin of germinal disc.]

It is clear from the above that an immense accumulation of food-yolk at the vitelline pole necessarily causes a partial segmentation. It is equally clear that the part of meroblastic ova which does not undergo segmentation is not a new addition absent in other cases. It is on the contrary to be regarded merely as a part of the ovum in which the yolk-spherules have attained to a very great bulk as compared with the protoplasm; sometimes even to the complete exclusion of the protoplasm.

An ordinary meroblastic ovum consists then of a small disc at the formative pole, known as the germinal disc, composed mainly of protoplasm in which comparatively little food-yolk is present. This graduates into the remainder of the ovum, being separated from it by a more or less sharp line. This remainder of the ovum, which almost always forms the major part, usually consists of numerous yolk-spherules, embedded in a very scanty protoplasmic matrix.

In some cases, _e.g._ the eggs of Elasmobranchii[46], the protoplasm is present in the form of a delicate network; in other and perhaps the majority of cases, too little protoplasm is present to be detected, or the protoplasm may even be completely absent. In some Osseous Fishes, _e.g._ Lota, the yolk forms a homogeneous transparent albuminoid substance containing a large globule at the pole furthest removed from the germinal disc. In this case the germinal disc is sharply separated from the yolk. In other Osseous Fishes the separation between the two parts is not so sharp[47]. In these cases we find adjoining the germinal disc a finely granular material containing a large proportion of protoplasm; this graduates into a material with very little protoplasm and numerous yolk-spherules, which is in its turn continuous with an homogeneous albuminoid yolk substance. In Elasmobranchii we find that immediately beneath the germinal disc there is present a finely granular matter, rich in protoplasm, which is continuous with the normal yolk.

[46] _Vide_ Schultze, _Archiv. f. mikr. Anat._ Vol. XI.;
and F. M. Balfour, _Monograph on the Development of
Elasmobranch Fishes_.

[47] _Vide_ Klein, _Quart. Journal of Micr. Science_,
April, 1876. Bambeke, _Mem. Cour. Acad. Belgique_, 1875.
His, _Zeit. für Anat. u. Entwicklung_. Vol. I.

The Elasmobranch ovum may conveniently serve as type for the Vertebrata. The ovum is formed of a spherical vitellus without any investing membrane. The germinal disc is recognizable on this as a small yellow spot about 1-1/2 millimetres in diameter. In the germinal disc a furrow appears bisecting the disc, followed by a second furrow at right angles to the first. Thus after the formation of the second furrow the disc is divided into four equal areas. Fresh furrows continue to rise, and eventually a circular furrow, equivalent to the equatorial furrow of the frog's ovum, makes its appearance, and separates off a number of smaller central segments from peripheral larger segments. In the later stages the smaller segments at first divide more rapidly than the larger, but eventually the latter also divide rapidly, and the germinal disc becomes finally formed of a series of segments of a fairly uniform size. So much may be observed in surface views of the segmenting ovum, and it may be noted that there is not much difference to be observed between the segmentation of the germinal disc of the Fowl's ovum and that of the Elasmobranchii. Indeed the figure of the former (fig. 44) would serve fairly well for the latter. When however we examine the segmenting germinal discs by means of sections, there are some differences between the two types, and several interesting features which deserve to be noticed in the segmentation of the Elasmobranchii. In the first stages the furrows visible on the surface are merely furrows, which do not meet so as to isolate distinct segments; they merely, in fact, form a surface pattern. It is not till after the appearance of the equatorial furrow that the segments begin to be distinctly isolated. In the subsequent stages not only do the segments already existing in the germinal disc increase by division, but fresh segments are continually being formed from the adjacent yolk, and added to those already present in the germinal disc. (Fig. 46.) This fact is one out of many which prove that the germinal disc is merely part of the ovum characterized by the presence of more protoplasm than the remainder which forms the so-called food-yolk. During the latest stages of segmentation there appear in the yolk around the blastoderm a number of nuclei. (Fig. 46, _nx´._) These are connected with a special protoplasmic network (already described) which penetrates through the yolk. Towards the end of segmentation, and during the early periods of development which succeed the segmentation, these nuclei become very numerous. (Fig. 47 A, _n´_.) Around many of them a protoplasmic investment is established, and cells are thus formed which eventually enter the blastoderm.

[FIG. 46. SECTION THROUGH GERMINAL DISC OF A PRISTIURUS EMBRYO
DURING THE SEGMENTATION.

_n._ nucleus; _nx._ nucleus modified prior to division; _nx´._
modified nucleus of the yolk; _f._ furrow appearing in the yolk
adjacent to the germinal disc.]

The result of segmentation is the formation of a lens-shaped mass of cells lying in a depression on the yolk. In this a cavity appears, the homologue of the segmentation cavity already spoken of. It lies at first in the midst of the cells of the blastoderm, but very soon its floor of cells vanishes, and it lies between the yolk and the blastoderm. (Fig. 47 A.) Its subsequent history is given in a future Chapter.

Segmentation proceeds in Osseous Fishes in nearly the same manner as in Elasmobranchii. In some cases the germinal disc is small as compared with the yolk, in other cases it is almost as large as the yolk. The only points which deserve special notice are the following: (1) Nuclei, precisely similar to those in the Elasmobranch yolk, appear in the protoplasmic matter around the germinal disc; (2) After the deposition of the ova there is present in some forms a network of protoplasm extending from the germinal disc through the yolk[48]. At impregnation this withdraws itself from the yolk. It is to be compared to the protoplasmic network of the Elasmobranch ovum.

[48] _Vide_ Bambeke, _loc. cit._

[FIG. 47. TWO LONGITUDINAL SECTIONS OF THE BLASTODERM OF A
PRISTIURUS EMBRYO AT STAGES PRIOR TO THE FORMATION OF THE MEDULLARY
GROOVE.

_ep._ epiblast; _ll._ lower layer cells; _m._ mesoblast; _hy._
hypoblast; _sc._ segmentation cavity; _es._ embryo swelling; _n´_.
nuclei of yolk; _er._ embryonic rim.]

There are two types of meroblastic ova. In one of these (Aves, Elasmobranchii) the germinal disc is formed in the ovarian ovum. In the second type the germinal disc is formed after impregnation by a concentration of the protoplasm at one pole. This concentration is analogous to what has already been described for Anodon and other Molluscan ova (p. 100).

The ova of some Teleostei are intermediate between the two types.

The ovum of the wood-louse, Oniscus murarius[49], may be taken as an example of the second type of meroblastic ovum. In this egg development commences by the appearance of a small clear mass with numerous transparent vesicles. This mass is the protoplasm which has become separated from the yolk. It undergoes segmentation in a perfectly normal fashion. Examples of other cases of this kind have been described by Van Beneden and Bessels[50] in Anchorella, and in Hessia by Van Beneden[51]. It appears from their researches that the protoplasm collects itself together, first of all in the interior of the egg, and then travels to the surface. It arrives at the surface after having already divided into two or more segments, which then rapidly divide in the usual manner to form the blastoderm.

[49] _Vide_ Bobretzky, _Zeitschrift für wiss. Zoologie_,
Vol. XXIV., 1874.

[50] _Loc. cit._

[51] _Bulletins de l'Acad. Belgique_, Tom. XXIX., 1870.

There are some grounds for thinking that the cases of partial segmentation in the Arthropoda are not really quite comparable with those in other groups, but more probably fall under the next type of segmentation to be described. The grounds for this view are mentioned in connection with the next type.

In most if not all meroblastic ova there appear during and after segmentation a number of nuclei in the yolk adjoining the blastoderm, around which cells become differentiated. (Figs. 46 and 47.) These cells join the part of the blastoderm formed by the normal segmentation of the germinal disc. Such nuclei are formed in all craniate meroblastic ova[52]. In Cephalopods they have been found by Lankester, and in Oniscus by Bobretzky. They have been by some authors supposed to originate from the nuclei of the blastoderm, and by others spontaneously in the yolk.

[52] Though less obvious in the ovum of the fowl than in that of
some other types, they may nevertheless be demonstrated there
without very much difficulty.

Some of the earliest observations on these nuclei were made by Lankester[53] in the Cephalopods. He found that they appeared first in a ring-like series round the edge of the blastoderm, and subsequently all over the yolk in a layer a little below the surface. He observed their development in the living ovum and found that they "commenced as minute points, gradually increasing in size like other free-formed nuclei." A cell area subsequently forms around them.

[53] _Quart. Journ. of Micr. Science_, Vol. XV. pp. 39, 40.

By E. van Beneden[54] they were observed in a Teleostean ovum to appear nearly simultaneously in considerable numbers in the granular matter beneath the blastoderm. Van Beneden concludes from the simultaneous appearance of these bodies that they develop autogenously. Kupffer at an earlier period arrived at a similar conclusion. My own observations on these nuclei in Elasmobranchii on the whole support the conclusions to be derived from Lankester's, Kupffer's and Van Beneden's observations. As mentioned above, the nuclei in Elasmobranchii do not appear simultaneously, but increase in number as development proceeds; and it is possible that Van Beneden may be mistaken on this point. No evidence came before me of derivation from pre-existing nuclei in the blastoderm. My observations prove however that the nuclei increase by division. This is shewn by the fact that I have found them with the spindle modification (fig. 46, _nx´_), and that in most cases they usually exhibit the form of a number of aggregated vesicles[55], which is a character of nuclei which have just undergone division. It should be mentioned however that I failed to find a spindle modification of the nuclei in the later stages. Against these observations must be set those of Bobretzky, according to which the nuclei in Oniscus are really the nuclei of cells which have migrated from the blastoderm. Bobretzky's observations do not however appear to be very conclusive.

[54] _Quart. Journ. of Micr. Science,_ Vol. XVIII. p. 41.

[55] At the time when my observations on Elasmobranchii were
carried out, this peculiar condition of the nucleus had not been
elucidated.

It must be admitted that the general evidence at our command appears to indicate that the nuclei of the yolk in meroblastic ova originate _spontaneously_. There is however a difficulty in accepting this conclusion in the fact that all the other nuclei of the embryo are descendants of the first segmentation nucleus; and for this reason it still appears to me possible that the nuclei of the yolk will be found to originate from the continued division of one primitive nucleus, itself derived from the segmentation nucleus.

The existence of these nuclei in the yolk and the formation of a distinct cell body around them is a strong piece of evidence in favour of the view above maintained, (which is not universally accepted,) that the part of the ovum of meroblastic ova which does not segment is of the same nature as that which does segment, and differs only in being relatively deficient in active protoplasm.

The following forms have meroblastic ova of the first type: the Cephalopoda, _Pyrosoma_, Elasmobranchii, Teleostei, Reptilia, Aves, Ornithodelphia (?). The second type of meroblastic segmentation occurs in many Crustacea, (parasitic Copepoda, Isopoda _Mysis_, etc.). It is also stated to be found in _Scorpio_.

The ova of the majority of groups in the animal kingdom segment according to one of the types which have just been described. These types are not sharply separated, but form an unbroken series, commencing with the ovum which segments uniformly, and ending with the meroblastic ovum.

It is convenient to distinguish the ova which segment uniformly by some term; and I should propose for this the term alecithal[56], as implying that they are without food-yolk, or that what little food-yolk there is, is distributed uniformly.

[56] For this term as well as for the terms telolecithal and
centrolecithal I am indebted to Mr Lankester.

The ova in which the yolk is especially concentrated at one pole I should propose to call telolecithal. They constitute together a group with an unequal or partial segmentation.

The telolecithal ova may be defined in the following way: ova in which the food-yolk is not distributed uniformly, but is concentrated at one pole of the ovum. When only a moderate quantity of food-yolk is present the pole at which it is concentrated merely segments more slowly than the opposite pole; but when food-yolk is present in very large quantity the part of the ovum in which it is located is incapable of segmentation, and forms a special appendage known as the yolk-sack.

There is a third group of ova including a series of types of segmentation nearly parallel to the telolecithal group. This group takes its start from the alecithal ovum as do the telolecithal ova, and equally with these includes a series of varieties of segmentation running parallel to the regular and unequal types of segmentation which directly result from the presence of a greater or smaller quantity of food-yolk. The food-yolk is however placed, not at one pole, but _at the centre of the ovum_. This group of ova I propose to name centrolecithal. It is especially characteristic of the Arthropoda, if not entirely confined to that group.

Centrolecithal ova. As might be anticipated on the analogy of the types of segmentation already described, the concentration of the food-yolk at the centre of the ovum does not always take place before segmentation, but is sometimes deferred till even the later stages of this process.

Examples of a regular segmentation in centrolecithal ova are afforded by Palæmon (Bobretzky) and Penæus (Hæckel). A type of unequal segmentation like that of the Frog occurs in _Gammarus locusta_ (Beneden and Bessels), where however the formation of a central yolk mass does not appear to take place till rather late in the segmentation. More irregular examples of unequal segmentation are also afforded by other Crustaceans, _e.g._ various members of the genus _Chondracanthus_ (Beneden and Bessels) and by Myriapods. In all these cases segmentation ends in the formation of a layer of cells enclosing a central mass of food-yolk.

[FIG. 48. SEGMENTATION OF A CRUSTACEAN OVUM (PENÆUS). (After
Hæckel.)

The sections illustrate the type of segmentation in which the yolk
is aggregated at the centre of the ovum.

_yk._ central yolk mass.

1 and 2. Surface view and section of the stage with four segments.
In 2 it is seen that the furrows visible on the surface do not
penetrate to the centre of the ovum.

3 and 4. Surface view and section of ovum near the end of
segmentation. The central yolk mass is very clearly seen in 4.]

The peculiarity of the centrolecithal ova with regular or unequal segmentation is that (owing to the presence of the yolk in the interior) the furrows which appear on the surface are not continued to the centre of the egg. The spheres which are thus distinct on the surface are really united internally. Fig. 48, copied from Hæckel, shews this in a diagrammatic way.

Many ova, which in the later stages of segmentation exhibit the characteristics of true centrolecithal ova, in the early stages actually pass through nearly the same phases as holoblastic ova. Thus in _Eupagurus prideauxii_[57] (fig. 49), and probably in the majority of Decapods, the egg is divided successively into two, four and eight distinct segments, and it is not till after the fourth phase of the segmentation that the spheres fuse in the centre of the egg. Such ova belong to a type which is really intermediate between the ordinary type of segmentation and that with a central yolk mass. Eupagurus presents one striking peculiarity, viz. that the nucleus divides into two, four and eight nuclei, each surrounded by a delicate layer of protoplasm prolonged into a reticulum, before the ovum itself commences to become segmented. The ovum before segmentation is therefore in the condition of a syncytium.

[57] Mayer, _Jenaische Zeitschrift_, Vol. XI.

[FIG. 49. TRANSVERSE SECTION THROUGH FOUR STAGES IN THE SEGMENTATION
OF EUPAGURUS PRIDEAUXII. (After P. Mayer.)]

The segmentation of Asellus aquaticus[58] is very similar to that of Eupagurus, etc. but the ovum at the very first divides into as many segments (viz. eight) as there are nuclei.

[58] Ed. van Beneden, _Bull. d. l'Acad. roy. Belgique_,
2me série, Tom. XXVIII. No. 7, 1869, p. 54.

In Gammarus locusta the resemblance to ordinary unequal segmentation is very striking, and it is not till a considerable number of segments have been formed that a central yolk mass appears.