and thus each takes the form of an angular loop, a straight rod, or
a roundish, oval, or spherical body (Fig. 74, _C_, _chrs_). While
this is happening, we can see at the side of the nucleus, and closely
apposed to it, a pale longitudinally striped figure with a swelling,
similar to a handle, at both ends--the so-called nuclear spindle or
central spindle (_ksp_). This is the apparatus for the division of the
nucleus, and it was previously represented by a small body susceptible
to certain stains--the centrosome, which was surrounded by a halo-like
zone, the centrosphere or 'sphere.' This body was long overlooked,
but now the majority of investigators assume that, though it is often
inconspicuous and very difficult to make visible, it is nevertheless
present in every cell which is capable of division, and that it is
therefore a permanent and indispensable constituent of the cell (Fig.
74, _A_ and _B_, _csph_).
When a cell is on the point of dividing, this remarkable cell-organ,
which has hitherto seemed no more than an insignificant, pale, little
sphere, now becomes active. First of all, often before the formation
of the chromatin coil, it doubles by division (_A_ and _B_ _csph_), at
first only as regards the centrosome, and then as regards the sphere
also (_B_); and while division is going on fine protoplasmic filaments
issue from the dividing sphere and radiate like rays from a sun into
the cell-substance. As they only retain their connexion with each other
at the surfaces of the dividing halves of the sphere which are turned
towards each other, we might almost say that fine threads are drawn out
between the two halves as they separate, and these become longer the
further apart the halves diverge. In this manner the much-talked-of
'spindle figure' arises, which was first described in the seventies
through the researches of A. Schneider, Auerbach, and Bütschli, but the
significance and origin of which have claimed the labours of many later
investigators down to our own day.
The processes now to be described do not always take place in exactly
the same manner, but the gist of the business is everywhere the same,
and it consists in this, that the two ends or 'poles' of the spindle
diverge further and further apart, and between them lies the nucleus
whose membrane now disappears (_C_, _km_) while the spindle threads
traverse its interior. Sometimes the membrane is retained, but
nevertheless the spindle threads penetrate into the interior of the
nucleus. But the chromosomes always range themselves quite regularly
in the 'equatorial plane' of the spindle (_D_, _aeq_)--a process the
precise mechanism of which is by no means clearly understood, and
indeed the play of the forces in the whole process of nuclear division
is still very imperfectly revealed to our intelligence.
Thus we have now before us a pale, spindle-shaped figure, which takes
only a faint stain, with the 'suns' (_cs_) at its 'poles,' and in its
equatorial plane the loop- or rod-shaped, or spherical chromosomes
(_chrs_). The whole is designated the 'karyokinetic,' the 'mitotic,' or
the 'nuclear division figure.'
The meaning and importance of this, at first sight, puzzling figure
will at once become clear from what follows. It may be observed at this
stage, if not even long before, that each of the chromatin rods or
loops has split along its whole length like a log of wood, and that the
split halves are beginning slowly and hardly noticeably to move away
from each other, one half towards one, the other towards the other pole
of the spindle (Fig. _D_ and _F_). Directly in front of the centrosome
they make a halt, and now the material for the two daughter-nuclei
is in its proper place (_F_, _chrs_). These develop quickly, each
chromosome group surrounding itself with a nuclear membrane (Fig.
_G_) within which the chromosomes gradually become transformed again
into a nuclear network. Within the chromatin substance proper this is
scattered about in small roundish or angular granules, lying especially
at the intersecting points of the network. It may be stated at once,
though the full significance of the statement can only be appreciated
later, that we may assume with probability that this breaking up of the
chromosomes is only apparent, and that these rods or spheres really
continue to exist in the nuclear network, only in a different form,
greatly spread out, somewhat after the manner of a Rhizopod which
stretches out fine processes in all directions. These processes branch
and anastomose, so that the body, which previously seemed compact,
now appears as a fine network. In point of fact, it can be directly
observed that the chromosomes, after the nucleus is completely divided
into two daughter-nuclei, send out pointed processes (_F_ and _G_)
which gradually increase in length and branch, while the body of the
chromosome itself becomes gradually smaller. It is thus probable
that, when such a daughter-nucleus is on the point of dividing anew,
it may, by a drawing together of the processes or pseudopodia of the
chromosomes, produce the same rods or spheres as those which previously
gave rise to the network. More definite reasons for this interpretation
will be adduced later on. In any case, the chromosomes, even in
their compact rod-like state, consist of two kinds of substance,
the chromatin proper, which stains deeply, and the linin, which is
difficult to stain; and it is the latter which, by breaking up, forms
the pale part of the nuclear network.
Thus we can understand that the number of chromosomes remains the same
in every cell-generation throughout development, as it is the same
in all the individuals of a species. The numbers are known for many
species: in some worms there are only two or four chromosomes, while
in other related worms there are eight; in the grasshopper there are
twelve, and in a marine worm, _Sagitta_, eighteen; in the mouse, the
trout, and the lily there are twenty-four; in some snails thirty-two;
in the sharks thirty-six, and in _Artemia_, a little salt-water
crustacean, 168 chromosomes. In Man the chromosomes are so small that
their normal number is not certain--sixteen have been counted. This
counting can only be done during the process of nuclear division, for
afterwards the chromosomes flow indistinguishably together, or rather
apart, only to reappear, however, in the old form and number whenever
the nucleus again begins to divide.
It remains to be told what becomes of the centrosphere in
cell-division. As soon as the formation of the daughter-nuclei has
been brought about by the divergence of the split halves of the loops,
the spindle figure begins to retrograde, its threads become pale and
gradually disappear, as does the whole radiate halo of the centrosphere
(Fig. _F_ and _G_). The cell-body has by this time also divided in the
equatorial plane of the nuclear spindle, and the centrosome remains
usually as a very inconspicuous pale body lying in the cytoplasm close
to the nucleus, reawakening to renewed activity when cell-division is
about to recommence (_G_, _csph_).
These, briefly, are the remarkable processes of nuclear division. Their
net result is obvious; the chromatin substance is divided between the
daughter-nuclei with the greatest conceivable accuracy.
It is not so easy to understand the mechanism of this partition, and
there are various divergent theories on this point. According to the
older idea of Van Beneden, the spindle fibres work like muscles, and
by contracting draw the halves of the chromosomes which adhere to them
towards the pole, while the rest of the fibres radiating out from
the polar corpuscles act as resisting and supporting elements. This
view, with many modifications however, has still its champions, and M.
Heidenhain in particular has made a notable attempt to establish it
and to work it out in detail. Opposed to it stand the views of those
who, like O. Hertwig, Bütschli, Häcker, and others regard the rays not
as specific elements which were pre-formed in the cell, but as the
expression of the orientation of certain protoplasmic particles--an
orientation evoked by forces which have their seat within the central
corpuscles, and act in the manner of magnetic or electric forces. That
the central corpuscles are centres of attraction seems to me hardly
open to doubt, and I cannot regard the regular arrangement of the
chromosomes in the equatorial plane of the spindle as due to a mere
adhesion to contractile threads. Some still unknown forces--chemotactic
or otherwise--must be at work here. Later on we shall study the
phenomenon of the migration of the sperm-nucleus into the ovum, when it
is accompanied by its central body and its halo of rays. Häcker seems
to me justified in inferring from this phenomenon alone that the sudden
origin of the rays is due to forces resident in the central corpuscle.
But undoubtedly even this 'dynamic' explanation of karyokinesis is
still only at the stage of hypothesis and reasoning from analogy, and
is far removed from a definite knowledge of the forces at work.
For the problems with which we are here chiefly concerned, the problems
of heredity, it is enough to know that the cells of multicellular
organisms possess an extremely complex apparatus for division, whose
chief importance lies in the fact that through it the chromatin units
of the nucleus are divided into precisely equal parts, and so separated
from each other that one half forms one daughter-nucleus, the other
half the other. It is not merely that there is an exact division of the
whole chromatin in the mass, which could have been effected much more
simply, but that there is _a regulated distribution of the different
qualities of the chromatin_, as we shall see later.
It must here be emphasized that the splitting of the chromosomes does
not depend on external forces, but on internal ones involved in their
organization, and in the definite attractions and repulsions of their
component particles which come about in the course of growth. The
chromosomes do not split like a trunk that has been broken open with
an axe, but rather like a tree burst apart by the frost, that is, by
the freezing of the water within itself. I consider it very important
that we should recognize this, even though we do not yet know what
the forces are that have control in this case, because it leads us to
conclude that the structure of the chromosomes is extremely complex,
that they are, so to speak, a world in themselves, that they possess an
infinitely complex and delicate though invisible organization, in which
intrinsic chemico-physical forces produce the regulated succession
of changes which we observe. We shall afterwards see that we are led
to the same conclusion from another direction--that is, from the
phenomena of inheritance. We shall then recognize that the rod- or
loop-shaped chromosomes cannot be simple elements, but are composed
of linear series of 10, 20, or more globular single-chromosomes, each
of which represents a particular kind of chromatin or hereditary
substance. If we consider this carefully, we shall see that it would
hardly be possible to think out a mode of nuclear division which would
so exactly and securely fulfil the purpose of conveying these many
kinds of chromatin to the two daughter-nuclei in like proportions as
does the mechanism of distribution actually brought about by nature.
The longitudinal splitting of the rods halves the chromosomes, and the
spindle apparatus secures the proper distribution of the halves between
the two daughter-nuclei.
So much, at least, is certain, that no such complicated mechanism for
'mitotic' division would have arisen if the very precise division of
a substance _of the highest importance_ had not been concerned, and
in this conclusion lies the first hint of the interpretation of the
chromatin substance as the bearer of the hereditary qualities.
We are now familiar with the cell-nucleus and the apparatus for
its division, and we are thus fully prepared to begin the study of
the phenomena of 'fertilization.' Here also the processes depend
essentially on the behaviour of the cell-nuclei, for even the first
observations made by O. Hertwig on the behaviour of the spermatozoon
after it has penetrated into the ovum led to the suggestion that the
essential fact is the union of two nuclei; and numerous later, more and
more deeply penetrating researches have furnished abundant evidence
that the so-called 'fertilization' _is essentially a nuclear fusion_.
Let us begin with O. Hertwig's observations on the ovum of the
sea-urchin. Eggs of this animal, which have been taken out of the ovary
of the female, may easily be fertilized artificially by pouring over
them spermatic fluid taken from a male, and diluted with sea-water.
Before this is done only one nucleus can be observed in the ovum, but
shortly afterwards two nucleus-like structures of unequal size can
be seen within the ovum, and the smaller is surrounded by a circle
of rays. Hertwig rightly interpreted this smaller nucleus as the
modified remains of the penetrating spermatozoon, which then slowly
approaches the nucleus of the egg, and ultimately fuses with it to form
a 'segmentation nucleus.' From this starts the so-called 'segmentation'
of the ovum, that is, the series of repeated divisions resulting in the
formation of an ordered mass of cells, which by continued division of
cells builds up the embryo.
Simple as this process of nuclear conjugation may seem, it was by no
means so easy to recognize, and several investigators, especially
Auerbach, Schneider, and Bütschli, had seen stages of the process at
an earlier date without arriving at the true interpretation of the
phenomena. This was chiefly due to the fact that, in addition to the
phenomena of fertilization proper, which we have briefly sketched,
other nuclear changes take place in the maturing ovum, and these are
not very easy to distinguish from the former; we refer to the phenomena
of the so-called 'maturation of the ovum.' When the ovum-cell has
attained its full size within the ovary it is not yet capable of being
fertilized, but must first undergo two processes of division, to the
right understanding of which Hertwig's investigations, and afterwards
those of Fol, have contributed much.
For a long time it had been a familiar observation that small
refractive corpuscles were extruded from one pole of the ovum shortly
before the beginning of embryonic development. These were called 'polar
bodies,' because it was believed that they marked the place which would
afterwards be intersected by the first plane of division; it was only
known at that time that they had to be extruded from the egg, but no
one had the remotest idea of their real nature.
We now know that they are cells, and that their origin depends on a
twice repeated division of the egg-cell; but it is a very unequal
division, for these 'directive cells' or 'polar bodies' are always much
smaller than the ovum, and indeed are usually so small that it is easy
to understand why their cellular nature was for so long overlooked.
Yet they have always a cell-body, and in many ova, for instance those
of certain marine Nudibranchs, this is quite considerable; and they
have likewise always a nucleus, which, notwithstanding the smallness of
the cell-body, is in all cases exactly of the same size as the sister
nucleus which remains behind in the ovum after division--a fact which
is in itself enough to indicate that we have here to do essentially
with readjustments and changes in the nucleus of the ovum.
Long before the polar or directive divisions were recognized as
divisions of the egg-cell it was known that the nucleus of the ovum
disappeared as soon as the latter attained to its full size within
the ovary. It was also known that this nucleus--the large so-called
'germinal vesicle' lying in the middle of the ovum--left its central
position and moved to the upper surface of the ovum, there to become
paler and paler, and ultimately to disappear altogether from the
sight of the observer. By many it was believed that it broke up, and
that the 'segmentation nucleus,' which is afterwards obvious, is a
new formation. The truth is that the germinal vesicle, at the time
of its disappearance, is transformed into a division figure which
is invisible without the aid of artificial staining. The nuclear
membrane breaks up; the centrosome of the ovum, which, although hardly
visible, had previously lain beside the germinal vesicle, divides
into two centrosomes and their centrospheres, and these now form the
'mitotic figure' by moving away from each other and sending out their
protoplasmic rays. This nuclear spindle soon ranges itself at right
angles to the surface of the egg, which at the same time arches itself
into a protuberance, and soon two daughter-nuclei are formed, one of
them lying within the protuberance (Fig. 75, _A_, _Rk1_). This soon
separates itself off from the ovum, surrounded by a small quantity
of cell-substance. The other daughter-nucleus remains within the
ovum, but neither of them remains in a state of rest; both are again
transformed into a spindle and divide once more; the minute first
'polar body' dividing into two 'secondary polar bodies' of half the
size (_B_, _Rk1_), while the nuclear spindle within the egg brings
about a second division of the ovum (_B_, _Rk2_) whose unequal products
are the second polar cell and the mature ovum--that is, the ovum ready
for fertilization. The process is now complete; the egg-cell, which
has lost very little plasmic material through the 'polar bodies' and
has not become visibly smaller, has now a nucleus (_B_, _Eik_) which
has become considerably smaller through the two rapidly successive
divisions, and, as we shall see later, has also undergone internal
changes. In this state it is 'ripe,' that is, it is ready to enter into
conjugation with the nucleus of a male cell, and this we have already
recognized as the essential element in the process of fertilization.
These processes of 'maturation of the ovum' are common to all animal
ova which require fertilization, and they follow almost the same
course, only that in many cases the second division of the first
polar body does not take place, so that only two polar bodies in all
are formed. All these processes have nothing directly to do with
fertilization, but it is only through them that the ovum becomes
capable of fertilization. This does not prevent the spermatozoon from
previously making its way into the ovum, for this is usually the case
(Fig. 75, _A_, _sp_); there it waits until the second 'directive
division' of the ovum has been accomplished, utilizing the time to
become transformed in the manner necessary for the conjugation of the
two nuclei. Only in a few species, for example in the sea-urchin, does
the egg complete its polar divisions within the ovary, therefore before
it has come into contact with the sperm at all.
[Illustration: FIG. 75. Process of fertilization in _Ascaris
megalocephala_, the thread-worm of the horse, adapted from Boveri and
Van Beneden. _A_, ovum in process of the first directive division;
_Rk_ 1, first polar body; _sp_, spermatozoon with two chromosomes in
its nucleus, attaching itself to the ovum, and about to penetrate
into it; a protrusion of the egg-protoplasm is meeting it. _B_, the
second directive division has been completed; _Rk2_, the second polar
body; _Eik_, the reduced nucleus of the ovum. The first polar body
(_Rk_ 1) has divided into two daughter-cells, _spk_; the nucleus of
the spermatozoon remains visible with its two centrospheres (_csph_).
_C_, the sperm nucleus (♂_k_) and the ovum nucleus (♀_k_) have grown,
each has two loop-like chromosomes; only the male nucleus has a
centrosphere, which has already divided into two (_csph_). _D_, the two
nuclei lie apposed between the poles of the nuclear spindle. _E_, the
four chromosomes have split longitudinally; the spindle for the first
division of the ovum (the segmentation spindle, _fsp_) has been formed.
_F_, divergence of the daughter-chromosomes towards the two poles;
division of the ovum into the first two cleavage cells or embryonic
cells.]
That we may be able to penetrate still more deeply into the processes
of fertilization, the best illustration to take seems to me to
be, as yet, the ovum of the thread-worm of the horse (_Ascaris_
_megalocephala_), which has become famous through the classical
observations of Ed. van Beneden. Many favourable circumstances
unite in this case to make the essentials of the process clearly
recognizable. Fertilization takes place within the body of the female,
in an enlarged portion of the oviduct, within which a number of the
remarkable sperm-cells are always found in a mature female. They are
remarkable in being not thread-like, but rather spheroidal cells,
bearing, however, a small protuberance something like a pointed horn
(Fig. 75, _A_, _sp_). When such a sperm-cell comes in contact with the
upper surface of an ovum a swelling forms at the place touched, and the
sperm-cell attaches itself firmly to this, and is drawn by it into the
ovum. Without doubt, amœboid movements on the part of the sperm-cell
itself play some part in this, as can be most plainly seen in the
large sperm-cells of many Daphnids which we have already discussed. In
the egg of the thread-worm the whole sperm-cell with its nucleus can
soon be detected within the substance of the ovum, and it then changes
rapidly. Its main body fades more and more completely, until at last
it disappears altogether, while the nucleus becomes vesicle-like and
soon attains a considerable size (Fig. 75, _B_, _spk_). Meanwhile the
residue of the germinal vesicle which remained behind in the ovum after
the second directive division (_B_, _Eik_) has changed into a large
vesicle-like nucleus (_C_, ♀ _k_), which in the ovum of _Ascaris_, as
well as in the spermatozoon, at first contains a nuclear reticulum with
irregular fragments of chromatin. Later on, these form a spiral coil in
the manner we have already described, and finally this breaks up into
two large and relatively thick angular loops or chromosomes (Fig. 75,
_C_ and _D_, _chr_).
At the same time a nuclear division apparatus has formed in the space
between the two nuclei--the so-called male and female 'pronuclei'
(♂ _k_, ♀ _k_)--two centrospheres (_csph_) become visible, at first
lying close together, but afterwards moving apart (_D_) to form the
poles of a nuclear spindle, in the equatorial plane of which the four
chromosomes of the male and female pronuclei are now arranged. The
nuclear membranes disappear, and the two nuclei now unite to form one,
the segmentation nucleus (_D_). A dividing spindle then develops and
brings about the first embryonic cell-division (_E_), and thus the
beginning of the 'segmentation' of the ovum; each of the four chromatin
loops splits longitudinally, and each of the split halves migrates, one
to one, the other to the other daughter-nucleus (_F_). As this same
method of distribution of the chromatin substance is repeated at every
successive cell-division throughout embryogenesis, and indeed through
the whole of development, it follows that the result of fertilization
is, that all the cells of the body of the new animal which develops
from the ovum contain an equal quantity of paternal and of maternal
chromatin. If we are right in regarding the chromatin substance as the
hereditary substance, it becomes immediately apparent that this equal
division is of the most far-reaching importance, for it shows us that
the so-called process of fertilization is the union of equal quantities
of hereditary substance of paternal and maternal origin.