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==Chapter VI The Germinal Layers==


By the germ-layers we understand certain groups of cells which
contain in themselves the materials for certain definite groups
of organs and tissues. These groups of cells are definitely
separated from one another at an early period of development,
and the process of their separation is spoken of as the formation
of the germinal layers.


The germinal layers in a Vertebrate are three in number, the
=Chapter IV The Germ-Cells (continued)=
ectoderm, the endoderm, and the mesoderm. The ectoderm is
that group of cells which contains within itself the material for
the formation of the epidermis and epidermal derivatives like
hair, feather, skin-glands, the enamel of the teeth, the nervous
system both central and peripheral, and the sense organs, and
further the stomodaeum and proctodaeum, or entrances to the
mouth and anus ; the endoderm contains the material for the
lining epithelium of the alimentary canal and its outgrowths,
such as gill-slits, thyroid, thymus, lungs, liver, pancreas, bladder ;
while from the mesoderm-  with which we include the notochord
- skeleton and connective tissues, muscles, blood and vascular
system, coelom and urogenital organs will be derived.


The germ-layers are thus definable by thek fate in development. They may also be defined with reference to their position
in the embryonic body when they have been definitely segregated
from one another, for then the ectoderm is the outside layer,
the endoderm the inside layer, while the mesoderm with the
notochord is in between. Prior to that moment, however, it is
difficult if not impossible, to give generaUy valid definitions of
these sets of cells by their position, since the method of theur
origin from the different cells into which the substance of the
ovum is divided by cleavage varies in the several groups.


In a Vertebrate the germinal layers are segregated durmg
==III. The Maturation of the Germ-cells==
a process which is known as the formation and closure of the blastopore, or in an older terminology ' gastrulation the ' gastrula ' being the name bestowed on this stage in which a new
cavity, the ' archenteron ' or primitive gut, is formed and is in
communication with the exterior by an aperture, the blastopore.


This opening, and -ndth it the germinal layers, is from the first
===A. In the male===
bilaterally symmetrical. This is true of all Vertebrates, but in
the method of its origin the phylum must be divided into two
great groups, those in which the blastopore arises at the edge
of the blastoderm -  ^the Anamnia -  and those in which it appears
inside the blastoderm -  the Amniota. By the help of the Gymnophiona, however, the gap between the two may be bridged.


Anamnia
The Urodelous Amphibia have always been a favourite object for the study of these changes, and may conveniently be taken by us as a type.


We shall begin with the Anamnia, in which the conditions are much simpler.  
It will be recalled that duriag the spermogonial divisions the full somatic number of chromosomes is seen. The mitosis is of the ordmary character (Fig. 32). The granules of chromatm increase, run together m the form of beaded rows, which become the V-shaped chromosomes. The nuclear membrane has m the meantime broken down, the centrosome has divided, and around each daughter centrosome an aster is appearmg. The chromosomes then undergo longitudmal fission and, so spht, are placed on the equator of the spmdle now developed between the two centrosomes. The daughter chromosones are then puUed apart by the spmdle-fibres attached to them to the opposite spmdle poles, and there passmg through the same series of changes in the reverse order become the daughter nuclei. Meanwhile a celldivision has occurred m the equatorial plane of the spmdle, m which process the mtermediate bodies-thickenings of the spmdle fibres-  play an important part.  


As a type we shall take the common English frog {Eana
iemporaria) .


The first sign of the formation of the germ-layers is given as
All the features of an ordmary mitosis are here : the chromatm is the only part of the nucleus to be divided ; for that purpose it is thrown mto the form of chromosomes, which ^ht lengthways mdependently of any external agency , a division a^tatuLasters and spindle-is ^onst^f ?? e centrosomes and probably by them, the function of pull apart the halves of the aheady divided chromosomes and to LL?the division of the cell. When the W ceased dividing they enter upon a time the nucleus passes through complex changes, whlch are reallty the prophases of the fost of the two maturation divisions. This first division is of a very different character to an ordinary mitosis. There ensues the second division. This, with one important exception, resembles the mitoses of the spermogonia.  
soon as segmentation is at an end by the appearance of the  
structure known as the dorsal lip of the blastopore (Fig. 61).
This is a short, deeply-pigmented rim bounding a groove, placed
parallel to the equator, and a little below it (about 25°) at that
point in the boundary between the pigmented and unpigmented
regions of the egg where the latter area is most extensive. This  
is the side on which the grey crescent was formed and the original
unpigmented area so increased. The plane which includes the
egg-axis and the dorsal lip will shortly become the median
longitudinal or sagittal plane of the embryo ; it coincides evidently with the plane of symmetry of the unsegmented ovum.  


The egg is still in the position into which it turned at the time
of insemination with its axis vertical, and the heavy white pole
below. The changes that now take place as seen from this
vegetative pole are as follows. The rim of the groove begins
to travel downwards over the surface of the egg towards the
vegetative pole, the area over which it passes becoming covered
by cells which are as deeply pigmented as those of the animal
portion of the egg. At the same time the rim elongates, becoming
crescentic ; in other words, the processes of rim formation and overgrowth are extended to the right and left along the margin
of the pigmented area, and the lateral lips of the blastopore come into being. As the dorsal lip (the middle region of the rim)
continues on its course towards the vegetative pole, and as
continually fresh parts are drawn into the process at the sides,
the blastoporic lip becomes first semicircular, and then three parts of a circle, until finally, when that part which is diametrically opposite to the dorsal lip, namely the ventral lip, also
begins to grow down, it attains the form of a circle enclosing
the still uncovered portion of the vegetative hemisphere, the
yolk-plug. The dorsal lip has now moved down to or a little
beyond the vegetative pole.




Fig. 32. -  Stages in the karyokinetic division of the spermogonia of the newt.




The first maturation division (Figs. 33, 34). In the nucleus of a spermogonium the chromatin is in the form of fairly coarse lumps uniformly distributed over a wide achromatic reticulum. As the growth of the cell and its nucleus begin the chromatin becomes subdivided mto finer granules, which soon arrange themselves m rows or filaments ; in each row the granules are connected by threads of the achromatic reticulum, while similar threads pass from one filament to another. This is the narrow thread or leptotene'^ stage. As the nucleus enlarges still more


Fig. 61. -  Diagrams of the closure of the blastopore in the egg of the
common frog {R. temporaria). In a-d the egg is viewed from the vegetative pole, in E, F from below. The dorsal lip is at the top of the figures.
In D the ventral lip has just been formed and the blastopore is circular.
In E the rotation of the whole egg has begun, and in f is complete.




Fig. 33.-  Prophases of the heterotype division in the male Axolotl. 1, Nucleus of spermogonium or young spermocyte ; 2, Early leptotene ; 3, Transition to synaptene; 4, Synaptene with the double filaments converging towards the centrosome ; 5, Contraction figure ; 6, 7, Pachytene ; 8, Early, 9, Later diplotene ; 10, The heterotypic double chromosomes ; the nuclear membrane is disappearing.


At this moment the whole egg begins to rotate about a horizontal axis in the opposite direction to that in which the dorsal
it is seen that on one side some of the filaments are arranged in pairs, and converge towards one point, the point where the centrosome m its centrosphere is placed. On the other side of  
lip moved ; and this rotation continues -  the circle of the blastopore becoming smaller all the time -  until the dorsal lip has
returned, rather beyond the point from which it started, to the
new equator, or horizontal plane through the centre of the egg.
The end now occupied by the blastopore is posterior. The angle
subtended by the arc traversed by the dorsal lip -  both before
and during the rotation -  is 75°, and the angle through which
the whole egg rotates is 100°. It follows that the vertical line
now drawn through the centre of the egg, which will be the
dorso-ventral line of the embryo, makes the same angle of 100°
with the original egg-axis ; that the animal pole is situated below
what will be the anterior end of the embryo (Fig. 62, f), since
the blastopore is posterior ; and that the antero -ventral haK of  
the embryo is developed over the animal, the postero -dorsal half
over the vegetative hemisphere of the egg. The dorsal and ventral
lips are now actually dorsal and ventral.


It is clear that the lip of the blastopore which is thus formed
1 These and the followmg terms were first proposed by von Winiwarter in his classical work on the oogenesis of the rabbit.  
and closed arises along the whole of the boundary between small
pigmented and large yolk-cells, and that the process is bilateral,
taking place, as it does, first and most rapidly at the dorsal lip,
last and least rapidly at the ventral lip, and at an intermediate
rate at the lateral lips in between.  


The examination of sections (Fig. 62) will now show us that
the closure involves (1) a movement of the yolk-cells into the
segmentation cavity together with (2) an overgrowth and ingrowi^h of cells at the blastoporic lip, resulting in the formation
of a new cavity, the ' archenteron ' ; and that during the process
the material for the germinal layers is brought into position and
laid down.


A sagittal section of the egg passing through the dorsal lip at its first appearance shows the groove placed about 25° below
the nucleus the filaments pass into the general network. This is the paired thread or synaptene stage.  
the equator in the zone of intermediate cells. The radial disposition of the cells immediately about the groove marks the
beginning of a process of overgrowth and ingrowth which becomes
more obvious a little later, when it is seen that a fold of small
cells has grown over a certain area of yolk-cells. This fold consists naturally of two sheets, an outer and an inner. The cells
of the outer sheet resemble closely the small pigmented cells of
the animal hemisphere into which they are uninterruptedly continued ; like the latter, they are arranged in about four layers,
the outermost of which is epithelial. At the lip of the blastopore
the outer passes into the inner sheet, the cells in the outermost
layer of the former being gradually turned over into the innermost layer of the latter. This inner sheet also consists of several
layers of cells, the innermost of which is pigmented and epitheUal, the remainder being more irregularly disposed. The inner
sheet forms the outer, or, as it will be when the egg has rotated,
the upper wall of the slit-like cavity between itself and the yolksurface now covered up. This cavity is the archenteron and the
inner sheet of the fold is its roof ; the original vegetative surface
of the egg forms its floor.  


This overgrowth and ingrowth of cells, with consequent formation of an archenteric cavity, takes place in an exactly similar
By coalescence of the component granules the filaments become shorter and thicker : at the same time in each pair the filaments approach one another so closely that only a narrow slit is left between them. On one side the pairs of filaments still converge towards the centrosome, but on the other are inextricably coiled and tangled together into a bunch which is withdrawn some little way from the nuclear membrane. The pairing of the filaments can, however, be seen in the tangle. The several pairs are still imited by achromatic threads, the filaments being toothed at each point of insertion of such a thread. A few threads stretch across the empty space between the tangle and the membrane. This is the contraction figure.  
fashion at the lateral (Fig. 63, a) and ventral lips. By the time  
the latter has appeared the archenteric cavity is much enlarged,
first by its being extended in an anterior direction into the yolkcells that have meanwhile been pushed up into the segmentation
cavity on the dorsal side, and secondly in a lateral and finaUy
a ventral direction by a movement of the mass of yolk-cells
towards these regions of the egg also. The segmentation cavity
is thus first reduced to a small space upon the ventral side and
then obliterated altogether. In a small percentage of cases
however, the segmentation cavity communicates with the front
end of the archenteron, is surrounded by yolk-cells, and mcorTiorated in the front end of the gut.  


' t the shifting of the heavy yolk-cells to the ventral s.d
tZ alters the eentre of gravity and so ca,.es the rotafou of
the egg until equilibrium is regained.




Fig. 34. -  First maturation division in the male. 2, Salamander, the remainder Axolotl. 1, 2, The heterotypic chromosomes on the spindle (metaphase) ; 3, Anaphase ; 4, 5, Telophase ; 6, Resting nuclei ; 4-6, Celldivision into two secondary spermocytes.




The members of each pair of filaments now unite throughout their length, so that the longitudinal sHt disappears. The thick filaments still converge towards the centrosome side, where apparently they end against the nuclear membrane. There is, therefore, not one continuous filament or spireme, but several.


The other ends of the filaments pass into the tangle, which is still retracted from the nuclear membrane, but becoming looser as the nucleus enlarges. The coil is soon still more unravelled and occupies the whole of its side of the nucleus. This is the pachytene stage.


arch
The several filaments now separate from one another, so that the polar convergence is lost, and coil in various directions through the nucleus. At the same time the longitudinal sUt reappears in each, and the filaments are once more paired, so reaching the diplotene condition. Their surfaces are still toothed where the connecting achromatic threads are inserted. Soon, however, these cross threads disappear and the filaments become smooth. At the same time the members of the several pairs begin to separate a little from one another, in places if not throughout their length.


The nuclear membrane now breaks down and disappears, the pairs of filaments shorten and thicken, and assume the most various shapes and sizes. A pair may be in the form of two straight parallel rods, or two curved parallel rods, either V-shaped, or C -shaped, or two rods parallel at one, divergent at the other extremity, and so -||--shaped ; or the sht between them may be expanded in two or more places, and then the two may be tAvisted over one another mto a figure of g or or by expansion of the whole sht, while the rods are united at the ends, may be ring-shaped, while finally the ring may be pushed in in four places and assume the form of a cross, =[}:. These bizarre double bodies are the chromosomes of the first maturation division. It seems clear that they are derived from the separate paired filaments of the diplotene stage, these from the thick filaments of the pachytene stage, and these again from the paired filaments of the synaptene nucleus. The origm of these we shall have to discuss later on.


The number of the double chromosomes, and therefore of the several double filaments in the earUer diplotene, pachytene, and synaptene stages, is one-haK that seen in the spermogoma. The reduction from the somatic number {2n) to the germnumber (n) has already taken place. It seems that this half number must be estabhshed in the synaptene nucleus.


mes
The actual division now occurs (Fig. 34). A spindle is formed in the ordinary way, and the double chromosomes are thrown upon its equator in such a way that the two ends of each member of a pair he in the equatorial plane. This is easily seen where the pair retains the original form of two closely-parallel rods separated by a longitudinal slit, and can often be made out in the ring- and cross-shaped and other chromosomes.


mes. Y.  
The members of the pairs now come apart and travel to opposite spindle poles, where they coalesce and pass into the condition of resting nuclei. The cell, meanwhile, has divided and the two secondary spermocytes have been formed. The nucleus of each of these, it is clear, contains only one -half of the ordinary number of chromosomes.  


The division which we have just witnessed is unlike an ordinary mitosis in at least two respects. First, the number of chromosomes is reduced from the somatic to the germ number, and second, the chromosomes are double and frequently of extraordinary shape. For these reasons the division is spoken of as heterotypic, or xmlike the usual type. The term meiotic or reducing, also appHed to it, refers to the numerical lessening of the chromosomes.


We have now to inquire whether this division is or is not like an ordinary mitosis in another respect, the manner ia which the chromosomes are divided. Ordinarily, as we know, the chromosomes are longitudinally divided ; but on this occasion it is held by many observers that the division, albeit in appearance longitudinal, is in reahty transverse.


Fig, After rotation ^'*''^°P°'^'^- before rotation ; e, During rotation ;  
The uiterpretation of the nuclear changes is a matter of considerable difficulty, and very diverse opinions are entertained (1) as to the origin of the double filaments seen in the synaptene and later stages of the prophase, and (2) as to the mode of formation of the ring-shaped chromosomes seen in the actual mitosis ; different combination of these diverse opinions has led to the formulation of three principal views.


archenteron; y p! yolk fri^f/-'^ segmentation cavity; arcL,  
I. It is held that (I) the double filaments of the synaptene stage arise by longitudinal fission of the filament, that the longitudinal split disappears, but reappears (2) to form the cavity of the rings. Hence the actual division is longitudinal (Meves). This is illustrated in the accompanying diagram (Fig. 35, I).


formed below th;;^! ^ ^'^"'^ = mesoderm
For the sake of simpUcity we will suppose that the full number of chi'omosomes is four, the reduced number two. We will further suppose ttot these four chromosomes are really diff^^' Tne another though apparently identical. Let us caU them 7 A' B, and B: In the prophase of the mitosis two mstead of' four filaments appear. We may suppose that each of th«a consists of two ordinary chromosomes muted end to end say aTo 4' and B to B'. Each filament becomes then spht lengthwa^ll 1). the sHt widens out until each filament assumes a ling shape (I, 2), and the rings are then so placed on the equator of the spindle that the ends of the chromosomes lie in the equator (I, 3). Hence, smce each half ring consists of an A and an A', or of a 5 and a jB', when the halves are separated and travel towards the spindle poles, each daughter nucleus of a secondary spermocyte will receive a chromosome of each kind, A, A', B, and B'.


pushed into the segment; Sty."""^"'" ''""^'^ '^'^"^ "^'^ ^-^k-cella




With the exception of the yolk-plug the outer surface of the
egg is now covered by a sheet of small cells, disposed in about
four layers, the outermost of which is epithelial and pigmented.
This sheet is the ectoderm. In part it comes from the original
animal cells which formed the roof of the segmentation cavi. r â–  but
part of it is derived from the outer sheet of the blastoporic fold.


Fig. 35.-Diagram to illustrate three interpretations of the first matoasee text.)




Fig 63 - Transverse sections of the frog's egg. A, During the closure
of The blastopJie • B, After, mes. 2, mesoderm differentiated from the
yoM Tus^^^^^^^ segmentation cavity (in B these are seen to be


Central); U., lateral lip of the blastopore; n.c/^., notoehord.  
II. On the second view (von Winiwarter, Schreiner, Agar), (1) while the paired filaments of the sjmaptene stage are believed to arise, not by longitudinal fission of the leptotene, but by apposition of distinct chromatin filaments (that is, chromosomes), the formation (2) of the rings from these double ' filaments is in accordance with the first view.  


The notochord and the dorsal mesoderm are differentiated out
The diagram (Fig. 35, II, 1) shows the four chromosomes united in pairs by their entire length, though presenting every appearance of longitudinally spht rods : A is paired with A', and B with B'. The chromosomes of each pair then separate to form rings, remaining united only by their ends, and then are placed on the spindle ua such a way that these ends lie in the equator. It follows that A and B face towards one. A' and B' towards the opposite pole, and hence that each nucleus of a secondary spermocyte receives not all tour chromosomes, but only two, say A and B, ov A' and B'.  
of the roof of the archenteron (Figs. 63 B, 64). The latter sheet
of cells becomes split into (1) a thin layer next the cavity (this
will be the roof of the ahmentary canal) and (2) a layer next
the outside. This outer layer is divided into (a) a median strip
or rod, which is the notochord, and (6) two lateral shee s, the
dorsal mesoderm. The notochord is not separated miti after
the sheets of mesoderm have been detached. The separation o
both notochord and mesoderm begins at the anterior end and
proceeds backwards. At the lip of the blastopore there is thus
for a time an undiiferentiated mass of tissue in which ectoderm
notochord, mesoderm, and roof of the alimentary ^canal are all
continuous (Fig. 62, e). It will be remembered that the front
end of the archenteron arises by an extension of that cavity
into the yolk-cells ; here, therefore, yolk-cells form the roof, and  
it is from them that the anterior portions of notochord and dorsal mesoderm are formed. The posterior portions, however,
arise in that part of the archenteric roof which comes into position
as the inner sheet of the blastoporic fold.  


The ventral mesoderm (Fig. 63, b) has a similar double origin.
The division, therefore, is not really but only apparently longitudinal : the result is the same as though A and A' (and B and B') had been united end to end, and then separated by a transverse division of the double chromosome so formed.
In front the floor of the archenteron is formed of the yolk-cells
pushed into the segmentation cavity ; the cells next the ecto




III. On the third view (Farmer, Montgomery), (1) the double thread of the synaptene and pachytene is formed by the longitudinal sphtting of the chromatm filament ; but (2) the rings do not arise by the opening out of the spUt. The longitudmal division disappears, and the filament is first gathered up into half as many loops as there are chromosomes in the spermogonia, and these loops then separate as the n ring-shaped chi omosomes. The rings are therefore open at one end only, and the cavity of the ring arises, not by the opening out of the longitudinal split (for that has disappeared), but by the bending of the two halves, united end to end, of each double chromosome upon one another (Fig. 36, III). That is, the filament, consisting of Aj A', B, and B', is first gathered up into two loops, A being bent on A', and B on B', and then the loops separate. In the mitosis (III, 3) the rings are so placed on the spindle that A becomes separated from A' and B from B', so that one secondary spermocyte receives A and B, the other A' and B' (or, of course, A and B', A' and B).




Fig. 64.-  Three stages in the differentiation of the roof of the archenteron
in the irog. arch, archenteron; n.ch., notochord ; mes., dorsal mesoderm.


derm subdivide and become mesoderm. Behind mesoderm arises
Fig. 36.-  Second maturation division in the male (Axolotl). 1, Prophase (split spireme); 2, The homoeotypic spht chromosomes on the spindle; 3, Polar view of the same ; 4, Anaphase ; 5, Telophase ; 6, Resting nuclei and completion of cell-division ; in each spermatid the centrosome has divided, and the sphere has become detached.  
from the inner sheet of the fold at the ventral lip. At the sides
of the embryo dorsal and ventral mesoderm pass continually
into one another. The middle layer, therefore, taken as a whole,  
arises anteriorly and ventrally from the yolk-cells, posteriorly
and dorsally from the blastoporic overgrowth ; the former is in the  
onginal animal, the latter in the original vegetative hemisphere.  


Smce mesoderm is formed also at the lateral lips, the two
The result is therefore the same as on the second hypothesis.  
sheets of this tissue which flank the notochord aje necessarily continuous, around the blastopore (Fig. 64*), with the mesoderm
at the ventral lip (Fig. 62, e) ; only at the dorsal lip, where the
notochord is formed, is there an interruption in the middle layer.
The endoderm or lining of the gut cavity is what is left of the
roof and floor of the archenteron, the roof of the gut being
the thin layer left when the notochord and mesoderm have been
detached, the floor the bulky mass of yolk-cells after the separation of the ventral mesoderm.  


It must be remembered that though the differentiation of  
Considering the diversity of opinion, it would be rash to dogmatize, but it may be pointed out that the evidence on the whole is agamst the mode of formation of the rings adopted by the third view. It does seem as though the rings were made by the opening out of the double filaments. We are left, therefore, with the choice between the first and second hypotheses. We can only say that the way in which the members of the pairs of filaments diverge into the general network in the fourth stage (Fig. 33, 4) suggests apposition rather than fission, and this involves ultimately a transverse division of double chromosomes, and that the phenomena of maturation observed in a number of Invertebrate forms corroborate this view.  
these germ-layers is only completed when the blastopore has
closed, it has in reality been in progress during the earlier stages.  


Before discussing the theoretical significance of this mode of division, we shall describe the second maturation division (Fig. 36).


The nucleus of the secondary spermocyte soon emerges from the resting condition, and a chromatic filament appears. This filament becomes longitudinally split and then divided into a number of V-shaped chromosomes, themselves therefore split lengthways. The number of chromosomes is the half somatic, n. A spindle is developed, the spht chromosomes are placed on its equator, and division takes its ordinary course, resulting in two spermatids, the nucleus of each of which therefore possesses n chromosomes. La the V-shape of the chromosomes, as well as in their longitudinal division, this second mitosis is of the ordinary type. Hence it is called homoeotypic. Each spermatid becomes metamorphosed into a spermatozoon in the fashion already described.


The phenomena of maturation in the male are, as far as is known, similar in other forms {Myxine, Elasmobranchs, Mammaha). Each ripe male cell, therefore, is provided with only haK the number of chromosomes seen in the spermogonia and in the tissue cells of the body. Whether the n chromosomes in all the spermatozoa are or are not alike depends upon the interpretation placed on the first maturation division, as well as upon our views of the nature of the chromosomes.


===B. In the female===


Fro 64* - Horizontal section of an older stage showing the sheets of
While m the male the first or heterotypic division follows immediately upon the prophases, in the female the two episodes - prophase and division- are separated by an interval, sometimes of great length, a year or more-  during which the yolk is deposited in the cytoplasm to the accompaniment of complex nuclear changes.  
mesoderm passing back into the lateral Ups of the blastopore (b.j).).  


It still remains for us to discuss very briefly the origm of  
Prophases of the heterotypic division. The oogonial divisions come to an end at a fairly early period, and growth of the oocyte begins almost at once. The prophases of the heterotype are therefore usually found only in very young animals- in the tadpole of the frog, or the new-born or embryonic Mammal.  
the cells from which the blastoporic fold is derived, that is, the
origin of parts of each of the three germ-layers. The inner layer
of the fold is certainly derived neither wholly from the smaU
cells of the animal hemisphere, nor whoUy from the large cells
of the vegetative hemisphere, but from the region about the
egg-equator, in which the cells are of a character intermediate
between these two (Fig. 62, i, z.). The outer layer of the fold
comes from the same source, and from an extension of the roof
of the segmentation cavity. These intermediate cells divide
rapidly and give rise to the fold, which, as we have seen, contains ectodermal, endodermal, and mesodermal elements.  




To sum up, the ectoderm of the frog comes partly from the
These two afford good examples. The nuclear changes which are readily seen in the tadpole's ovary (Fig. 37) are obviously closely j)arallel to what we have observed in the other sex.  
cells of the animal hemisphere, partly from the intermediate
cells ; the endoderm in part from the latter, in part from the
yolk-cells, while the mesoderm and notochord have a similar
double origin ; and the materials for these layers are brought
into their definitive positions during the bilateral closure of the
blastopore, which arises all along the line separating animal from
vegetative cells.  


We shall see that a similar statement may be made for the  
A stage in which the chromatin is in the form of scattered granules is followed by one in which the granules run together to form the leptotene filament. Then comes the synaptene, with parallel filaments, followed by the contraction figure. The paired filaments emerge from the tangle to converge to one pole, the tangle itself beiiag withdra-WTi from the other side of the nucleus. The pachjiiene and diplotene follow in due course. A remarkable change now occurs in the straining capacity of the chromatm filaments. Up to the diplotene stage they behave in the usual way, showing great affinity for chromatin stains (carmine, haematoxylin, and basic aniline dyes) ; but from now onwards they lose this faculty and stain only with the acid plasma dyes. Meanwhile, the number of nucleoh (these also stain in acid dyes) is increasing, and presently it is seen that granules of chromatin (that is, granules which are coloured by the ordinary chromatin dyes) begin to settle upon (? be precipitated round) the nucleoh. By what appears to be a continuation of this process the nucleoh become converted into highly chromatic bodies.  
remaining Anamnia.  


The filaments (chromosomes) persist for a while, but will eventually disappear.


Precisely similar phenomena are seen in the young Mammahan ovary (Fig. 38), and only one or two points require to be mentioned. There is a very obvious centrosphere with included centrosome on one side of the nucleus (this usually goes by the name of the yolk-body of Balbiani), towards which the filaments of the synaptene and pachytene converge. In the early stage of contraction the paired filaments are seen to emerge from the rather open tangle on this side, while on the other a few filaments, also paked, stretch out to the nuclear membrane. In the later contraction figure the latter are retracted and the tangle, much closer, hes wholly on the side of the centrosphere.


After the diplotene stage the ruig-shaped figures of eight and other forms of double chromosomes are seen, but then the chromosomes break up into their constituent granules and range themselves along the achromatic threads which make a network through the nucleus. This is the diciyale condition, and in this the nucleus remains through the growth period until the moment of maturation arrives.


Fig. 65. -  ^Formation of the germ-layers in Petrornyzon. (After Scott.)
A, Sagittal section ; b, c. Transverse sections of two stages ; arch., archenteron ; d.l., dorsal lip of the blastopore ; n.ch., notochord ; d.m., dorsal
mesoderm ; v.m., ventral mesoderm ; m.t., medxillary tube (here a solid
wedge of cells).






==Cyclostomata==
Fig. 37. -  Prophases of the heterotypic division in the female (ovary of tadpole). 1, Nucleus of oogonium ; 2, Leptotene ; 3, Synaptene ; 4, 5, Contraction figures ; 6, Pachytene ; 7, Later pachytene, multiplication of nucleoli ; 8, 9, Diplotene : the chromatin filaments are becoming achromatic ; granules of chi'omatin are being deposited on the nucleoli.


In Petrornyzon (Fig. 65) the formation and closure of the
blastopore, the origin and extension of the archenteron, resemble
the same processes in the frog, with the exception that a ventral
lip is never developed. The ventral mesoderm is diflferentiated
from the yolk-cells pushed into the segmentation cavity, as in
the frog, and these latter cells form the floor of the gut. They
give rise, however, to much more than that, since the roof of
the archenteron is converted wholly into the notochord and the
gut is then completed by the upgrowt.h of yolk-cells from the
sides and underneath the notochord. The dorsal mesoderm arisea
m connexion with the overgrowth at the lip of the blastopore




In the Myxinoids (Fig. 66) segmentation produces a blastoderm at one end of the elHpsoid egg. At one point in the edge
of this blastoderm a dorsal blastoporic lip appears, and the
material for the germ-layers of the embryo is laid down during
the bilateral overgrowth and ingrowth of cells in this region.
The yolk is not wholly covered by this process, but as soon as




Fig. 38. -  Prophases of the heterotypic division in the female (Mammals). 1-6, Kitten three days old ; 7, Mouse embryo shortly before birth ; 8, Mouse eight days old.


1, Nucleus of oogonium or young oocyte ; 2, Leptotene ; 3, Synaptene ; 4, Contraction figure ; 5, Pachytene ; 6, Diplotene ; 7, Heterotypic clnomosomes ; 8, Dictyate.


Fig, 66- Bdellostoma. Overgrowth of the posterior edge or dorsal lip
In 2-5 the centrosphere and centrosome (volk-body of Balbiani) are shown with the chromatic filaments of the nucleus converging towards them.  
of the blastoderm over the yolk, d.l, dorsal lip (posterior edge) ; v.l,
ventral lip (anterior edge) ; op.r., operculum of the shell. (After Bashford
Dean.)




Fig. 38*.-  Small ovarian egg of the frog surrounded by its foUicle (/.) and theca (th.), which is continued into the pedicle {p.). b.v., a bloodvessel between follicle and theca ; v.rn., vitelline membrane ; ch., clu'omatin filaments, now achi'omatic ; n., chromatic nucleoli, ejected from the nucleus (n'.) and becoming achromatic (w".).


the body of the embryo is formed all parts of the edge of the
blastoderm grow down and the blastopore eventually closes at
the vegetative pole.




The period of growth and deposition of yolk. The nuclear changes accompanying the deposition of the yolk in the oocyte have only been studied in the Amphibia, to which we must now accordingly return (Fig. 38*).


Elasm obe anohh
We have seen that after the prophases the chromatin filaments become achi-omatic, while the nucleoH increase in number and become chi-omatic. The filaments gradually break up into a number of small granules, which disappear, or at least become indistinguishable from the general ground substance -  or magma -  of the nucleus.  
Germ-layer formation begins with the appearance at one point
in the edge of the blastoderm of a fold or overturning of cells
of the superficial layer. This point is, as will appear, in the
middle line and at the posterior end. The fold, the rim of which  
is the dorsal lip of the blastopore, is slightly raised and covers
over a space-  the beginning of the archenteron-  between itself
and the yolk (Figs. 67, 68). By the continued backward growth
of the fold and by the ingrowth of its under layer the archenteron
attains a considerable length. The floor of the archenteron is formed of yolk, into which yolk-nuclei subsequently make their
way ; its roof consists of a columnar epithelium derived in
part from the overturning of cells at the lip of the blastopore,
in part possibly from the posterior marginal cells of the lower
layer.  


The nucleoli become more numerous, larger, and more chromatic. They pass into the cytoplasm in one of two ways : either they are bodily ejected from the nucleus, lose their staining capacity and break up into small fragments, or else they disintegrate inside the nucleus, the products of their disintegration then passing out -  either in the form of small particles or in solution -  through the nuclear membrane into the cytoplasm. The nucleoli consist of nucleo -protein, and the result of their transference to the cytoplasm is that the latter first acquires an affinity for the chromatin stains, and then begins to secrete yolk-granules. There is thus a direct connexion between the nucleo-protein of the nucleoli and that which, as we have seen, is demonstrable in the yolk.


It appears that this cycle of changes is repeated many times during the growth of the oocyte, fresh nucleoli being formed, moving to the centre of the nucleus, and there disintegrating.


This passage of material from the nucleus to the cytoplasm of the egg-cell during the time of growth and yolk-formation is of constant occurrence in animals. The material may be solid and bodily ejected or liquid and diffusible, it may be chromatic or achromatic, but it is always given off and is always concerned m yolk-secretion. The chemical changes are, unfortunately, not fully understood.


Fig. 67. -  Overgrowth of the lip of the blastopore and formation of the
The material is known generally as ' yolk-nucleus '. It has sometimes been confounded with another quite distinct structure, the sphere and centrosome. In Mammalian ova the sphere has indeed long been known as the yolk-body of Balbiani (Pig. 39). In the Mammals the sphere usually divides into two or more bodies, which persist for some time, but disappear (in the bat) when there are two or three cell-layers in the follicle.  
embryo in Elasmobranchs. (a-c after Riickert, d-f after Ziegler.) c.s.,
caudal swelling ; l.L, lateral lip. In r the formation of a lip has extended
almost to the anterior edge. In d, e, the medullary folds are still open, in  
r they are closed.  


In some Mammals {Cavia, Vespertilio) chromatoid bodies are found in the cytoplasm. These may be of nuclear origin and correspond to the yolk-nucleus of Amphibia.


The yolk-nucleus is a very important contribution made by the nucleus to the structure of the cytoplasm : a second contribution has still to be made.


But while this process is taking place at the dorsal lip, that is,
With the growth of the oocyte the nucleus has been enlarging pari passu, and by the time growth is completed is of considerable size. It lies in the axis, but excentrically, near the surface in the animal half of the egg. The oocyte is now ready for the first maturation division.  
at the median posterior margin of the blastoderm's edge, it is
also being extended, though in a far less degree, to the neighbouring regions, the lateral lips, on the right and on the left.  
The archenteron thus comes to assume a crescentic shape, with
a median anterior prolongation ; the latter underlies the embryonic portion of the blastoderm, while the crescentic part is
wholly extra-embryonic, and remains very shallow, though it is
subsequently prolonged to the right and left round the edges of  
the blastoderm until a slight overgrowth is formed even at the  
anterior margin.  


With the overgrowth at the lips of the blastopore the material
for the germinal layers is laid down (Fig. 69). The superficial
layer is now the ectoderm. The mesoderm consists of two
parts : (1) two sheets of cells lying one on each side of the
middle line over the embryonic portion of the archenteron ;
posteriorly these sheets pass into the caudal swellings-  two
thickenings at the edge of the blastoderm, one on each side of
the middle line -  where they are continuous with the roof of the
archenteron, out of which they have been differentiated ; (2) the
formation of mesoderm is, however, not limited to the parts
immediately adjacent to the dorsal lip, but is carried on at the
lateral lips, and, as these extend forwards round the whole edge
of the blastoderm, at the anterior edge as well. This extraembryonic mesoderm is naturally continuous in the caudal
swellings with the embryonic mesoderm first described ; it takes
part only in the formation of the area vasculosa.


The notochord is formed from a median strip of cells which
Maturation. The nuclear membrane breaks down and disappears. From a very small part of the achromatic substance of the nucleus a spindle -  the first polar spindle -  is formed (Fig. 41), and on this are placed the heterotypic chromosomes, of which we shall speak in a moment. The whole of the rest of the contents of the nucleus -  chromatic nucleoli and achromatic granular ' magma ' -  are cast into the cytoplasm. This is the second contribution made by the nucleus to the cytoplasmic structure, and it is of considerable importance, since on it in part depends the diflEerence between animal and vegetative hemispheres.
is cut out of the roof of the archenteron ; the process, like the  
differentiation of the mesoderm, takes place from before backwards. With the separation of the notochord and mesoderm
the remainder of the archenteric roof is endoderm, and gives rise
to the alimentary canal, the front end and sides bending dovm


As we have already had occasion to observe, there is a definite relation between the polar structure and symmetry of the egg and the structure of the embryo which is to come out of it, inasmuch as the anterior end is always developed near the animal, the posterior end near the vegetative pole. The structm-e of the embryo is at this moment bemg predetermmed in the egg, by the dispersal of the contents of the nucleus.


This is a fact of universal occurrence. When the germinal vesicle breaks down, only a smaU part of it is utihzed m the formation of the chromosomes which take part in the maturation mitosis. The remainder is given to the cytoplasm, of which it forms henceforward a definite and integral part. Experiment has sho^vn that that part is causally related to the development of certain organs, is therefore a vehicle of inheritance. It will be noticed that this process is without parallel in the male sex.


VJ
We return to the first polar spindle and its chromosomes.


The chromosomes appear first, as beaded filaments of heterotypic form -  wrings, crosses, figures of eight, curved rods, and so on (Fig. 40). Their number is the half -somatic or germ-number n. It has been disputed whether these chromosomes are identical with those which were formed at the end of the prophases, in the young oocyte.


It must be remembered, in discussing this question, that the hypothesis of the individuahty of the chromatin A does not necessarily involve 'p^-'\X^ that of the individuality of the chromosomes. We have so™1-from^?SS''i? th?o»y?; seen elsewhere that there is of the Axolotl (Siredon) just before reason for beheving that the membrane breaks down.


THE GERMINAL LAYERS
chromatia of the nucleus comprises a number of quahtatively unlike bodies-  not merely that the chromosomes are different, but that they are composed of individually different granules. It is also probable, to say the least, that chromosome formation is a nratter of precipitation from solution, for there is certainly much more chromatin in a dividing than in a resting nucleus, and the chromatin often disappears from view in the latter condition. But a body endowed with certain properties wiH retain those properties in solution and emerge from solution with the same, and in a mixture of unlike bodies each wiU retain its own properties in solution and exhibit them afresh when reprecipitated. The chromatin granules are such bodies, and wo may weU suppose that they do retain their properties in spite of their disappearance. It does not foUow, however, that the granules are associated always in the same order to form chromosomes, though that may be so. Hence the chromatin granules may well retam their individuaUty while the chromosomes do not.




The chromatin, therefore, of those heterotypic cliroinosomes that now apj)ear may, on this view, bo regarded as identical with the chromatin of the prophases.


123








Fig. 42.-  The maturation divisions in the female (Axolotl). 1, First polar spindle with heterotypic chromosomes ; 2, Extrusion of first polar body ; 3, Appearance of second polar spindle ; longitudinal division of chromosomes in egg and in first polar body ; 4, Second polar spindle radial ; homoeotypic chromosomes on equator (metaphase) ; 5, Polar view of the same ; 6, Anapliase ; 7, Extrusion of second polar body ; 8, Second polar body with resting nucleus ; 9, Female pronucleus in resting condition, closely surrounded by yolk-granules.


Fig. 69.-  Five successive transverse sections through the hinder
When the spindle is formed the chromosomes are placed on it and shorten and thicken (Fig. 41). The spindle then moves to the surface at the animal pole, Avhere it takes up a radial position, closely surrounded by yolk-granules. The actual maturation divisions now occur.  
(embryonic) portion of the blastoderm of the dog-fish during tlie formation
ot the germinal layers a is posterior, cutting the two caudal sweUings ;
E, Anterior through the head of the embryo, arch., archenteron; mes
mesoderm; e.m., embryonic mesoderm; ex.m., extra-embryonic mesoderm ; w.c^., notochord ; lateral lip of the blastopore






124


Fig. 41. -  Germinal vesicle of the oocyte of the frog just before maturation (after Carney). The nuclear membrane has disappeared. The first polar spindle, bearing the heterotypic cliromosomes, is seen in the middle of the nucleus {p.s.). n., nucleoli ; v.m., vitelline membrane ; /., follicle ; th., theca.




THE GERMINAL LAYERS






VI




Fig. 42. -  Oocyte of mouse with heterotypic spindle from the Fallopian tuDe. ihe oocyte is still surrounded by the cumulus of follicle-cells.


and meeting to form the ventral wall. The yolk in the floor of
the archenteron plays no part in this process (Fig. 70).


Up to the present it is the posterior edge or dorsal lip which
has been principally active, but now the anterior and lateral
margins of the blastoderm become exceedingly vigorous and
begin to grow over the yolk, the overgrowth being accompanied,
as stated above, by a slight marginal invagination ; and eventu




The first maturaiion division (Fig. 42, 1, 2). The heterotypio chromosomes are placed upon the spmdio in the same way as in the male -  that is, Avitli the extremities of the half -rings in the equators. The half-rmgs break away from one another and pass to the spindle poles. Cell-division now occurs. This is extremely unequal. The outer group of chromosomes, with a small quantity of cytoplasm, is cut off as the first polar body from the egg ; it lies in a depression at the surface. The inner group of chromosomes remain in a clear area in the egg, now the secondary oocyte.


The first polar spindle is found (in Siredon, and generally in Amphibia, also in Bnds) in the egg as it passes mto the oviduct. Li Mammalia -  ^where it is also known to be heterotypic (Fig. 43) -  ^it may be formed while the egg is in the ovary, or after it has passed into the Fallopian tube.


y.n.  
The first maturation division in the female evidently involves similar changes to those seen in the male : the prophases, the number and form of the chromosomes are all exactly the same. The interpretation of the manner of division of the chromosomes -  ^whether longitudinal or transverse -  which is adopted for the one, may therefore be applied to the other.  


Fig. 70.- Two stages in the formation of the gut of the dog-fish by the  
The second maturation division (Fig. 42, 3-9). Without passing into a resting condition the V-shaped chromosomes in the egg undergo longitudinal fission, as also do those in the first polar body. A number of parallel fibres, tangentially placed, now appear -  the second polar spindle. The spindle is soon rotated into a radial position and the V-shaped chromosomes, already split, are thrown upon its equator with their apices towards the spindle axis, as in the male. Their number is, of course, n. The halves of the chromosomes then separate and pass to the spindle poles. Another miequal cell-division now occurs. The outer group of chromosomes, together with a httle cytoplasm and one or two yolk-granules, is extruded as the second polar body, while the inner group remain in the now mature ovum as the female nucleus, or rather pronucleus, to employ the more usual term.  
bending down and fusion of the edges of the roof of the archenteron.  
y.n., yolk-nuclei.  


ally the anterior edge makes the whole ckcuit of the yolk, passmg
In both the second polar body and the ovum the chromosomes break up, a membrane is formed round them, and the nucleus passes into the resting condition.  
round the vegetative pole and reappearmg behind the embryo
as the ventral lip of the small ' yolk-blastopore ' (Fig. 71). At
the dorsal hp backgrowth of the caudal sweUings is responsible
for the posterior elongation of the body of the embryo alone,
the body being raised above the surface of the yolk. Where the
body passes into the hinder edge of the blastoderm growth of
the latter ceases, but the lateral edges immediately adjacent
to this point swing backwards untU they bound a narrow
median strip of yolk by which alone the aperture at the dorsal
lip now communicates with the rest of the blastopore.  




Since the chromosomes are V-shaped, are longitudinally divided, and are present in half the normal number, this division is evidently homoeotypic, as in the male.


as  
The second polar spindle is formed as the egg passes down the glandular region of the oviduct (in Siredon and most other Amphibia). In the uterus the polar spindles are in metaphase (with the chromosomes in the equator). The division is not completed until after the egg has been fertilized (which is just after the egg is laid).


Where fertihzation is internal (Elasmobranchs, Birds, Reptiles, Mammals) the second polar body is extended while the egg is in the oviduct.


Although the chromosomes of the first polar body have divided, cell-division (in Siredon) does not usually follow. In other cases the first polar body does divide.


B
A centrosphere -  if not an actual centrosome -  is present at the poles of both the first and second spindles. In the mature ovum there is, however, no trace of it. The female pronucleus is immediately surrounded by yolk-granules (Fig. 42, 9).


Nature of the reducing division. We have already assumed for the purposes of illustration that the several chromosomes of a nucleus are genuinely different from one another. We may now add that there is experimental evidence (which we cannot discuss here) in support of this ; it is further probable that the granules of which each chromosome is composed are again of different values. Secondly, there are cases where the chromosomes are of different sizes (certain Insects), and in these cases they are found in pairs (in tissue- and in yomig germ-cells), the two members of a pair being of the same size. In the heterotypic division of maturation the members of the paks get separated from one another, so that each secondary spermocyte (and consequently each spermatid after the second homoeotypic division) receives a similar set of different-sized chromosomes.


Attention has akeady been called to the difference in size of the ring-shaped chromosomes in Siredon.


a.e = i/.l.  
Now when a row of granules (or chromosome) is divided lengthways each half contains its due portion of each granule, and hence each daughter nucleus receiving half of each chromosome receives ipso facto a specimen of each different gi'anule. The two daughter nuclei are therefore alike and a longitudinal division of the chromosomes is merely quantitative.  


C. D.  
If, on the other hand, the row of granules (or chromosome) is transversely divided, or, what is the same thing, if two different chromosomes are separated from one another, each daughter nucleus will not receive a specimen of each different granule or chromosome, but only one-half, the remainder passing to the other nucleus, and the division is qualitative.  


f M^*^" Extension of the blastoderm over the yolk after formation and
The first condition may be represented by some such formula as this (where a-h are the quahtatively different granules in a chromosome, A,A',B, B', &c., whole chromosomes) : abcdefgh A A' B B' G C D D' abcdefgji' A A' B B' G C D D'' the line being the division, while the second condition will be represented by
loldmg ofiE of the embryo in an Elasmobranch. a, The lateral lips liave
swung back parallel to one another behind the dorsal lip, so enclosino- a
narrow strip of yolk. B, Side view of the same, c, The anterior ed-o
(«.e.) has passed beyond the vegetative pole, and in d it appears behind the  
embryo as the ventral lip (yi.) ; y.b., yolk- blastopore.


abed A B G D or  efgh A' B' G' D' Ordinary somatic mitoses are therefore quantitative, and so is the second homoeotypic maturation division. If, however, we adopt the view that in the heterotypic mitosis a transverse division of the chromosomes is involved, then we must further beheve that the division is quahtative, and consequently that the secondary spermocytes, and eventuaUy the spermatozoa, receive chromosomes of different kinds. Of every four spermatozoa produced from a single primary spermocyte, therefore, two wiU be aUke of one kind (containing, say, A, B, Sec), while two will be alike of another kind (containing A', B', &c.).


But it is evident from the foregoing that identical nuclear changes occur during maturation in the two sexes. The prophases of the first division-  with the leptotene, synaptene, pachytene, and diplotene stages-  are the same, and whatever view is taken of these phenomena must hold good for both sexes. In the female the growth period intervenes between the prophases and the actual division, but when this division occurs it is of the same form as in the male, heterotypic. The second division is homoeotypic in both sexes.


P. 124
While, however, the cell-divisions are equal in the male-  resulting in four spermatozoa -  in the female they are unequal -  giving one large ovum which receives practically the whole of the cytoplasm and the yolk, and three small polar bodies. The similarity of the nuclei shows that in spite of their small size these polar bodies are in reality potential ova, and there are cases where they are large -  as large as the ovum -  and can be fertilized and develope.  


Like the spermatozoa, the ovum (and polar bodies) receives only one-half the somatic number of chromosomes. As we shall see more fully in the next section, these chromosomes form a complete set, as do those of the male. If-  as is probably the case-  there are varietal differences between hidividual spermatozoa in respect of these chromosomes, the same will be true of the ova.^


But what the further significance of these difEerences is, if they exist, we do not know. The chromosomes of the spermatozoon and ovum are certainly vehicles of inheritance -  ^that is, concerned in the transmission of at least some of the inheritable characters of the species from one generation to the next. But since every spermatozoon or ovum can perform this function as well as every other, we are driven to conclude that each one possesses a complete set of the necessary specific chromosomes ; but that in different spermatozoa or ova the chromosomes may be of different varieties-  that is, be concerned in the transmission of different varieties of the same inheritable character. This may be expressed by the followmg scheme. A B, G, D, Sec, are the n different specific chromosomes. In the tissue-cells and young germ-cells there are 2n, each kind bemg represented by two slightly different varieties, namely, A and A', B and B', G and G', &c. In the prophases of the heterotype division A and A' unite,^ and so B and B', G and G'.


VI
In the actual heterotype division A and A', B and B', G and G' are separated from one another, so that each secondary spermocyte or oocyte has A or A', B or B', and so on.


1 Provided of course that priraary oocytes differ inter se in the arrangement and distribution of the heterotypic chromosomes.


2 If the union is by parallel apposition it is further possible to suppose that the individual granules of"^ which 4 and^' are composed pair ofi each with each, namely a with a', b with b', and so on.


THE GERMINAL LAYERS


The homoeotypic division is quantitative, hence each spermatozoon or ovum obtains A or A', B or B' , and so on ; that is, a complete set of the various kinds of chromosomes.


In only one respect are there chromosomal differences between the two sexes. In certain forms (Insecta), and possibly in others also, there is an accessory chromosome or heterochromosome (often paired), which not only differs in size and behaviour from the ordinary chromosomes, but is not the same in spermatozoon and ovum. The variations in the behaviour of this body or bodies are too complex to be discussed here, but those who have investigated it beheve it to be concerned in the determination of sex. Apart from the heterochromosomes and the varietal differences of the ordinary chromosomes, the germ-nuclei are exactly alike.


125
We have now to see how the two nuclei -  each containing one-half the somatic number of chromosomes -  are brought together when the germ-cella unite in the act of fertilization.




==IV. Fertilization==


Teleostei
The Axo\otl- 8iredon- wi\\ serve as a type (Fig, 44). The spermatozoon-  which is of the same form as that of the newt and salamander -  after passing through the mucin jelly surrounding the egg, reaches the surface of the latter. It approaches the egg with its anterior end-  acrosome -  and always in the pigmented animal hemisphere, sometimes near the equator, but more usually near the animal pole.


The processes are essentially the same as in the Elasmobranchs.  
The acrosome pierces the surface-layer of the egg-cytoplasm, and immediately the egg reacts in a remarkable manner. From all sides there begins to flow towards the acrosome what appears to be a watery albuminous fluid : it is hyaline, but coagulable. This becomes concentrated round the acrosome in the form of a conical plug, the base of which projects at the surface, the apex towards the interior of the ovum (Fig. 44, a). This plug is the entrance-funnel, its base bemg known as the entrancecone (' cone of attraction ' is an erroneous expression, as it is not formed prior to the contact of the sperm with the egg). The entrance-funnel enlarges and extends more and more . into the interior of the ovum, being directed usually towards the axis : it carries in with it a number of the superficial pigment granules and the spermatozoon. The latter, therefore, after moving actively up to the surface of the ovum and penetrating it with its acrosome, is passively carried in by the inflow of the entrance-funnel ; this movement is apparently due to a difference in surface tension between the entrance-funnel and the surrounding cytoplasm. The acrosome presently gets caught in the side of the entrance-funnel, but the substance of the latter, still moving on, carries the head and tail of the sperm with it. The result is that the anterior end of the head now faces outwards, while the posterior end Kes at the bottom of the funnel, where the head is bent on the tail, and the whole sperm-head has been rotated through 180°. Between the head and the tail -  and therefore now at the inner end of the funnel -  is the large anterior centrosome (Fig. 44, c).
Blastopore formation begins at the posterior edge, where the  






Fig. 44. -  Fertilization in the Axolotl.


A and B. Meridional sections of the whole egg. a, Formation of entrancefunnel (first part of sperm-path), b, Formation of sperm-sphere and aster ; o3 male pronucleus ; ? female pronucleus ; p.b the t^^^ polar bodies.


c Formation of the sperm-sphere round the middle piece (anterioi centrosome) ; narts only of the head (black) and tail are sho^vn.


Fig. 72.-  Growth of the blastoderm over the yolk after the formation of
X. Formation of the sperm-aster. The centrosome has disappeared ; the head besinning to be vacuolated, is separated from the tail.  
the material for the embryo in the Teleostean fish Serramis. (After Wilson )
d.L, dorsal hp of the blastopore (posterior edge of the blastoderm) â–  a e
anterior edge of the blastoderm or ventral lip {v.l.) of the blastopore •
s.c, segmentation cavity ; o.g., oil-globulc. '


backward growth of the dorsal lip with concomitant development of an archenteric cavity gives rise to the body of the
T'FuSr shortening and vacuolation of the sperm-nucleus. There is still no centrosome.  
embryo, but the process is extended to the lateral and anterior
edges, where there is a slight invagination. By the growth of






Fig. 73.-  Sagittal sections through the blastoderm of Serranm during
F, Appearance of the definitive centrosome. g, h. Division of thn centrosome.  
the formation of the germinal layers. (After Wilson.)


A, Beginning of overgrowth at dorsal lip {d.l.).  
(In c-H the arrow marks the direction of entrance of tlie spermatozoon.) I, Approach of the two pronuclei. Formation of spindle-fibres J, i?ormation of asters, elongation of spindle, further enlargement of pronuclei, and appearance of clu'omosomes.  


B, Overgrowth at anterior edge.
K, Further elongation of spindle, and formation of a ccntrosnhere


c, Later stage of posterior edge. t u i
irfhe'Toindr^r""" ^he pronudear men.branes are breakSg 5ow,x ana trie spindle-hbres passing in.  


D, The anterior edge has become theventraUip {v.l.); n.cA., notochord ;
L, The fully.formed fertilization spindle. In the equator are the chromoomes, now longitudinally split, and attached to large spiiidle fi'bre Tu each centrosome the centriole has divided.  


end., endodorm ; m.p., medullary plate ; par., parablast (periblast) ; y.f.,


yolk-plug ; K.v., Kuppfer's vesicle.




The entrance-funnel soon disappears, but the pigment carried in by it remains for some time as a streak, usually known as the first part of the sperm-path (Fig. 44, b).


A clear, yolk-free area now appears round the centrosome ; this is the sperm-sphere (Fig. 44, c). Very soon radial fibres or processes of some kind begin to pass out from the sphere amongst the yolk-granules ; this is the sperm-aster (Fig. 44, d). Meanwhile the head or sperm-nucleus has become detached from the tail, and the centrosome which was between them has totally disappeared. It seems that the formation of the sperm-sphere and aster-  like that of the entrance-funnel-  is due to the extraction of water from the cytoplasm, in the case of the entrancefimnel by the acrosome, in the present case by the centrosome ; and that the centrosome is completely used up, in fact dissolved, in the process.


Fig. li.- Serra^ms. Transverse sections showing differentiation of the  
The tail of the spermatozoon will not concern us : it degenerates and vanishes. The head of course remains to become the sperm-nucleus or male pronucleus. It shortens and thickens : as it does so it becomes vacuolated. By further shortening and vacuolation it becomes transformed into an ordinary nucleus (Fig. 44, E). It Hes on the outside of the sperm-aster.  
roof of the archenteron into notochord
(n.ch.), mesoderm (mes.), and cndodprm
{end.); j^ar., parablast (periblast). (After
Wilson.)


It is at this moment that the definitive centrosome makes its appearance (Fig. 44, v). On the side towards the sperm-aster the nuclear membrane breaks down, and through the aperture something comes out of the nucleus which appears, when outside, as a rounded granular body. This is the definitive centrosome. It is not preformed in the sperm-nucleus and then ejected, but, probably, is due to a precipitation of the albumins of the cytoplasm by the nucleic acid of the sperm-nucleus. But, whatever interpretation be put upon the process, the centrosome is of male origm.


The male pronucleus, preceded by its centrosome and aster, now advances to meet the female pronucleus which has aheady left its position at the animal pole and is retm-ning towards the centre of the egg. The line in which the male pronucleus is now moving is knomi as the second part of the sperm-path. This does not necessarily lie in the same straight line, nor even in the same meridional plane as the first or entrance part of the path. This depends in part on the position of the female pronucleus (Fig. 46).


aic. 3
The first or entrance part of the path is usually directed towards some point in the egg axis, that is, it Hes in a meridional plane of the egg. If, as also is usual, the female pronucleus hes in the axis, it is evident that the second part of the sperm-path or line of union of the two pronuclei will he in the same plane. In that case it may be in the same straight line with the first part, or, more usually, make an angle with it, smce the pomt in the axis at which the pronuclei meet is at a fairly constant distance from the animal pole, while the point of entrance o the spermatozoon in the animal hemisphere is variable. If, however while the first part of the path is in a meridional plane the female pronucleus is not in the axis, then the sperm -nucleus must turn out of its meridional plane to meet the female pronucleus at some point which is not in the axis. The converse of this is seen when the entrance-path is not m a meridional plane while the female pronucleus is in the axis ; m this case also the sperm must turn aside. Thirdly, both sperm-entrance path and female pronucleus may be out of their normal direction


Fig. 75. - Serranus. Formation of the gut (a/.c.) by the  
Afterwords, the meridional plane which includes or is parallel to the entrance-path does not necessarily coincide with the meridional plane which includes or is parallel to the line of union of the pronuclei.  
bending down of tlie sides of the
roof of the archenteron. s.n.ch.,
sub-notocliordal rod; aid., cndoderm. (After Wilson.)


During the advance of the sperm-nucleus the centrosome divides (Fig. 44, g, h) at right angles to the direction in which the sperm-nucleus is travelKng, that is, to the second part of the sperm-path, and also to the meridional plane in which the path lies. The daughter centrosomes therefore lie in a plane parallel to the equator of the egg. Hence, when the pronuclei have met, they lie together between the daughter centrosomes, which lie in a plane parallel to the equator of the egg.


The two pronuclei are now closely apposed, but not fused, inside the sperm-sphere and aster. Next, the centrosomes send out fine fibres in all directions (Fig. 44, i, j). On the one hand these impinge upon the pronuclear membranes -  ^these are the begimiing of the fertilization spindle ; on the other hand they radiate out until they pass into the radiations of the original aster inside which they He.


VI
The pronuclei enlarge, and presently in each granules of chromatin appear and run together in rows to form chromosomes (Fig. 44, j). The number of these in each pronocleus is the same as that which entered into it at the close of maturation, namely n, the germ-number. Meanwhile the asters round each centrosome have been growing larger, the spindle-fibres longer, and the latter now break through the pronuclear membranes to meet their fellows from the opposite pole (Fig. 44, k). The membranes, achromatic network, and nuclei are now all dispersed, and the two sets of chromosomes, paternal and maternal, are placed side by side on the equator of the fertilization spindle, where they undergo longitudinal fission as in ordinary mitosis (Fig. 44, l). Hence, when the daughter chromosomes pass to the spindle poles, each daughter nucleus will receive a complete set of paternal, and a complete set of maternal chromosomes. The full somatic number, 2 n, is now restored, and with each repetition of nuclear and cell-division each cell in the body comes to possess 2 w chromosomes, one-half of which are derived from the father, one-half from the mother. With the apposition of the two sets of chromosomes in the equator of the division apparatus -  asters and spindle -  the act of fertilization may be said to be complete.


The whole falls into two periods. In the first the spermatozoon is carried into the egg by means of the entrance-funnel, which in turn is due to a stimulus of some kind imparted to the egg cytoplasm by the acrosome ; the acrosome is the modified centrosphere. In the second the definitive centrosome is formed from the male pronucleus and the division apparatus made between its two halves while the pronuclei meet. The mechanisms involved in both periods are therefore centrosomal.


The details of fertilization have been studied in many animals, including several Vertebrates. In Vertebrates it is a rule for the sperm to enter during the second maturation division of the ovum, as in the Axolotl {Petromyzon, Salmo, Triton, Mus), but in other cases it may enter at an earlier or later period. The tail may be left outside {Mus), but is more often taken in : it always degenerates.


THE GERMINAL LAYERS
The pronuclei may fuse to form a segmentation nucleus, from which 2 n chromosomes arise {Pristiurus, Salmo, Petromyzon) ; but the newt and the mouse resemble the Axolotl in the separate formation of the chromosomes in each pronucleus.


It is certain that in all cases the female centrosome disappears. Whether the definitive cleavage centrosome is identical with the centrosome seen in the spermatozoon, that. is, in the spermatid, or is, as in the Axolotl, a new formation from the sperm-nucleus, is not certainly known, but there is little doubt that it is invariably a male centrosome.


As a rule only one spermatozoon enters the egg, and the presence of more than one leads to serious derangements of development (pathological polyspermy).! in what is known as physiological polyspermy, however, two or more, sometimes a great number, normally get in, as in some Amphibia (including the Axolotl), Reptiles, Birds, and Elasmobranch fishes, in which last they are very numerous and known as 'merocytes' (Riickert). In these cases only one of the sperm-nuclei fuses with the egg-nucleus. The remainder lie about in the yolk, each develops its own centrosome and aster, and may divide (with n chromosomes) many times. Ultimately the accessory sperm-nuclei degenerate without contributing to any embryonic structure.


127
1 As in the sea-urchin, where the several nuclei fuse and their chromosomes become irregularly distributed. Where, however as m the frog Srseveral nuclei remain apart the polyspermy need not cause abnormal ^eveToprenHM. Herlant, Arch, de Biol. xxvi. 1911) although the superfluous sperm-nuclei do take part in the edification of the embryo.




It remains for us to discuss the significance of fertilization.


these extra-embryonic edges the yolk is finally enclosed and the  
It has commonly been supposed that its essence is to be found in the union of the pronuclei of the germ-cells, both nuclei being held to be necessary for the development of a normal individual. This view is based partly on the phenomena of conjugation in certain Infusoria, but also very largely on the assumption that the nuclei of the germ-cells are the sole vehicles for the transmission of inheritable characters ; this again rests upon the fact that it is only in their nuclei that the germ-cells are alike, while in every other respect they differ, and upon the supposition that the paternal and maternal contributions to the total inheritance are equal.  
anterior margin is then the ventral lip (Fig. 72). Notochord
and mesoderm are differentiated in the roof of the embryonic
part of the archenteron, the rest of this layer giving rise to
the alimentary canal, as in Elasmobranchs. Extra-embryonic
mesoderm arises at the remaining edges of the blastoderm
(Figs. 73-75).  


Now, whatever view we may take of the parts played by nucleus and cytoplasm respectively in the handing on of the characters of the species, it is most assuredly certain that for the production of a normal individual both pronuclei are not a necessity. In the first place, there is the phenomenon of parthenogenesis, natural and artificial. In the former the ovum develops without fertilization by the sperm and without artificial assistance (as in Aphidae and some other Insects, and in certam Crustacea). In the latter the stimulus usually given by the sperm is replaced experimentally by some physical or chemical agent. Thus the ovum of a sea-urchin or Mollusc may be stimulated by treatment with hypertonic sea-water, or butyric acid or other substance, or by mechanical shock, or a lowering of the temperature ; in the case of the frog it is sufficient to pierce the egg with a fine needle. In all these instances some physical or chemical alteration (or both) IS produced in the egg, as a result of which it begins to segment and develop. The process, if care is taken, may be perfectly normal, and the individual reach the adult condition A sexually mature (male) sea-urchin has been reared in this way In all cases of parthenogenesis only the female pronucleus is


The converse is seen in what is called merogony, where the egg (of a sea-urchin, Worm, or Mollusc) is divided into two halves, only one of wlucli contains the nucleus. Both halves can be fertilized, the nucleate and the enucleate, and will develop into normal larvae. In the latter case only the male pronucleus is present.


On the other hand, a nucleus must of course be present, and actual experiment has shown that what is really necessary for normal development is the presence in the ovum, and ultimately in every cell of the body developed from it, of a complete set of the n unlike chromosomes characteristic of the species.


v.l.  
Hence, both male and female pronuclei are not necessary, and we must look elsewhere for the significance of fertilization.  


As we know already, the germ-cells of both sexes pass through two maturation divisions, and two only, after which their capacity for reproducing themselves is lost. The first effect, or almost the first effect, of their union is that their product, the fertilized ovum, begins to segment and continues to do so. In other words, the power of reproduction by cell-division which was previously lost is in fertilization restored. It is mutually restored.


That the ovum regains the power of nuclear and cell-division is obvious : we see the maternal chromosomes undergo longitudinal fission, as they lie on the spindle, and subsequently we see the egg cytoplasm divide. In the case of the male Ave see the male chromosomes divide in ordinary fertilization as they lie alongside the female ; in the fertilization of enucleate eggfragments the stimulus imparted by the female cytoplasm to the male chromosomes is still more evident.


Fig. 76.-  Formation of the germ-layers in Ganoid fishes, a, b, in the  
A study of fertilization reveals the mechanism by which this stimulation is effected. For ordinary nuclear and cell-division an apparatus is necessary, the spindle with its asters ; this apparatus is made by the centrosomes in the cytoplasm, the two centrosomes proceeding from the division of one, and its function is first to pull apart the halves of the divided chromosomes, and second, to ensure cell-division by the cell-plate or intermediate bodies developed in the equator.  
Sturgeon {Acipenser) (after Bashford Dean) ; c, d, in Amia (after Sobotta) ;
arch.^ archenteron ; d.l, dorsal lip ; v.l., ventral lip ; n.ch., notochord ;
mes., mesoderm.  


Ganoidei
The mature ovum possesses no centrosome : the mature spermatozoon possesses little cjrtoplasm, and that only in the tail. In fertilization the centrosome is either introduced by the male cell or made by it after entering the egg : the necessary cytoplasm in which this centrosome can divide and make the asters and spindle is provided by the female. The wholly different structures of the two germ-cells are therefore mutually complementary in the stimulation by which the lost power of cell-division is restored, and this is the significance of fertilization.


Our knowledge of the differentiation of the germinal layers is
The experiments on artificial parthenogenesis suggest that a physico-chemical expression may be found for this stimulus.  
very slight, but it is known that the closure of the blastopore
is bilateral, and that mesoderm is formed at its lips, the notochord in the middle dorsal line (Fig. 76).  


This is not, however, its only effect. A very common, if not universal, result of the approach of the sperm is the exudation by the ovum of a peri vitelline fluid. In some cases (for instance, the sea-urchin) a membrane which prevents the entry of more spermatozoa is secreted at the same time and pushed out by the peri vitelline fluid. In the frog it remains as a thin fluid layer between the ovum and the jelly ; it is the exudation of this fluid which enables the egg previously adherent to the mucin jelly to turn over till its axis is vertical and the white pole below : this occurs shortly after insemination.


Of greater importance than this is the change in the cytoplasmic structure of the egg brought about at this time.


128 THE GERMINAL LAYERS VI
A few hours after insemination there appears in the frog's egg a crescentic grey patch on one side along the border of the pigmented area (Fig. 45). The grey crescent is due to the immigration of pigment from the surface into the interior, and this in turn is caused by the entrance of the spermatozoon. The grey crescent always appears on the side of the egg opposite to that on which the sperm has entered. We know that a watery fluid flows towards the sperm from the cytoplasm (the entrancefunnel flrst, and later the sperm-sphere, are due to this), and we may suppose that this streaming movement drags the pigment granules away from the surface on the opposite side, whence the grey crescent.


The grey crescent is actually opposite to-  that is, in the same meridional plane as-  the first or entrance part of the sperm-path (Fig. 46). Hence it does not necessarily lie in the same meridional plane as that which includes the line of union of the pronuclei.


We shall see in the next chapter that the meridional plane of the first division always includes the line of union of the pronuclei, and hence does not always coincide with the meridional plane of the grey crescent.


DrPNOi


The holoblastic egg of Ceralodus resembles that of the frog
It is clear that, whereas the unfertilized egg was radially symmetrical about its axis, it can now be divided into similar halves by only one plane, that which includes the axis and the middle point of the grey crescent. About this plane it is bi-laterally symmetrical. The greatest interest attaches to this alteration of symmetry, since the side of the grey crescent will become the dorsal side of the embryo, the side on which the sperm entered its ventral side. Since the animal and vegetative poles mark respectively the future anterior and posterior ends (approximately), it follows that the plane of symmetry of the fertiUzed but unsegmented egg coincides with the median longitudmal or sagittal plane of the future embryo. The whole bilateral symmetry of the embryo is now predetermined in the cytoplasmic structure of the egg.  
very closely in the development of its archenteron. The roof








Fig. 77. -  Formation of the germ-layers in Dipnoi. A, B, in Ceralodus
(after Semon) ; c, D, in Lepidosiren (after Graham Kerr), arch., archenteron-; d.l, dorsal lip ; n.ch., notochord ; ines., dorsal mesoderm.


of this cavity, however, takes no part in the formation of the gut,  
Fig. 45. -  Formation of the grey crescent in the frog's egg (R. ternporaria). a, b from the side ; c, d from the vegetative pole. In A, c there is no crescent, in b, d a part of the border of the pigmented area has become grey.  
but is differentiated simply into median notochord and lateral
plates of mesoderm. The yolk-cells then grow up to complete
the dorsal wall of the aUmeutarx, canal (Fig. 77, A, u).  




That the blastodisc has a bilateral structure in Birds and Elasmobranch fishes also seems to foUow from the fact that the cells in both these cases are larger at one end of the blastoderm than at the other. Further, this structure is definitely related to that of the embryo since the large-celled end becomes anterior.


VI
Whether the change from the original radial to the definitive bilateral symmetry is in these cases also brought about by the spermatozoon, future researches must show.


In the Teleostei the concentration of the superficial cytoplasm (periblast) to form the blastodisc is an effect of fertihzation.




THE GERMINAL LAYERS




Fig.  Diagrams to show the relation between the first and second parts of the sperm-paths. The paths are projected on a plane perpendicular to the axis. In a the two parts are in the same meridional plane, in B m different meridiona.1 planes. 1, First part of the sperm-path ; 2. Second part; o^, male pronucleus ; ?, female pronucleus ; ^.c, grey crescent : on the opposite side (side of entrance of the sperm) the superficial pigment Z rS; ' «,<^^ has divided in a plane perpendicular to thf a^
at right angles to the second part of the path. '


129


In conclusion we may attempt to estimate the parts played by the cytoplasm and the nucleus of the germ-cells in inheritance.




Lepidosiren resembles the frog in all respects, except that the  
That some at least of all the inheritable characters of the species-  and not only specific but varietal and individual characters as well-  can be inherited from the father as readily as from the mother is obvious. Since the nucleus, beside the centrosome which is merely an organ of cell-division, and the acrosome which merely provides for the entrance, is the only part of the male cell which is always incorporated in the fertilized ovum for the tail may be left outside, we are obliged to regard the nucleus, that is, the chromosomes, as the vehicles by which tliese characters are transmitted.  
yolk is more vokimmous and that a ventral lip is never developed
(Fig. 77, c, D).  


Urobelotjs Amphibia


The method of germ-layer separation is here practically identical with that which is observed in the frog, except in one
The chromosomes of the nuclei of the germ-cells -  which, as we have already pointed out, are different from one another -  are in some sense the determinants of inheritance in the offspring : on their presence depends the ultimate appearance in the offspring of certain characters, and, in respect of their capacity for transmitting these characters, the two germ-cells are similar : each possesses a full set of the necessary chromosomes. In ordmary sexual reproduction the offspring receives two such sets, but one will suffice, as in parthenogenesis and merogony.  
important respect. In the bilateral closure of the blastopore,
the presence of a ventral as well as of a dorsal lip (Fig. 78, A)
and the formation of the mesoderm from a double source, the
two groups closely resemble one another ; but while in the frog
the under layer of the roof of the archenteron persists as the  
dorsal lining of the alimentary tract, in the Urodeles the roof
of the archenteron becomes wholly converted into the notochord,  
as in Petromyzon, and the gut must be completed dorsally by
an ingrowth of yolk-cells from the sides (Fig. 78, b, c).  


The Anurous Amphibia, such as the toad, generally resemble
It does not, however, follow that the determinants for the whole of the inheritance are located in the nucleus.  
the frog in this matter, but in one case the notochord is described
as being formed from the middle streak of the whole thickness
of the roof, and even in the frog such a procedure may be
experimentally instigated by subjecting the embryos to the
influence of cane sugar and other substances.  


A comparison of these processes in the small-yolked and the  
As we have just seen, the material for the different parts of the body of the embryo is present in the cytoplasm of the fertilized but unsegmented egg ; to that structure the spermatozoon has . contributed nothing, beyond the rearrangement of material, the substitution of a bilateral for a radial symmetry. Experiment teaches us that the various parts of this structure are so many organ-forming substances, causally related to the development of certain organs, and therefore determinants of a part of the whole inheritance ; and recent researches on heterogeneous hybridization show clearly what this part is. The ovum of a sea-urchin, if the proper precautions are taken, may be fertilized by the sperm of a starfish, a feather-star (both of which of course are, like the urchin, Echinoderms), or even of a Mollusc or Worm. The result is always the same. A typical sea-urchin larva is developed. Even an enucleate egg-fragment will develop a little way when so fertilized, and exhibits the maternal characters alone. The spermatozoon employed does nothing but convey to the egg a stimulus, which sets the process in action ; its chromosomes sometimes persist, sometimes do not.
large-yolked types shows that :


1. The blastoderm of the large-yolked corresponds to the
animal region of the small-yolked egg, the yolk to the vegetative
part, and that the edge of the blastoderm in the former is equivalent to the boundary between animal and yolk cells in the latter.


2. In both this bounding line becomes in its entirety the lip
Hence the characters, the determinants of which reside in the cytoplasm, are the large characters which put the animal in its proper phylum, class and order, which make it an Echinoderm and not a Mollusc, a Sea-urchin and not a Starfish ; and these large characters are transmitted through the cytoplasm and therefore through the female alone. The smaller characters -  generic, specific, varietal, individual -  are equally transmissible by both germ-cells, and the determinants of these are in the chromosomes of their nuclei.  
of the blastopore (except where the ventral lip is absent), the  
posterior point of the edge in the large-yolked being equivalent
to the dorsal lip of the small-yolked, the anterior point to the  
ventral lip.  


3. In both the germinal layers are laid down during the
bilateral closure of this blastopore, the notochord stretching in
front of the dorsal lip, the mesoderm springing from the lateral
lips in two sheets which are continuous with one another behind
the ventral lip.


1355 T
And yet the cytoplasm of the egg-cell is indebted very largely for its structure to the activity of the nucleus. As we have seen, the nucleus makes two contributions to the cytoplasm, first, the so-called ' yolk-nucleus ', the substances concerned in the deposition of the yolk, and second, the contents of the germinal vesicles dispersed when the latter breaks down at matm'ation. These processes are perhaps independent of the chromosomes. Further, they find no parallel in the male sex.


Even if, therefore, the cytoplasmic determinants are ultimately to be assigned to the nucleus, the share taken by the female in the transmission of the whole heritage is greater than the part played by the male.




130
==Literature==




W. E. Agab. The spermatogenesis of Lepidosiren paradoxa. Quart. Journ. Micr. Set. Ivii, 1911.


THE GERMINAL LAYERS
G. Belikens. Die Reifung und Befruchtung des Forelleneies. Aiiat. Hefte, x; 1898.


J. B. Caenoy et H. Lebeun. La v6sicule germinative et les globules polaires chez les Batraciens, La Cellule, xii, xiv, 1897, 1898.


J. B. Farmer and J. E. S. Moore. On the meiotic phase in animals and plants. Quart. Journ. Micr. Sci. xlviii, 1905.


VI
K. Herfort. Die Reifung und Befruchtung des Eies von Pelromyzon fluviatilis. Arch. miJcr. Anat. Ivii, 1901.


'J. W. Jenkinson. Observations on the maturation and fertilization of the egg of the Axolotl. Quart. Journ. Micr. Sci. xlviii, 1904.


E. KoRSCHELT u. K. Heider. Vergleichende Entwicklungsgeschichte der wirbellosen Tiere. Allg. Th., Lief. 2, Jena, 1903.


4. The principal points of difference are two. First, the closure
F. Meves. Ueber die Entwicklung der mannlichen Geschlechtszellen von Salamandra mactdosa. Arch. mikr. Anat. xlviii, 1896.
of the blastopore in Elasmobranchs, Myxinoids, and Teleostei,
is effected in two periods ; during the first the overgrowth is
almost confined to the dorsal lip and produces the material for


F. Meves. Es gibt keine parallele Conjugation der Chromosomen ! Arch. Zellforsch. i, 1908.


T. A. Montgomery. The heterotypic maturation mitosis in Amphibia and its general significance. Biol. Bull, iv, 1903.


A. Oppel. Die Befruchtung des Reptihencies. Arch. mikr. Altai, xxxix,


-piQ. 78 - Formation of the germ-layers in the Axolotl.  
J. RijOKEET. Zur Bofruchtung des Selacliioroies. Ami. Am. vi, 189L A. u. K. E, SoHEEiNER. Uio Rcif ung der maiinlichen Geschlechtszelleu


A, Sagittal section after completion of the blastopore and rotation of
von Salamandra maculosa, Spinax niger und Myxine gluHnosa. Arch, de Biol, xxii, 1906.  
the egg.  


B, Transverse section of the same stage. i. u a
J. SoBOTTA. Dio Befruchtung und Furohung des Eies der Maus. Arch, mikr. Anat. xlv, 1895.
c Dorsal part of a transverse section of a later stage, n.ch., notochord;


d.l.', dorsal hp ; v.l, ventral lip ; mes.v., mesoderm formed ^Wentral hp ;
E. B. Wilson. The cell in development and inheritance. New York, 1902.  
mei.l, dorsal mesoderm ; mes.2, ventral mesoderm (from the yolk-cellh
pushed into the segmentation cavity) ; end., endoderm.  


the formation of the embryo ; in the second the yolk is gradually
H. VON WiNrwABTER. Rechcrches sur I'ovogentee et I'organogcneso de I'ovaire des Mammif^rea. Arch, de Biol, xvii, 1901.
covered by an extension of the blastoderm in which the lateral
and anterior margins are alone concerned. Secondly, in these
cases a part only of the blastoporic hp is involved in the formation of the embryo, the lateral and ventral lips remaining wholly
extra-embryonic.  


VI
THE GERMINAL LAYERS
131
Gymnophiona
In this group the egg is so laden with yolk that in it segmentation nearly approaches the meroblastic type and results in a
blastoderm lying on a partially divided yolk. This blastoderm
consists of a superficial epithelium of columnar cells, covering
Fig. 79.-  Formation and closure of the blastopore in the Gymnophiona.
A-D, Surface views of the blastoderm of Hypogeophis. The lateral lips are
seen to meet behind, and so form the ventral lip ; y.p., yolk-plug (after
Brauer). e. Embryo of Ichthyophis lying on the partially segmented yolk
which is still uncovered by the blastoderm. (After the brothers Sarasin.)
several irregular layers of scattered cells which are more abundantly supplied with yolk. The cavities between these cells are
equivalent to the ordinary segmentation cavity. Below these
again is the yolk, divided at its surface into cells, and containing
nuclei scattered through its substance. Immediately round the
blastoderm the surface of the yolk is also partially segmented.
At one point-  the posterior middle point-  of the edge of this
I 2
132
THE GERMINAL LAYERS
VI
blastoderm the dorsal lip appears (Fig. 80) ; it exhibits the
usual radiate arrangement of cells. The lip quickly grows back
and so produces a long archenteron which comes to open into
80.-  Formation of the germ-layers in Hyi)0!7eoi)te. (After Br auer.)
A-c Sagittal sections of three successive stages, d, Transverse section
Lough^he blastopore and yolk-plug {y-v.) ; s.c, ^^tlX /f
into which in B and o the archenteron {aroh.) opens ; U., dorsal hp , l.L,
lateral lip ; and vX., ventral Up.
the segmentation cavity in front. The roof of the archenteron
which seems to be derived entirely from the superficial layer of
the blastoderm, consists of a plate of columnar cells, its floor of
the partially segmented yolk.
VI
THE GERMINAL LAYERS
133
The process of overgrowth is not limited to the dorsal lip,
but extends to the immediate right and left. Surface views
(Fig. 79, a-d) show that the transversely placed li]p soon becomes
crescentic, and that the horns of the crescent then grow not
only backwards, but towards the middle line as well, approaching
one another until they meet and so form what is the ventral
lip of the now circular blastopore. In section it is seen that
there is a slight ingrowth at the lateral and at the ventral lips
of a plate of cells continuous with the similarly formed jolate
Â¥iG. 81.-  Transverse sections of HypogeopMs showing the differentiation
ot the root of the archenteron into notochorcl (nxh.) and mesoderm, and
Z fXf (Iftttrarxg"' ^"'^-^
which forms the roof of the archenteron in front ; beneath the
plate is a slit-like space, also, of course, archenteric ; in the midst
of the blastopore is the projecting typically Amphibian yolk-plug.
But m spite of this resemblance there is a very serious difference between the ventral lip of the Gymnopliiona and that of
ail other Anamnia. For while in the latter the whole of the edge of
the blastoderm or small-celled area is converted into a blastoporic
hp, the posterior point being the dorsal, the diametrically
opposite anterior point becoming sooner (in small-yolked eggs)
or la er (m large-yolked eggs) the ventral lip, and while consequently the whole of the vegetative surface of the egg is covered
up when the blastopore closes, in the former the anterior and
134
THE GERMINAL LAYERS
VI
a large part of the two lateral edges take no iiart in this process,
which is confined to the posterior and immediately adjacent
portions of the edge ; this small portion gives rise to the dorsal
and two lateral lips, which latter by their fusion produce the â– 
remarkable similitude of the ventral lip of other forms. As
a result the vegetative hemisphere is still uncovered when the
blastopore has become circular (Fig. 79, d, e). The importance
of this fact for the correct understanding of the relations of the
blastopore to the blastoderm in the Amniota cannot possibly be
over-emphasized.
To return to the germinal layers. The superficial layer is now
the ectoderm. The roof of the archenteron becomes divided
into a median strip-  the notochord, and two lateral sheets-  the
mesoderm which are continuous with one another behind the
yolk-plug by means of the cell-plate invaginated at the lateral
and ventral hps (Fig. 81). The mesoderm has in fact precisely
the same relations as in other Anamnia at this stage. The
notochord passes back into the dorsal lip. No additions are
made to either notochord or mesoderm from any other source.
The roof of the gut (endoderm) is completed by upgrowth and
ingrowth of vegetative cells underneath the midcUe layer.
Amniota
Whereas in the Anamnia the blastoporic lip appears at the
edge of the blastoderm, in the Amniota it hes wholly within the
latter. The blastopore leads into an archenteron, and with the
formation of these structures the materials for the germinal
layers are laid down. Only in the more primitive forms is the
archenteric cavity well developed ; usually it is much reduced
and represented only by the ' neurenteric ' passage or ' chordacanal In primitive forms the upper and lower layers are still
united at the point where the blastopore and archenteron arise,
and both layers may perhaps be said to share in their formation ;
but in most cases all these parts are derived from the upper
layer of the blastoderm alone, the subsequent fusion with the
lower layer being purely secondary. The edge of the blastoderm,
which is entirely independent of the blastopore, grows steadily over
the surface of the yolk, finally enclosing it at the vegetative pole.
i
IPiG 82 - Three stages in the formation of the blastopore at the hinder
end of the embryonic shield of a Reptile {PlaUjdacUjlus). Sm'face views.
(After Will.)
P. 1S5
II
VI
THE GERMINAL LAYERS
136
The Reptiles will be considered first as the whole process is
far clearer in them than in the other two groups.
Rbptilia
There is distinguishable in the blastoderm at the close of
segmentation a circular or oval area placed excentrically towards
the posterior end ; this area is the embryonic shield. The upper
layer of the blastoderm consists of cyHndrical cells in the embryonic shield, of flat cells in the surrounding region ; below it
is the segmentation cavity. The lower layer is an irregular sheet
of scattered rounded cells, not arranged at present in an epithelium, and is constantly being reinforced by the addition of
cells from the nucleated yolk beneath. Between the lower layer
and the yolk is a shallow cavity, the subgerminal cavity. In
some forms, such as Platydactylus and Lacerta, there is one point
in the margin of the embryonic shield where upper and lower
layers are continuous ; this is the primitive plate, and it is
situate at what will be the hinder end (Fig. 83, a). The lower
layer cells before long arrange themselves in a flat epithelium.
Meanwhile a depression has appeared in the primitive plate ;
this is the beginning of the archenteron, and its anterior margin
is the dorsal lip of the blastopore. Seen from the surface (Fig. 82)
the dorsal lip presents the appearance of a transverse rim bounding a groove at the hinder edge of the embryonic shield. The
rim rapidly becomes crescentic, the horns of the crescent turn
back, meet, and fuse behind the primitive plate which now
corresponds exactly to the Gymnophionan yolk-plug.
During the backgrowth of the horns of the crescent, which
are the lateral blastoporic lips, the cavity of the archenteron
has rapidly extended until it reaches the anterior end of the
embryonic shield (Fig. 83) ; the cavity is broad. The roof consists of a layer of columnar cells which at the dorsal lip turn
over in the ordinary way into the cells of the upper layer. The
floor is in front distinct from the lower layer, and here it consists of a single layer of cubical cells ; behind the dorsal lip -  in
the primitive plate-  it is much thickened, and from this thickenmg there proceeds backwards a narrow tongue of cells between
the upper and the lower layers.
136
THE GERMINAL LAYERS
VI
A transverse sectign (Figs. 84, a ; 86) through the blastopore
shows the mass of cells of the primitive plate flanked on each
side by a projecting blastoporic lip and sending out between
the upper and lower layers two lateral sheets of cells.
Fig. 83. -  Sagittal sections of the blastopore and archenteron in the
Gecko Platydadylus. (After Will.) a-e, Successive stages ; jj.^p., primitive
plate ; pd., lower layer or paraderm ; s.g.c, subgerminal cavity ; arch.,
archenteron ; d.l, dorsal lip ; y.p., yolk-plug ; mes.v., mesoderm formed
at the ventral lip.
The resemblance between these structures and those in the
Amphibian, and particularly the Gymnophionan egg when the
blastopore has become circular, is sufficiently obvious. The
dorsal and lateral lips (there is no ventral lip in the Reptiles)
clearly correspond in the two cases ; the mass of cells in the
primitive plate embraced by these lips is the yolk-plug ; the
Fio. 84. -  Four successive transverse sections through the blastopore
and archenteron of Plalydactylus. (After Will. )
A, Posterior section through the yolk-plug {y.f.) ; l.l, lateral lip ; 2^(1. ,
lower layer ; mes., mesoderm springing from the lateral lips.
B is more anterior, just behind the dorsal lip.
c is just in front of the dorsal lip, where the floor of the archenteron
{arch.) is still intact, and
D more anterior, where the archenteron communicates with the subgerminal cavity.
c
arc?-..
c
138
THE GERMINAL LAYERS
VI
cavity of invagination is the archenteron in which floor corresponds to floor and roof to roof ; lastly, the sheets of cells
projecting beneath the upper layer at the sides of and behind
the blastopore are the equivalents of the mesoderm formed at
the lateral and ventral lips in the Amphibia.
From this comparison it follows of course that cells which are
the morphological equivalents of the yolk-cells of the Amphibia
are to be found in the upper layer of the Reptihan blastoderm.
That layer, therefore, cannot be termed the ectoderm until the
process of invagination is complete.
The floor of the archenteron now fuses throughout with the
lower layer, and as soon as the fusion is completed perforations
-PTP 86 - Transverse section of the blastopore and yolk-plug (y.p.) of
thTS^itoise (Trionyx). (After Mitsukuri.) U., lateral lip ; > m/so^erm
produeed at the lateral lips ; pel., lower layer not yet detaehed from the
yolk (stippled).
begin to appear in tte fused layers (Figs. 83, E ; 84, e). They
seem to be unable to keep pace with the general gro^vth of the
blastoderm and to become first stretched and then fenestrated.
But to whatever causes the perforation may be due, the floor
of the archenteron with the underlying lower layer completely
disappears, and the archenteron then communicates freely with
the subgerminal cavity. The roof of the archenteron is now
inserted by its edges into the surrounding lower layer.
The median strip of the roof next thickens to form the notochord (Fig 85), and separates from the two lateral portions which
then become the mesoderm. The notochord passes posteriorly
into the dorsal lip. the plates of mesoderm into the latei^hps
of the blastopore, and here the latter are perfectly contmuous
with the mesoderm produced at the sides of and behind the
I
Fig 87*.-  Area pellucida of the lien's egg. a, After 12 hours , b, After
18 hours' incubation, as seen by transmitted light. J5r.£/., prnnitive groove ;
n.ch., notochord ; pr.am., pro-amnion.
P. 139
VI THE GERMINAL LAYERS 139
blastopore (Figs. 84, a, b ; 86). The mesoderm thus exhibits
all the relations which it has in the Anamnia.
The Uning epithelium of the alimentary canal (endoderm) is
derived from the lower layer, which grows in from the sides
below the mesoderm and notochord (Fig. 85, c, d). From this
layer the gut is subsequently folded off, the remainder being
yolk-sac epithelium. In several cases the lip of the blastopore
is not the only source of origin of notochord and mesoderm,
both receiving additions in front, and the mesoderm at the sides
also, from the lower layer.
Fig. 87. -  ^Formation of the primitive streak and groove of the chick by
proliferation of cells of the upper layer. Transverse sections.
A, At 10 hours. There is at present no sign of the primitive groove ;
the lower layer {'pd.) takes no part in the proliferation.
B, At 15 hoiu's. The primitive groove has appeared. It is occupied
by a projecting mass of cells, tlie yolk-plug {y.'p.), and bounded by the
lateral hps {U.). The proliferated cells spread out on cacli side as the
lateral sheets of mesoderm {mes.).
The conditions observed in the Birds are very readily derived
from and very easily understood in the light of those which
obtain in the Reptiles.
There appears in the posterior region of the blastoderm a proliferation of cells in the upper layer (Fig. 87, a) ; this rapidly
extends in the median line, and along it there appears a narrow
groove. The cell proliferation is the ' primitive streak ', the groove the "primitive groove" (Fig. 87*).
Fig. 90. -  ^Anterior (a) and posterior (b) halves of a sagittal section
through the primitive streak and associated structures of the sparrow.
(After Schauinsland.) There is a sUght cavity, archenteron, below the
dorsal Hp (d.i.), and a well-marked ventral lip {vL). n.ch., notochord ;
p.s., primitive streak ; 7nes.v., mesoderm behind the ventral lip ; p.a.,
lower layer.
Fig. 91. - Transverse section of the anterior end of the blastoderm of the chick "at 15 hours showing the formation of anterior notochord (n.ch.) and
mesoderm (mes.) directly from the lower layer {end.) ; ec., ectoderm.
This primitive groove is simply an elongated and laterally
compressed blastopore. In front of the anterior end -  the dorsal
lip -  the notochord is produced (Figs. 88, 89) ; to right and left
of the notochord are the sheets of mesoderm which, springing
from the sides -  the lateral lips -  of the groove (Fig. 87, b), are
continued into one another behind its posterior end, where there
may be an actual ventral lip (Fig. 90). The archenteric cavity has, however, in most cases disappeared, though a vestige of it is
sometimes to be seen (Fig. 90) . Between the sides of the groove- 
which still exhibit the structure characteristic of blastoporic hps, is
merely a mass of cells-  representative of the yolk-plug (Fig. 87, b)
- fused with the lower layer. The so-called ' neurenteric canal ' ,
which appears later, is the sole remnant of the archenteron
together with the communication which we have seen to become
established between it and the subgerminal cavity in Reptiles.
The primitive streak and groove invariably originate in the upper Icayer, fusion with the lower layer being merely secondary ;
only after the germ-layers have been formed can the upper layer
be described as ectoderm.
The notochord and mesoderm receive increments in front from
the lower layer (Fig. 91).
The gut (endoderm) is formed as in Reptiles.
Mammaiaa
In the Monotremata there is a long archenteron with a much
reduced lumen produced from the upper layer. The blastopore
is an elongated ' primitive groove '. The notochord and mesoderm have the usual relations to these structures. The interpretation put by Wilson and Hill on their observations -  namely,
that the dorsal lip and archenteron are derived from the ' primitive plate ' while the primitive streak and groove are of distinct
origin -  is probably erroneous. We may accept Assheton's
explanation that the ' primitive plate ' of the authors is simply
the point of final enclosure of the yolk by the blastoderm,
a precociously rapid process in this form, and that archenteron
and primitive groove are, as everywhere else, parts of one and
the same structure (Fig. 92).
We are still in ignorance of the formation of the germinal layers
in Marsupials, though we may hazard the conjecture that the embryonic area of the blastocyst wall will be found to behave like the
embryonic shield in Reptilia, that a blastopore and archenteron will
be developed near its posterior edge in connexion with which the
notochord and mesoderm will arise in the usual way, that the
archenteron will break through into the subgerminal cavity below
the lower layer, and that this layer will give rise to the gut.
This indeed is what occiurs in the Placental Mammals, the
only diflference being that here the embryonic area is from the
first enclosed in the sac of the trophoblast as part of the embryonic knob. This knob, as we have already seen, is, together
with the lower layer, differentiated from the original inner mass.
The embryonic area (Fig. 92*), derived from the embryonic
knob, behaves precisely as the embryonic shield of the upper layer
in Reptiles, giving rise to an archenteron and blastopore ; this event
is, however, postponed until after the amnion has been formed.
trek a
Fig. 132. Diagiain of the egg of Ornithorhynchus after formation of the germinal layers. (After Assheton's modification of Wilson and Hill.) x, the
point at which the blastoderm has finally enclosed the yolk ; here the
upper layer (double line) and lower layer (broken line) are continuous with
one another and with the yolk. This is the ' primitive plate ' of Wilson and
Hill, a to p, primitive streak ; a, anterior end (dorsal lip) ; p., posterior
end. In front of a. is the archenteron (arch.), behind p. the mesoderm of
the ventral lip {mes.v.).
Fig. 92.-  Embryonic shield of the dog. (After Bomiet.) In the embryonic shield, where the cells are columnar, the nuclei are more closely packed
than in the surrounding trophoblast, where the cells are flat. At tne
posterior end is a notch, the blastopore (lower end in the fagure).
When the archenteron has been developed it behaves in the
manner we are already acquainted with. Its floor fuses with
the lower layer, and then the two break away so that the archenteron comes to communicate with the subgerminal or yolk-sac
cavity (Fig. 93). The notochord is differentiated out of its
roof, the mesodermal sheets pass into the lateral lips and are
Fig. 93. -  a. Longitudinal section of the embryonic shield and blastopore
of the bat, VesperlUio. (After Van Beneden.) The archenteron (arch.) has
broken through into the subgerminal cavity [s.g.c.) or cavity of the blastocyst. Below tlie dorsal lip (d.l.) is the blastopore (so-called neurcnteric
canal), and behind this the yolk- plug {ij.j}.). (With this should be compared
Fig. 138, which shows a human embryo in the same stage.)
B, Transverse section showing the origin of the notochord [n.cli.) from
the roof of the rudimentary archenteron in the mouse. The floor of the
archenteron has already disappeared, mes., mesoderm ; 'pd., lower layer.
Above is the ectoderm of the medullar}^ plate.
continuous with one another behind the blastopore. Accessory
notochordal and mesoblastic material is proliferated in front from
the lower layer. After this the lower layer is endoderm, and
gives rise to the gut and yolk-sac, after growing in from the
sides underneath the notochord.
The archenteron may be well developed (as in VesperUlio), but
more usually is reduced to a narrow canal, the ' chorda-canal '
or, so called, ' ncurenteric ' passage.^
^ Neurenteric passage means properly the communication between the
medullary tube and the hmd end of the archenteron. See below, chap. vii.
The Relation between the Amniote and the Anamnian Blastopore
The facts we have now reviewed will have made it evident
that there are certain features common to the separation of the
germinal layers in all Vertebrates.
Thus in all cases the material for the germ-layers is laid down
during an overgrowth and ingrowth of cells which takes place
at the lip of the blastopore during the formation and closure
of the latter. This closure is always bilaterally symmetrical,
beginning at the dorsal lip and taking place most actively there,
less actively at the lateral lips, and least of all at the ventral
lip. It leads to the formation of a bilateral archenteron, the
extent of which is greatest anteriorly, least posteriorly. The
layer that now remains outside is the ectoderm. The notochord
is differentiated out of the roof of the archenteron in the middle
line in front of the dorsal lip, while the mesoderm sheets which
flank the notochord pass back to the lateral lips and are confluent with one another behind the ventral lip.
A, 1-3, The closure of the blastopore in such a form as the frog ; 1, 2,
before, 3, after rotation of the egg. The blastoderm, or small-celled area,
is heavily stippled. Its whole edge, which becomes the lip of the blastopore, is represented by a thick continuous line, d.l., dorsal, v.l., ventral lip.
B, 1-3, Three similar stages in such a form as Lepidosiren, where the
ventral lip is absent. Only that part of the edge of the blastoderm which
becomes converted into a blastoporic lip -  namely, the posterior and immediately adjacent parts -  is indicated by the thick continuous line, d.l,
dorsal lip. '
c, 1, 2, The condition seen in the Gymnophiona, where still less of the
edge of the blastoderm-  only a small part at the posterior end, represented
by the thick line-  becomes the lip of the blastopore, but the lateral lips
swing back, meet, and fuse to form the ventral lip, v.l. Thus the yolk
(white) remains uncovered.
D, 1, 2, The Amniote blastopore. The heavily stippled area is the
embryomc shield, the central portion only of the Amniote blastoderm
but the equivalent of the whole blastoderm of the Anamnia. From the
posterior part of its margin a blastoporic lip is formed (d.l, dorsal lip)
and by the bending back and union of the lateral lips a ventral lip (v I )
as m the Gymnophiona. i' v
The lightly stippled area outside this represents the extra- embryonic
Fâ„¢,"? blastoderm; which is equivalent to the yolk-cells immediately
surrounding the blastoderm of the Gymnophiona ^
xlr^l^^-^l the unsegmented yolk (white). Thus the blastopore of the
f^t lTfC formed inside its blastoderm, but at the edge of what is equiva
Th« blastoderm, namely, the embryonic shield. ^
lUe yolk is finally covered later on by the growth of the blastoderm
k2
148
THE GERMINAL LAYERS
VI
So far there is general agreement. There is, however, a very
serious difference between the two great groups of Vertebrates
in respect of the reh^.tion of the blastoporic lip to the blastoderm
-  the cap of cells produced at the end of segmentation in a largeyolked egg or the area of small cells in a small-yolked egg -  for
in the Anamnia the blastopore arises from the edge of this
blastoderm (Fig. 94, a), while in the Amniota it arises inside it
(Fig 94, d). By the help of the Gymnophiona, however, the
second condition may without difficulty be derived from the first.
In the Gymnophiona (Fig. 94, c) (1) the blastoderm is an
oval area of columnar cells resting upon and surrounded by
a partially segmented yolk. (2) Only a part of the edge of the
blastoderm is converted into a blastoporic lip, namely, a small
region at the posterior end. Here a dorsal lip is formed and
lateral lips quickly follow ; the lateral lips then turn back,
encircling a small area of the yolk, behind which they meet and
fuse to form a ventral lip to the now circular blastopore. In
this process the anterior margin of the blastoderm is wholly
unconcerned. (3) The archenteron opens into the segmentation
cavity, notochord and mesoderm are derived from its roof, the
endoderm from the yolk-cells which lie in its floor. The notochord stretches in front of the dorsal lip ; the mesoderm sheets
springing from the lateral lips are continuous with one another
behind the ventral lip.
As a result of this peculiarity in the formation of the ventral
lip the yolk remains uncovered. In all other Anamnia, however,
where the ventral lip is developed from the anterior edge of the
blastoderm, the yolk is necessarily covered up by the closure of
the blastopore.
We turn now to the Amniota, to the Reptiles for instance,
and find (1) that the embryonic shield is a circular or oval
area of columnar cells resting upon a lower layer, and surrounded
by a zone of flattened cells. (2) At the posterior margin of this
embryonic shield upper and lower layers are continuous. Here
a dorsal lip is formed and lateral lips quickly follow ; the lateral
lips turn back encircling a small area of the outer zone of cells- 
where these are continuous ^\'ith the lower layer-  behind which
they meet and fuse to form (a virtual, in some cases an actual)
VI
THE GERMINAL LAYERS
149
ventral lip to the now circular blastopore. In this process the
anterior margiji of the embryonic shield is wholly unconcerned.
(3) The archenteron opens into the subgerminal cavity, notochord
and mesoderm are derived from its roof, the endoderm from the
lower layer. The notochord stretches in front of the dorsal lip,
the sheets of mesoderm springing from the lateral lips are continuous with one another behind the ventral lip.
It seems clear, then, that the embryonic shield of the Amniota
is the representative of the blastoderm of the Gymnophiona
(and of all Anamnia), while the marginal zone of the upper
layer, together with the lower layer with which it is at one
point -  ^the primitive plate -  still united, represents the yolk-cells
or nucleated yolk.
In passing from the Gjrmnophiona to the higher Vertebrates
we have therefore to suppose that with the further increase of
yolk segmentation has become restricted not to the blastoderm
alone (as in Fishes), but to the blastoderm and those circumjacent and subjacent cells which in the Gymnophiona are partially
segmented from the yolk. In the most primitive Reptiles the
lower layer cells are still crowded with yolk and still retain
a connexion, in the primitive plate, with the marginal cells of
the upper layer. In other Reptiles, in Birds, and in Mammals this
primitive connexion is lost, and it is only secondarily, after the
formation of the primitive groove and streak, that the upper
fuses with the lower layer.
The Gymnophionan condition must in turn be derived from
some Anamnian blastopore in the formation of which the anterior
edge takes no part, in which consequently no ventral lip is formed.
Such a form may be found in Lepidosiren (Fig. 94, b), in which
the yolk is less abundant than in the Gymnophiona, but more
abundant than in the typical smaU-yolked egg. Here the formation of a blastopore is restricted to the dorsal and lateral Jips.
The absence of a ventral lip may be a very primitive feature,
smce none is found in Petromyzon.
It may also be noticed that the union of segmentation cavity
with archenteron occurs here and there in various Anamnia,
sometimes in Eana, and in Petromyzon, thus foreshadowing the
condition seen in Gymnophiona and the Amniota.
150
THE GERMINAL LAYERS
VI
In the Anamnia, indeed, the archenteron has a direct relation
to the endoderm in that, after notochord and mesoderm have
been differentiated, the aUmentary canal is formed from its roof,
or floor, or both. But as we pass up the series the archenteric
cavity loses this significance, its lumen dwindles and finally
disappears, and its function is reduced to the differentiation of
notochord and mesoderm alone. The endoderm is then derived
from the lower layer cells -  ^representative of yolk-cells -  ^which
line the segmentation cavity.
The same lower layer cells may contribute to the notochord
and mesoderm anteriorly, and this, as we have seen, is of constant occurrence in such small-yolked Anamnian types as the
Amphibia, and Petromyzon ; not, however, in the large-yolked
eggs of Fishes.
The Significance of the Gebminal Layers
It will have been repeatedly noticed that the same elementary
organ or germ-layer may come into being by different processes.
This is true of the front end of the notochord and mesoderm,
and still more obviously of the endoderm, for the lining epithehum
of the alimentary canal may be derived from the roof only of
the archenteron (Elasmobranchs and Teleostei), from the floor
only {Petromyzon, Urodela, Ceratodus), from both roof and floor
{Rana, Lepidosiren), from the yolk-cells in the floor and from
those in the segmentation cavity (Gymnophiona, occasionally
Rana), or from the lower layer (yolk-) cells of the segmentation
cavity alone (Amniota).
In considering such discrepancies in the mode of origin of
homologous structures-  and discrepancies of this kind are of
common occurrence, not only in development from the egg but
also in budding and regeneration-  it must be borne in mind
that experiment has shown the formation of the embryonic
organs-  such as the germ-layers-  to be dependent on the
presence of certain stuffs in the cytoplasm of the ovum, but
that these stuffs are not necessarily deposited in the situations
which will eventually be occupied by the organs to which they
give rise, nor even in the same position in the ova of animals
belonging to the same group. Thus they may occupy dissimilar
VI THE GERMINAL LAYERS 151
positions also in the segmented ovum, and again in the later
stage which we speak of as gastrulation or the closure of the
blastopore. The necessary materials -  now cut up into cells - 
have then to move into their definite positions, and thus we
witness the roof of the gut being formed by an upgrowth of
yolk-cells, or its floor by a bending down of the roof of the
archenteron.
The way in which an organ is developed is not, therefore,
necessarily a criterion of its homologies. Homologous structures,
that is, those derived, like the alimentary tract of the Vertebrate, from some common ancestral structure, may differ in their
origin during individual development. The stuffs on which their
differentiation depends are doubtless comparable, but the paths
by which that differentiation is achieved may be diverse.
LITERATURE
R. AsSHETON. Professor Hubrecht's paper on the early ontogenetic
phenomena in Mammals. Quart. Jotirn. Micr. Sci., 1909.
E. VAN Beneden. Untersuchungen iiber die Blatterbildung, den
Chordakanal und die Gastrulation bei Siiugetieren. Anat. Am. iii, 1888.
R. Bonnet. Beitrage zur Embryologie des Hundes. Anat. Hefte,
Abt. ix, 1897.
A. Beaueh. Beitrage zur Entwickelungsgeschichte der Gymnophionen.
Zool. Jahrb. x, 1897.
Bashfoed Dean. The early development of gar-pike and sturgeon.
Journ. Morph. xi, 1895.
Bashfoed Dean. On the embryology of Bdellostoma stouti. Festschr.
f. C. von Kupffer, Jena, 1899.
L. F. Hennequy. Embryog6nie de la truite. Journ. de VAnat. et de la
Phys. xxiv, 1888.
J. W. Jenkinson. Remarks on the germinal layers of Vertebrates and
on the significance of germinal layers in general. Mem,. Manchester Lit.
and Phil. Soc. I, 1906.
J. Geaham Keeb. The development of Lepidosiren paradoxa. Quart.
Journ. Micr. Sci. xlv, 1901.
K. MiTSUKUEi and C. IsmKAWA. On the formation of the germinal layers
in Chelonia. Quart. Journ. Micr. Sci. xxvii, 1886.
J. Ruckeet. Die erste Entwickelung des Eies der Elasmobranchier.
Festschr. f. C. von Kupffer, Jena, 1899.
H. ScHAUiNSLAND. Studien zur Entwickelungsgeschichte der Sauropsiden. Zoologica, xvi, 1903.
162
THE GERMINAL LAYERS
VI
11. Semon. Die Furchung und Entwickelung dcr Keimbliitter bei
Ceralodiis forstcri. Zool. Forschiivgsreise in Anslralien, 1901.
A. E. Shipley. The development of Pctromyzon jluvialilis. Quart.
Joum. Micr. Sci. xxvii, 1887.
J. SoBOTTA. Die Gastrulation von .4»n{aaZw. VerJtaiidl. Anat. Geaellsch.
Berlin, 1896.
C. O. Whitman and A. C. Eycleshymer. The egg of Aviia and its
cleavage. Journ. Mor]}h. xii, 1897.
L. Will. Die Entwickelungsgescliichte der Ileptilien. Zool. Jahrh. vi, ix.
H. V. Wilson. The embryology of the sea- bass {Scrramts alrarius).
Bull. U. S. Fish Commission, ix, 1889.
J. T. Wilson and J. P. Hill. Observations on the development of
Ornilhorhynchus. Phil. Trails. Roy. Soc, Series B, cxcix, 1907.
H. E. ZiEGLEE. Beitrage zur Entwickelungsgeschichte von Torpedo.
Arch. mikr. -Anal, xxxix, 1892.
i
Fig. 95. -  External features of the development of the tadpole of the
Frog.
a. Medullary plate, anterior end : the three divisions of the brain are
apparent.
h. The same embryo from the posterior end : the sides of the medullary
plate pass back on either side of the blastopore. The blastopore is now
reduced to a narrow slit by the approximation of the lateral lips ; at the
dorsal and ventral lips the aperture is rather wider.
c. Medullary folds and groove, anterior end : the three divisions of the
brain are readily seen, and the anterior part of what will be the spinal
cord. External to the inner medullary folds are the outer, and these pass
in front into the broad gill-plates, in front of which again are the senseplates.
d. Closure of medullary folds, but the suture is still visible : the gill-plate
is divided on each side into two, and in front of it is the sense-plate ; behind
the gill-plate is a slight constriction.
e. Anterior view of the same embryo : the medullary folds have not
quite closed in front. Beneath their anterior end is a depression, the
stomodaeum, and on either side of this the sense-plates ; the gill-plates can
just be seen behind these.
f. Posterior view of the same embryo : the medullary folds have closed
over the dorsal division of the blastopore (neurenteric canal) while the
ventral remains as the proctodaeum. The middle region of the blastopore
is marked by a very narrow suture.
g. Later embryo from below showing the stomodaeum, in front of the
V-shaped sucker, and posteriorly the proctodaeum at the base of the tailstump.
h. Older embryo from the right side. The tail is rather longer, the
proctodaeum at its base : the stomodaeum can be seen in front between
the two halves of the sucker. At the side of the head in front is the
nostril, behind the gill-slits.
i. Older embryo (ready to hatch) with well-developed tail and external
gills.
Betweenl52 andlG")




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Jenkinson JW. Vertebrate Embryology. (1913) Oxford University Press, London.

Vertebrate Embryology 1913: 1 Introduction | 2 Growth | 3 The Germ-Cells, their Origin and Structure | 4 The Germ- Cells, their Maturation and Fertilization | 5 Segmentation | 6 The Germinal Layers | 7 The Early Stages in the Development of the Embryo | 8 The Foetal Membranes of the Mammalia | 9 The Placenta | Figures
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Chapter IV The Germ-Cells (continued)

III. The Maturation of the Germ-cells

A. In the male

The Urodelous Amphibia have always been a favourite object for the study of these changes, and may conveniently be taken by us as a type.

It will be recalled that duriag the spermogonial divisions the full somatic number of chromosomes is seen. The mitosis is of the ordmary character (Fig. 32). The granules of chromatm increase, run together m the form of beaded rows, which become the V-shaped chromosomes. The nuclear membrane has m the meantime broken down, the centrosome has divided, and around each daughter centrosome an aster is appearmg. The chromosomes then undergo longitudmal fission and, so spht, are placed on the equator of the spmdle now developed between the two centrosomes. The daughter chromosones are then puUed apart by the spmdle-fibres attached to them to the opposite spmdle poles, and there passmg through the same series of changes in the reverse order become the daughter nuclei. Meanwhile a celldivision has occurred m the equatorial plane of the spmdle, m which process the mtermediate bodies-thickenings of the spmdle fibres- play an important part.


All the features of an ordmary mitosis are here : the chromatm is the only part of the nucleus to be divided ; for that purpose it is thrown mto the form of chromosomes, which ^ht lengthways mdependently of any external agency , a division a^tatuLasters and spindle-is ^onst^f ?? e centrosomes and probably by them, the function of pull apart the halves of the aheady divided chromosomes and to LL?the division of the cell. When the W ceased dividing they enter upon a time the nucleus passes through complex changes, whlch are reallty the prophases of the fost of the two maturation divisions. This first division is of a very different character to an ordinary mitosis. There ensues the second division. This, with one important exception, resembles the mitoses of the spermogonia.


Fig. 32. - Stages in the karyokinetic division of the spermogonia of the newt.


The first maturation division (Figs. 33, 34). In the nucleus of a spermogonium the chromatin is in the form of fairly coarse lumps uniformly distributed over a wide achromatic reticulum. As the growth of the cell and its nucleus begin the chromatin becomes subdivided mto finer granules, which soon arrange themselves m rows or filaments ; in each row the granules are connected by threads of the achromatic reticulum, while similar threads pass from one filament to another. This is the narrow thread or leptotene'^ stage. As the nucleus enlarges still more


Fig. 33.- Prophases of the heterotype division in the male Axolotl. 1, Nucleus of spermogonium or young spermocyte ; 2, Early leptotene ; 3, Transition to synaptene; 4, Synaptene with the double filaments converging towards the centrosome ; 5, Contraction figure ; 6, 7, Pachytene ; 8, Early, 9, Later diplotene ; 10, The heterotypic double chromosomes ; the nuclear membrane is disappearing.

it is seen that on one side some of the filaments are arranged in pairs, and converge towards one point, the point where the centrosome m its centrosphere is placed. On the other side of

1 These and the followmg terms were first proposed by von Winiwarter in his classical work on the oogenesis of the rabbit.


the nucleus the filaments pass into the general network. This is the paired thread or synaptene stage.

By coalescence of the component granules the filaments become shorter and thicker : at the same time in each pair the filaments approach one another so closely that only a narrow slit is left between them. On one side the pairs of filaments still converge towards the centrosome, but on the other are inextricably coiled and tangled together into a bunch which is withdrawn some little way from the nuclear membrane. The pairing of the filaments can, however, be seen in the tangle. The several pairs are still imited by achromatic threads, the filaments being toothed at each point of insertion of such a thread. A few threads stretch across the empty space between the tangle and the membrane. This is the contraction figure.


Fig. 34. - First maturation division in the male. 2, Salamander, the remainder Axolotl. 1, 2, The heterotypic chromosomes on the spindle (metaphase) ; 3, Anaphase ; 4, 5, Telophase ; 6, Resting nuclei ; 4-6, Celldivision into two secondary spermocytes.


The members of each pair of filaments now unite throughout their length, so that the longitudinal sHt disappears. The thick filaments still converge towards the centrosome side, where apparently they end against the nuclear membrane. There is, therefore, not one continuous filament or spireme, but several.

The other ends of the filaments pass into the tangle, which is still retracted from the nuclear membrane, but becoming looser as the nucleus enlarges. The coil is soon still more unravelled and occupies the whole of its side of the nucleus. This is the pachytene stage.

The several filaments now separate from one another, so that the polar convergence is lost, and coil in various directions through the nucleus. At the same time the longitudinal sUt reappears in each, and the filaments are once more paired, so reaching the diplotene condition. Their surfaces are still toothed where the connecting achromatic threads are inserted. Soon, however, these cross threads disappear and the filaments become smooth. At the same time the members of the several pairs begin to separate a little from one another, in places if not throughout their length.

The nuclear membrane now breaks down and disappears, the pairs of filaments shorten and thicken, and assume the most various shapes and sizes. A pair may be in the form of two straight parallel rods, or two curved parallel rods, either V-shaped, or C -shaped, or two rods parallel at one, divergent at the other extremity, and so -||--shaped ; or the sht between them may be expanded in two or more places, and then the two may be tAvisted over one another mto a figure of g or or by expansion of the whole sht, while the rods are united at the ends, may be ring-shaped, while finally the ring may be pushed in in four places and assume the form of a cross, =[}:. These bizarre double bodies are the chromosomes of the first maturation division. It seems clear that they are derived from the separate paired filaments of the diplotene stage, these from the thick filaments of the pachytene stage, and these again from the paired filaments of the synaptene nucleus. The origm of these we shall have to discuss later on.

The number of the double chromosomes, and therefore of the several double filaments in the earUer diplotene, pachytene, and synaptene stages, is one-haK that seen in the spermogoma. The reduction from the somatic number {2n) to the germnumber (n) has already taken place. It seems that this half number must be estabhshed in the synaptene nucleus.

The actual division now occurs (Fig. 34). A spindle is formed in the ordinary way, and the double chromosomes are thrown upon its equator in such a way that the two ends of each member of a pair he in the equatorial plane. This is easily seen where the pair retains the original form of two closely-parallel rods separated by a longitudinal slit, and can often be made out in the ring- and cross-shaped and other chromosomes.

The members of the pairs now come apart and travel to opposite spindle poles, where they coalesce and pass into the condition of resting nuclei. The cell, meanwhile, has divided and the two secondary spermocytes have been formed. The nucleus of each of these, it is clear, contains only one -half of the ordinary number of chromosomes.

The division which we have just witnessed is unlike an ordinary mitosis in at least two respects. First, the number of chromosomes is reduced from the somatic to the germ number, and second, the chromosomes are double and frequently of extraordinary shape. For these reasons the division is spoken of as heterotypic, or xmlike the usual type. The term meiotic or reducing, also appHed to it, refers to the numerical lessening of the chromosomes.

We have now to inquire whether this division is or is not like an ordinary mitosis in another respect, the manner ia which the chromosomes are divided. Ordinarily, as we know, the chromosomes are longitudinally divided ; but on this occasion it is held by many observers that the division, albeit in appearance longitudinal, is in reahty transverse.

The uiterpretation of the nuclear changes is a matter of considerable difficulty, and very diverse opinions are entertained (1) as to the origin of the double filaments seen in the synaptene and later stages of the prophase, and (2) as to the mode of formation of the ring-shaped chromosomes seen in the actual mitosis ; different combination of these diverse opinions has led to the formulation of three principal views.

I. It is held that (I) the double filaments of the synaptene stage arise by longitudinal fission of the filament, that the longitudinal split disappears, but reappears (2) to form the cavity of the rings. Hence the actual division is longitudinal (Meves). This is illustrated in the accompanying diagram (Fig. 35, I).

For the sake of simpUcity we will suppose that the full number of chi'omosomes is four, the reduced number two. We will further suppose ttot these four chromosomes are really diff^^' Tne another though apparently identical. Let us caU them 7 A' B, and B: In the prophase of the mitosis two mstead of' four filaments appear. We may suppose that each of th«a consists of two ordinary chromosomes muted end to end say aTo 4' and B to B'. Each filament becomes then spht lengthwa^ll 1). the sHt widens out until each filament assumes a ling shape (I, 2), and the rings are then so placed on the equator of the spindle that the ends of the chromosomes lie in the equator (I, 3). Hence, smce each half ring consists of an A and an A', or of a 5 and a jB', when the halves are separated and travel towards the spindle poles, each daughter nucleus of a secondary spermocyte will receive a chromosome of each kind, A, A', B, and B'.



Fig. 35.-Diagram to illustrate three interpretations of the first matoasee text.)


II. On the second view (von Winiwarter, Schreiner, Agar), (1) while the paired filaments of the sjmaptene stage are believed to arise, not by longitudinal fission of the leptotene, but by apposition of distinct chromatin filaments (that is, chromosomes), the formation (2) of the rings from these double ' filaments is in accordance with the first view.

The diagram (Fig. 35, II, 1) shows the four chromosomes united in pairs by their entire length, though presenting every appearance of longitudinally spht rods : A is paired with A', and B with B'. The chromosomes of each pair then separate to form rings, remaining united only by their ends, and then are placed on the spindle ua such a way that these ends lie in the equator. It follows that A and B face towards one. A' and B' towards the opposite pole, and hence that each nucleus of a secondary spermocyte receives not all tour chromosomes, but only two, say A and B, ov A' and B'.

The division, therefore, is not really but only apparently longitudinal : the result is the same as though A and A' (and B and B') had been united end to end, and then separated by a transverse division of the double chromosome so formed.


III. On the third view (Farmer, Montgomery), (1) the double thread of the synaptene and pachytene is formed by the longitudinal sphtting of the chromatm filament ; but (2) the rings do not arise by the opening out of the spUt. The longitudmal division disappears, and the filament is first gathered up into half as many loops as there are chromosomes in the spermogonia, and these loops then separate as the n ring-shaped chi omosomes. The rings are therefore open at one end only, and the cavity of the ring arises, not by the opening out of the longitudinal split (for that has disappeared), but by the bending of the two halves, united end to end, of each double chromosome upon one another (Fig. 36, III). That is, the filament, consisting of Aj A', B, and B', is first gathered up into two loops, A being bent on A', and B on B', and then the loops separate. In the mitosis (III, 3) the rings are so placed on the spindle that A becomes separated from A' and B from B', so that one secondary spermocyte receives A and B, the other A' and B' (or, of course, A and B', A' and B).


Fig. 36.- Second maturation division in the male (Axolotl). 1, Prophase (split spireme); 2, The homoeotypic spht chromosomes on the spindle; 3, Polar view of the same ; 4, Anaphase ; 5, Telophase ; 6, Resting nuclei and completion of cell-division ; in each spermatid the centrosome has divided, and the sphere has become detached.

The result is therefore the same as on the second hypothesis.

Considering the diversity of opinion, it would be rash to dogmatize, but it may be pointed out that the evidence on the whole is agamst the mode of formation of the rings adopted by the third view. It does seem as though the rings were made by the opening out of the double filaments. We are left, therefore, with the choice between the first and second hypotheses. We can only say that the way in which the members of the pairs of filaments diverge into the general network in the fourth stage (Fig. 33, 4) suggests apposition rather than fission, and this involves ultimately a transverse division of double chromosomes, and that the phenomena of maturation observed in a number of Invertebrate forms corroborate this view.

Before discussing the theoretical significance of this mode of division, we shall describe the second maturation division (Fig. 36).

The nucleus of the secondary spermocyte soon emerges from the resting condition, and a chromatic filament appears. This filament becomes longitudinally split and then divided into a number of V-shaped chromosomes, themselves therefore split lengthways. The number of chromosomes is the half somatic, n. A spindle is developed, the spht chromosomes are placed on its equator, and division takes its ordinary course, resulting in two spermatids, the nucleus of each of which therefore possesses n chromosomes. La the V-shape of the chromosomes, as well as in their longitudinal division, this second mitosis is of the ordinary type. Hence it is called homoeotypic. Each spermatid becomes metamorphosed into a spermatozoon in the fashion already described.

The phenomena of maturation in the male are, as far as is known, similar in other forms {Myxine, Elasmobranchs, Mammaha). Each ripe male cell, therefore, is provided with only haK the number of chromosomes seen in the spermogonia and in the tissue cells of the body. Whether the n chromosomes in all the spermatozoa are or are not alike depends upon the interpretation placed on the first maturation division, as well as upon our views of the nature of the chromosomes.

B. In the female

While m the male the first or heterotypic division follows immediately upon the prophases, in the female the two episodes - prophase and division- are separated by an interval, sometimes of great length, a year or more- during which the yolk is deposited in the cytoplasm to the accompaniment of complex nuclear changes.

Prophases of the heterotypic division. The oogonial divisions come to an end at a fairly early period, and growth of the oocyte begins almost at once. The prophases of the heterotype are therefore usually found only in very young animals- in the tadpole of the frog, or the new-born or embryonic Mammal.


These two afford good examples. The nuclear changes which are readily seen in the tadpole's ovary (Fig. 37) are obviously closely j)arallel to what we have observed in the other sex.

A stage in which the chromatin is in the form of scattered granules is followed by one in which the granules run together to form the leptotene filament. Then comes the synaptene, with parallel filaments, followed by the contraction figure. The paired filaments emerge from the tangle to converge to one pole, the tangle itself beiiag withdra-WTi from the other side of the nucleus. The pachjiiene and diplotene follow in due course. A remarkable change now occurs in the straining capacity of the chromatm filaments. Up to the diplotene stage they behave in the usual way, showing great affinity for chromatin stains (carmine, haematoxylin, and basic aniline dyes) ; but from now onwards they lose this faculty and stain only with the acid plasma dyes. Meanwhile, the number of nucleoh (these also stain in acid dyes) is increasing, and presently it is seen that granules of chromatin (that is, granules which are coloured by the ordinary chromatin dyes) begin to settle upon (? be precipitated round) the nucleoh. By what appears to be a continuation of this process the nucleoh become converted into highly chromatic bodies.

The filaments (chromosomes) persist for a while, but will eventually disappear.

Precisely similar phenomena are seen in the young Mammahan ovary (Fig. 38), and only one or two points require to be mentioned. There is a very obvious centrosphere with included centrosome on one side of the nucleus (this usually goes by the name of the yolk-body of Balbiani), towards which the filaments of the synaptene and pachytene converge. In the early stage of contraction the paired filaments are seen to emerge from the rather open tangle on this side, while on the other a few filaments, also paked, stretch out to the nuclear membrane. In the later contraction figure the latter are retracted and the tangle, much closer, hes wholly on the side of the centrosphere.

After the diplotene stage the ruig-shaped figures of eight and other forms of double chromosomes are seen, but then the chromosomes break up into their constituent granules and range themselves along the achromatic threads which make a network through the nucleus. This is the diciyale condition, and in this the nucleus remains through the growth period until the moment of maturation arrives.



Fig. 37. - Prophases of the heterotypic division in the female (ovary of tadpole). 1, Nucleus of oogonium ; 2, Leptotene ; 3, Synaptene ; 4, 5, Contraction figures ; 6, Pachytene ; 7, Later pachytene, multiplication of nucleoli ; 8, 9, Diplotene : the chromatin filaments are becoming achromatic ; granules of chi'omatin are being deposited on the nucleoli.



Fig. 38. - Prophases of the heterotypic division in the female (Mammals). 1-6, Kitten three days old ; 7, Mouse embryo shortly before birth ; 8, Mouse eight days old.

1, Nucleus of oogonium or young oocyte ; 2, Leptotene ; 3, Synaptene ; 4, Contraction figure ; 5, Pachytene ; 6, Diplotene ; 7, Heterotypic clnomosomes ; 8, Dictyate.

In 2-5 the centrosphere and centrosome (volk-body of Balbiani) are shown with the chromatic filaments of the nucleus converging towards them.


Fig. 38*.- Small ovarian egg of the frog surrounded by its foUicle (/.) and theca (th.), which is continued into the pedicle {p.). b.v., a bloodvessel between follicle and theca ; v.rn., vitelline membrane ; ch., clu'omatin filaments, now achi'omatic ; n., chromatic nucleoli, ejected from the nucleus (n'.) and becoming achromatic (w".).


The period of growth and deposition of yolk. The nuclear changes accompanying the deposition of the yolk in the oocyte have only been studied in the Amphibia, to which we must now accordingly return (Fig. 38*).

We have seen that after the prophases the chromatin filaments become achi-omatic, while the nucleoH increase in number and become chi-omatic. The filaments gradually break up into a number of small granules, which disappear, or at least become indistinguishable from the general ground substance - or magma - of the nucleus.

The nucleoli become more numerous, larger, and more chromatic. They pass into the cytoplasm in one of two ways : either they are bodily ejected from the nucleus, lose their staining capacity and break up into small fragments, or else they disintegrate inside the nucleus, the products of their disintegration then passing out - either in the form of small particles or in solution - through the nuclear membrane into the cytoplasm. The nucleoli consist of nucleo -protein, and the result of their transference to the cytoplasm is that the latter first acquires an affinity for the chromatin stains, and then begins to secrete yolk-granules. There is thus a direct connexion between the nucleo-protein of the nucleoli and that which, as we have seen, is demonstrable in the yolk.

It appears that this cycle of changes is repeated many times during the growth of the oocyte, fresh nucleoli being formed, moving to the centre of the nucleus, and there disintegrating.

This passage of material from the nucleus to the cytoplasm of the egg-cell during the time of growth and yolk-formation is of constant occurrence in animals. The material may be solid and bodily ejected or liquid and diffusible, it may be chromatic or achromatic, but it is always given off and is always concerned m yolk-secretion. The chemical changes are, unfortunately, not fully understood.

The material is known generally as ' yolk-nucleus '. It has sometimes been confounded with another quite distinct structure, the sphere and centrosome. In Mammalian ova the sphere has indeed long been known as the yolk-body of Balbiani (Pig. 39). In the Mammals the sphere usually divides into two or more bodies, which persist for some time, but disappear (in the bat) when there are two or three cell-layers in the follicle.

In some Mammals {Cavia, Vespertilio) chromatoid bodies are found in the cytoplasm. These may be of nuclear origin and correspond to the yolk-nucleus of Amphibia.

The yolk-nucleus is a very important contribution made by the nucleus to the structure of the cytoplasm : a second contribution has still to be made.

With the growth of the oocyte the nucleus has been enlarging pari passu, and by the time growth is completed is of considerable size. It lies in the axis, but excentrically, near the surface in the animal half of the egg. The oocyte is now ready for the first maturation division.


Maturation. The nuclear membrane breaks down and disappears. From a very small part of the achromatic substance of the nucleus a spindle - the first polar spindle - is formed (Fig. 41), and on this are placed the heterotypic chromosomes, of which we shall speak in a moment. The whole of the rest of the contents of the nucleus - chromatic nucleoli and achromatic granular ' magma ' - are cast into the cytoplasm. This is the second contribution made by the nucleus to the cytoplasmic structure, and it is of considerable importance, since on it in part depends the diflEerence between animal and vegetative hemispheres.

As we have already had occasion to observe, there is a definite relation between the polar structure and symmetry of the egg and the structure of the embryo which is to come out of it, inasmuch as the anterior end is always developed near the animal, the posterior end near the vegetative pole. The structm-e of the embryo is at this moment bemg predetermmed in the egg, by the dispersal of the contents of the nucleus.

This is a fact of universal occurrence. When the germinal vesicle breaks down, only a smaU part of it is utihzed m the formation of the chromosomes which take part in the maturation mitosis. The remainder is given to the cytoplasm, of which it forms henceforward a definite and integral part. Experiment has sho^vn that that part is causally related to the development of certain organs, is therefore a vehicle of inheritance. It will be noticed that this process is without parallel in the male sex.

We return to the first polar spindle and its chromosomes.

The chromosomes appear first, as beaded filaments of heterotypic form - wrings, crosses, figures of eight, curved rods, and so on (Fig. 40). Their number is the half -somatic or germ-number n. It has been disputed whether these chromosomes are identical with those which were formed at the end of the prophases, in the young oocyte.

It must be remembered, in discussing this question, that the hypothesis of the individuahty of the chromatin A does not necessarily involve 'p^-'\X^ that of the individuality of the chromosomes. We have so™1-from^?SSi? th?o»y?; seen elsewhere that there is of the Axolotl (Siredon) just before reason for beheving that the membrane breaks down.

chromatia of the nucleus comprises a number of quahtatively unlike bodies- not merely that the chromosomes are different, but that they are composed of individually different granules. It is also probable, to say the least, that chromosome formation is a nratter of precipitation from solution, for there is certainly much more chromatin in a dividing than in a resting nucleus, and the chromatin often disappears from view in the latter condition. But a body endowed with certain properties wiH retain those properties in solution and emerge from solution with the same, and in a mixture of unlike bodies each wiU retain its own properties in solution and exhibit them afresh when reprecipitated. The chromatin granules are such bodies, and wo may weU suppose that they do retain their properties in spite of their disappearance. It does not foUow, however, that the granules are associated always in the same order to form chromosomes, though that may be so. Hence the chromatin granules may well retam their individuaUty while the chromosomes do not.


The chromatin, therefore, of those heterotypic cliroinosomes that now apj)ear may, on this view, bo regarded as identical with the chromatin of the prophases.



Fig. 42.- The maturation divisions in the female (Axolotl). 1, First polar spindle with heterotypic chromosomes ; 2, Extrusion of first polar body ; 3, Appearance of second polar spindle ; longitudinal division of chromosomes in egg and in first polar body ; 4, Second polar spindle radial ; homoeotypic chromosomes on equator (metaphase) ; 5, Polar view of the same ; 6, Anapliase ; 7, Extrusion of second polar body ; 8, Second polar body with resting nucleus ; 9, Female pronucleus in resting condition, closely surrounded by yolk-granules.

When the spindle is formed the chromosomes are placed on it and shorten and thicken (Fig. 41). The spindle then moves to the surface at the animal pole, Avhere it takes up a radial position, closely surrounded by yolk-granules. The actual maturation divisions now occur.



Fig. 41. - Germinal vesicle of the oocyte of the frog just before maturation (after Carney). The nuclear membrane has disappeared. The first polar spindle, bearing the heterotypic cliromosomes, is seen in the middle of the nucleus {p.s.). n., nucleoli ; v.m., vitelline membrane ; /., follicle ; th., theca.




Fig. 42. - Oocyte of mouse with heterotypic spindle from the Fallopian tuDe. ihe oocyte is still surrounded by the cumulus of follicle-cells.



The first maturaiion division (Fig. 42, 1, 2). The heterotypio chromosomes are placed upon the spmdio in the same way as in the male - that is, Avitli the extremities of the half -rings in the equators. The half-rmgs break away from one another and pass to the spindle poles. Cell-division now occurs. This is extremely unequal. The outer group of chromosomes, with a small quantity of cytoplasm, is cut off as the first polar body from the egg ; it lies in a depression at the surface. The inner group of chromosomes remain in a clear area in the egg, now the secondary oocyte.

The first polar spindle is found (in Siredon, and generally in Amphibia, also in Bnds) in the egg as it passes mto the oviduct. Li Mammalia - ^where it is also known to be heterotypic (Fig. 43) - ^it may be formed while the egg is in the ovary, or after it has passed into the Fallopian tube.

The first maturation division in the female evidently involves similar changes to those seen in the male : the prophases, the number and form of the chromosomes are all exactly the same. The interpretation of the manner of division of the chromosomes - ^whether longitudinal or transverse - which is adopted for the one, may therefore be applied to the other.

The second maturation division (Fig. 42, 3-9). Without passing into a resting condition the V-shaped chromosomes in the egg undergo longitudinal fission, as also do those in the first polar body. A number of parallel fibres, tangentially placed, now appear - the second polar spindle. The spindle is soon rotated into a radial position and the V-shaped chromosomes, already split, are thrown upon its equator with their apices towards the spindle axis, as in the male. Their number is, of course, n. The halves of the chromosomes then separate and pass to the spindle poles. Another miequal cell-division now occurs. The outer group of chromosomes, together with a httle cytoplasm and one or two yolk-granules, is extruded as the second polar body, while the inner group remain in the now mature ovum as the female nucleus, or rather pronucleus, to employ the more usual term.

In both the second polar body and the ovum the chromosomes break up, a membrane is formed round them, and the nucleus passes into the resting condition.


Since the chromosomes are V-shaped, are longitudinally divided, and are present in half the normal number, this division is evidently homoeotypic, as in the male.

The second polar spindle is formed as the egg passes down the glandular region of the oviduct (in Siredon and most other Amphibia). In the uterus the polar spindles are in metaphase (with the chromosomes in the equator). The division is not completed until after the egg has been fertilized (which is just after the egg is laid).

Where fertihzation is internal (Elasmobranchs, Birds, Reptiles, Mammals) the second polar body is extended while the egg is in the oviduct.

Although the chromosomes of the first polar body have divided, cell-division (in Siredon) does not usually follow. In other cases the first polar body does divide.

A centrosphere - if not an actual centrosome - is present at the poles of both the first and second spindles. In the mature ovum there is, however, no trace of it. The female pronucleus is immediately surrounded by yolk-granules (Fig. 42, 9).

Nature of the reducing division. We have already assumed for the purposes of illustration that the several chromosomes of a nucleus are genuinely different from one another. We may now add that there is experimental evidence (which we cannot discuss here) in support of this ; it is further probable that the granules of which each chromosome is composed are again of different values. Secondly, there are cases where the chromosomes are of different sizes (certain Insects), and in these cases they are found in pairs (in tissue- and in yomig germ-cells), the two members of a pair being of the same size. In the heterotypic division of maturation the members of the paks get separated from one another, so that each secondary spermocyte (and consequently each spermatid after the second homoeotypic division) receives a similar set of different-sized chromosomes.

Attention has akeady been called to the difference in size of the ring-shaped chromosomes in Siredon.

Now when a row of granules (or chromosome) is divided lengthways each half contains its due portion of each granule, and hence each daughter nucleus receiving half of each chromosome receives ipso facto a specimen of each different gi'anule. The two daughter nuclei are therefore alike and a longitudinal division of the chromosomes is merely quantitative.

If, on the other hand, the row of granules (or chromosome) is transversely divided, or, what is the same thing, if two different chromosomes are separated from one another, each daughter nucleus will not receive a specimen of each different granule or chromosome, but only one-half, the remainder passing to the other nucleus, and the division is qualitative.

The first condition may be represented by some such formula as this (where a-h are the quahtatively different granules in a chromosome, A,A',B, B', &c., whole chromosomes) : abcdefgh A A' B B' G C D D' abcdefgji' A A' B B' G C D D the line being the division, while the second condition will be represented by

abed A B G D or efgh A' B' G' D' Ordinary somatic mitoses are therefore quantitative, and so is the second homoeotypic maturation division. If, however, we adopt the view that in the heterotypic mitosis a transverse division of the chromosomes is involved, then we must further beheve that the division is quahtative, and consequently that the secondary spermocytes, and eventuaUy the spermatozoa, receive chromosomes of different kinds. Of every four spermatozoa produced from a single primary spermocyte, therefore, two wiU be aUke of one kind (containing, say, A, B, Sec), while two will be alike of another kind (containing A', B', &c.).

But it is evident from the foregoing that identical nuclear changes occur during maturation in the two sexes. The prophases of the first division- with the leptotene, synaptene, pachytene, and diplotene stages- are the same, and whatever view is taken of these phenomena must hold good for both sexes. In the female the growth period intervenes between the prophases and the actual division, but when this division occurs it is of the same form as in the male, heterotypic. The second division is homoeotypic in both sexes.

While, however, the cell-divisions are equal in the male- resulting in four spermatozoa - in the female they are unequal - giving one large ovum which receives practically the whole of the cytoplasm and the yolk, and three small polar bodies. The similarity of the nuclei shows that in spite of their small size these polar bodies are in reality potential ova, and there are cases where they are large - as large as the ovum - and can be fertilized and develope.

Like the spermatozoa, the ovum (and polar bodies) receives only one-half the somatic number of chromosomes. As we shall see more fully in the next section, these chromosomes form a complete set, as do those of the male. If- as is probably the case- there are varietal differences between hidividual spermatozoa in respect of these chromosomes, the same will be true of the ova.^

But what the further significance of these difEerences is, if they exist, we do not know. The chromosomes of the spermatozoon and ovum are certainly vehicles of inheritance - ^that is, concerned in the transmission of at least some of the inheritable characters of the species from one generation to the next. But since every spermatozoon or ovum can perform this function as well as every other, we are driven to conclude that each one possesses a complete set of the necessary specific chromosomes ; but that in different spermatozoa or ova the chromosomes may be of different varieties- that is, be concerned in the transmission of different varieties of the same inheritable character. This may be expressed by the followmg scheme. A B, G, D, Sec, are the n different specific chromosomes. In the tissue-cells and young germ-cells there are 2n, each kind bemg represented by two slightly different varieties, namely, A and A', B and B', G and G', &c. In the prophases of the heterotype division A and A' unite,^ and so B and B', G and G'.

In the actual heterotype division A and A', B and B', G and G' are separated from one another, so that each secondary spermocyte or oocyte has A or A', B or B', and so on.

1 Provided of course that priraary oocytes differ inter se in the arrangement and distribution of the heterotypic chromosomes.

2 If the union is by parallel apposition it is further possible to suppose that the individual granules of"^ which 4 and^' are composed pair ofi each with each, namely a with a', b with b', and so on.


The homoeotypic division is quantitative, hence each spermatozoon or ovum obtains A or A', B or B' , and so on ; that is, a complete set of the various kinds of chromosomes.

In only one respect are there chromosomal differences between the two sexes. In certain forms (Insecta), and possibly in others also, there is an accessory chromosome or heterochromosome (often paired), which not only differs in size and behaviour from the ordinary chromosomes, but is not the same in spermatozoon and ovum. The variations in the behaviour of this body or bodies are too complex to be discussed here, but those who have investigated it beheve it to be concerned in the determination of sex. Apart from the heterochromosomes and the varietal differences of the ordinary chromosomes, the germ-nuclei are exactly alike.

We have now to see how the two nuclei - each containing one-half the somatic number of chromosomes - are brought together when the germ-cella unite in the act of fertilization.


IV. Fertilization

The Axo\otl- 8iredon- wi\\ serve as a type (Fig, 44). The spermatozoon- which is of the same form as that of the newt and salamander - after passing through the mucin jelly surrounding the egg, reaches the surface of the latter. It approaches the egg with its anterior end- acrosome - and always in the pigmented animal hemisphere, sometimes near the equator, but more usually near the animal pole.

The acrosome pierces the surface-layer of the egg-cytoplasm, and immediately the egg reacts in a remarkable manner. From all sides there begins to flow towards the acrosome what appears to be a watery albuminous fluid : it is hyaline, but coagulable. This becomes concentrated round the acrosome in the form of a conical plug, the base of which projects at the surface, the apex towards the interior of the ovum (Fig. 44, a). This plug is the entrance-funnel, its base bemg known as the entrancecone (' cone of attraction ' is an erroneous expression, as it is not formed prior to the contact of the sperm with the egg). The entrance-funnel enlarges and extends more and more . into the interior of the ovum, being directed usually towards the axis : it carries in with it a number of the superficial pigment granules and the spermatozoon. The latter, therefore, after moving actively up to the surface of the ovum and penetrating it with its acrosome, is passively carried in by the inflow of the entrance-funnel ; this movement is apparently due to a difference in surface tension between the entrance-funnel and the surrounding cytoplasm. The acrosome presently gets caught in the side of the entrance-funnel, but the substance of the latter, still moving on, carries the head and tail of the sperm with it. The result is that the anterior end of the head now faces outwards, while the posterior end Kes at the bottom of the funnel, where the head is bent on the tail, and the whole sperm-head has been rotated through 180°. Between the head and the tail - and therefore now at the inner end of the funnel - is the large anterior centrosome (Fig. 44, c).


Fig. 44. - Fertilization in the Axolotl.

A and B. Meridional sections of the whole egg. a, Formation of entrancefunnel (first part of sperm-path), b, Formation of sperm-sphere and aster ; o3 male pronucleus ; ? female pronucleus ; p.b the t^^^ polar bodies.

c Formation of the sperm-sphere round the middle piece (anterioi centrosome) ; narts only of the head (black) and tail are sho^vn.

X. Formation of the sperm-aster. The centrosome has disappeared ; the head besinning to be vacuolated, is separated from the tail.

T'FuSr shortening and vacuolation of the sperm-nucleus. There is still no centrosome.


F, Appearance of the definitive centrosome. g, h. Division of thn centrosome.

(In c-H the arrow marks the direction of entrance of tlie spermatozoon.) I, Approach of the two pronuclei. Formation of spindle-fibres J, i?ormation of asters, elongation of spindle, further enlargement of pronuclei, and appearance of clu'omosomes.

K, Further elongation of spindle, and formation of a ccntrosnhere

irfhe'Toindr^r""" ^he pronudear men.branes are breakSg 5ow,x ana trie spindle-hbres passing in.

L, The fully.formed fertilization spindle. In the equator are the chromoomes, now longitudinally split, and attached to large spiiidle fi'bre Tu each centrosome the centriole has divided.



The entrance-funnel soon disappears, but the pigment carried in by it remains for some time as a streak, usually known as the first part of the sperm-path (Fig. 44, b).

A clear, yolk-free area now appears round the centrosome ; this is the sperm-sphere (Fig. 44, c). Very soon radial fibres or processes of some kind begin to pass out from the sphere amongst the yolk-granules ; this is the sperm-aster (Fig. 44, d). Meanwhile the head or sperm-nucleus has become detached from the tail, and the centrosome which was between them has totally disappeared. It seems that the formation of the sperm-sphere and aster- like that of the entrance-funnel- is due to the extraction of water from the cytoplasm, in the case of the entrancefimnel by the acrosome, in the present case by the centrosome ; and that the centrosome is completely used up, in fact dissolved, in the process.

The tail of the spermatozoon will not concern us : it degenerates and vanishes. The head of course remains to become the sperm-nucleus or male pronucleus. It shortens and thickens : as it does so it becomes vacuolated. By further shortening and vacuolation it becomes transformed into an ordinary nucleus (Fig. 44, E). It Hes on the outside of the sperm-aster.

It is at this moment that the definitive centrosome makes its appearance (Fig. 44, v). On the side towards the sperm-aster the nuclear membrane breaks down, and through the aperture something comes out of the nucleus which appears, when outside, as a rounded granular body. This is the definitive centrosome. It is not preformed in the sperm-nucleus and then ejected, but, probably, is due to a precipitation of the albumins of the cytoplasm by the nucleic acid of the sperm-nucleus. But, whatever interpretation be put upon the process, the centrosome is of male origm.

The male pronucleus, preceded by its centrosome and aster, now advances to meet the female pronucleus which has aheady left its position at the animal pole and is retm-ning towards the centre of the egg. The line in which the male pronucleus is now moving is knomi as the second part of the sperm-path. This does not necessarily lie in the same straight line, nor even in the same meridional plane as the first or entrance part of the path. This depends in part on the position of the female pronucleus (Fig. 46).

The first or entrance part of the path is usually directed towards some point in the egg axis, that is, it Hes in a meridional plane of the egg. If, as also is usual, the female pronucleus hes in the axis, it is evident that the second part of the sperm-path or line of union of the two pronuclei will he in the same plane. In that case it may be in the same straight line with the first part, or, more usually, make an angle with it, smce the pomt in the axis at which the pronuclei meet is at a fairly constant distance from the animal pole, while the point of entrance o the spermatozoon in the animal hemisphere is variable. If, however while the first part of the path is in a meridional plane the female pronucleus is not in the axis, then the sperm -nucleus must turn out of its meridional plane to meet the female pronucleus at some point which is not in the axis. The converse of this is seen when the entrance-path is not m a meridional plane while the female pronucleus is in the axis ; m this case also the sperm must turn aside. Thirdly, both sperm-entrance path and female pronucleus may be out of their normal direction

Afterwords, the meridional plane which includes or is parallel to the entrance-path does not necessarily coincide with the meridional plane which includes or is parallel to the line of union of the pronuclei.

During the advance of the sperm-nucleus the centrosome divides (Fig. 44, g, h) at right angles to the direction in which the sperm-nucleus is travelKng, that is, to the second part of the sperm-path, and also to the meridional plane in which the path lies. The daughter centrosomes therefore lie in a plane parallel to the equator of the egg. Hence, when the pronuclei have met, they lie together between the daughter centrosomes, which lie in a plane parallel to the equator of the egg.

The two pronuclei are now closely apposed, but not fused, inside the sperm-sphere and aster. Next, the centrosomes send out fine fibres in all directions (Fig. 44, i, j). On the one hand these impinge upon the pronuclear membranes - ^these are the begimiing of the fertilization spindle ; on the other hand they radiate out until they pass into the radiations of the original aster inside which they He.

The pronuclei enlarge, and presently in each granules of chromatin appear and run together in rows to form chromosomes (Fig. 44, j). The number of these in each pronocleus is the same as that which entered into it at the close of maturation, namely n, the germ-number. Meanwhile the asters round each centrosome have been growing larger, the spindle-fibres longer, and the latter now break through the pronuclear membranes to meet their fellows from the opposite pole (Fig. 44, k). The membranes, achromatic network, and nuclei are now all dispersed, and the two sets of chromosomes, paternal and maternal, are placed side by side on the equator of the fertilization spindle, where they undergo longitudinal fission as in ordinary mitosis (Fig. 44, l). Hence, when the daughter chromosomes pass to the spindle poles, each daughter nucleus will receive a complete set of paternal, and a complete set of maternal chromosomes. The full somatic number, 2 n, is now restored, and with each repetition of nuclear and cell-division each cell in the body comes to possess 2 w chromosomes, one-half of which are derived from the father, one-half from the mother. With the apposition of the two sets of chromosomes in the equator of the division apparatus - asters and spindle - the act of fertilization may be said to be complete.

The whole falls into two periods. In the first the spermatozoon is carried into the egg by means of the entrance-funnel, which in turn is due to a stimulus of some kind imparted to the egg cytoplasm by the acrosome ; the acrosome is the modified centrosphere. In the second the definitive centrosome is formed from the male pronucleus and the division apparatus made between its two halves while the pronuclei meet. The mechanisms involved in both periods are therefore centrosomal.

The details of fertilization have been studied in many animals, including several Vertebrates. In Vertebrates it is a rule for the sperm to enter during the second maturation division of the ovum, as in the Axolotl {Petromyzon, Salmo, Triton, Mus), but in other cases it may enter at an earlier or later period. The tail may be left outside {Mus), but is more often taken in : it always degenerates.

The pronuclei may fuse to form a segmentation nucleus, from which 2 n chromosomes arise {Pristiurus, Salmo, Petromyzon) ; but the newt and the mouse resemble the Axolotl in the separate formation of the chromosomes in each pronucleus.

It is certain that in all cases the female centrosome disappears. Whether the definitive cleavage centrosome is identical with the centrosome seen in the spermatozoon, that. is, in the spermatid, or is, as in the Axolotl, a new formation from the sperm-nucleus, is not certainly known, but there is little doubt that it is invariably a male centrosome.

As a rule only one spermatozoon enters the egg, and the presence of more than one leads to serious derangements of development (pathological polyspermy).! in what is known as physiological polyspermy, however, two or more, sometimes a great number, normally get in, as in some Amphibia (including the Axolotl), Reptiles, Birds, and Elasmobranch fishes, in which last they are very numerous and known as 'merocytes' (Riickert). In these cases only one of the sperm-nuclei fuses with the egg-nucleus. The remainder lie about in the yolk, each develops its own centrosome and aster, and may divide (with n chromosomes) many times. Ultimately the accessory sperm-nuclei degenerate without contributing to any embryonic structure.

1 As in the sea-urchin, where the several nuclei fuse and their chromosomes become irregularly distributed. Where, however as m the frog Srseveral nuclei remain apart the polyspermy need not cause abnormal ^eveToprenHM. Herlant, Arch, de Biol. xxvi. 1911) although the superfluous sperm-nuclei do take part in the edification of the embryo.


It remains for us to discuss the significance of fertilization.

It has commonly been supposed that its essence is to be found in the union of the pronuclei of the germ-cells, both nuclei being held to be necessary for the development of a normal individual. This view is based partly on the phenomena of conjugation in certain Infusoria, but also very largely on the assumption that the nuclei of the germ-cells are the sole vehicles for the transmission of inheritable characters ; this again rests upon the fact that it is only in their nuclei that the germ-cells are alike, while in every other respect they differ, and upon the supposition that the paternal and maternal contributions to the total inheritance are equal.

Now, whatever view we may take of the parts played by nucleus and cytoplasm respectively in the handing on of the characters of the species, it is most assuredly certain that for the production of a normal individual both pronuclei are not a necessity. In the first place, there is the phenomenon of parthenogenesis, natural and artificial. In the former the ovum develops without fertilization by the sperm and without artificial assistance (as in Aphidae and some other Insects, and in certam Crustacea). In the latter the stimulus usually given by the sperm is replaced experimentally by some physical or chemical agent. Thus the ovum of a sea-urchin or Mollusc may be stimulated by treatment with hypertonic sea-water, or butyric acid or other substance, or by mechanical shock, or a lowering of the temperature ; in the case of the frog it is sufficient to pierce the egg with a fine needle. In all these instances some physical or chemical alteration (or both) IS produced in the egg, as a result of which it begins to segment and develop. The process, if care is taken, may be perfectly normal, and the individual reach the adult condition A sexually mature (male) sea-urchin has been reared in this way In all cases of parthenogenesis only the female pronucleus is

The converse is seen in what is called merogony, where the egg (of a sea-urchin, Worm, or Mollusc) is divided into two halves, only one of wlucli contains the nucleus. Both halves can be fertilized, the nucleate and the enucleate, and will develop into normal larvae. In the latter case only the male pronucleus is present.

On the other hand, a nucleus must of course be present, and actual experiment has shown that what is really necessary for normal development is the presence in the ovum, and ultimately in every cell of the body developed from it, of a complete set of the n unlike chromosomes characteristic of the species.

Hence, both male and female pronuclei are not necessary, and we must look elsewhere for the significance of fertilization.

As we know already, the germ-cells of both sexes pass through two maturation divisions, and two only, after which their capacity for reproducing themselves is lost. The first effect, or almost the first effect, of their union is that their product, the fertilized ovum, begins to segment and continues to do so. In other words, the power of reproduction by cell-division which was previously lost is in fertilization restored. It is mutually restored.

That the ovum regains the power of nuclear and cell-division is obvious : we see the maternal chromosomes undergo longitudinal fission, as they lie on the spindle, and subsequently we see the egg cytoplasm divide. In the case of the male Ave see the male chromosomes divide in ordinary fertilization as they lie alongside the female ; in the fertilization of enucleate eggfragments the stimulus imparted by the female cytoplasm to the male chromosomes is still more evident.

A study of fertilization reveals the mechanism by which this stimulation is effected. For ordinary nuclear and cell-division an apparatus is necessary, the spindle with its asters ; this apparatus is made by the centrosomes in the cytoplasm, the two centrosomes proceeding from the division of one, and its function is first to pull apart the halves of the divided chromosomes, and second, to ensure cell-division by the cell-plate or intermediate bodies developed in the equator.

The mature ovum possesses no centrosome : the mature spermatozoon possesses little cjrtoplasm, and that only in the tail. In fertilization the centrosome is either introduced by the male cell or made by it after entering the egg : the necessary cytoplasm in which this centrosome can divide and make the asters and spindle is provided by the female. The wholly different structures of the two germ-cells are therefore mutually complementary in the stimulation by which the lost power of cell-division is restored, and this is the significance of fertilization.

The experiments on artificial parthenogenesis suggest that a physico-chemical expression may be found for this stimulus.

This is not, however, its only effect. A very common, if not universal, result of the approach of the sperm is the exudation by the ovum of a peri vitelline fluid. In some cases (for instance, the sea-urchin) a membrane which prevents the entry of more spermatozoa is secreted at the same time and pushed out by the peri vitelline fluid. In the frog it remains as a thin fluid layer between the ovum and the jelly ; it is the exudation of this fluid which enables the egg previously adherent to the mucin jelly to turn over till its axis is vertical and the white pole below : this occurs shortly after insemination.

Of greater importance than this is the change in the cytoplasmic structure of the egg brought about at this time.

A few hours after insemination there appears in the frog's egg a crescentic grey patch on one side along the border of the pigmented area (Fig. 45). The grey crescent is due to the immigration of pigment from the surface into the interior, and this in turn is caused by the entrance of the spermatozoon. The grey crescent always appears on the side of the egg opposite to that on which the sperm has entered. We know that a watery fluid flows towards the sperm from the cytoplasm (the entrancefunnel flrst, and later the sperm-sphere, are due to this), and we may suppose that this streaming movement drags the pigment granules away from the surface on the opposite side, whence the grey crescent.

The grey crescent is actually opposite to- that is, in the same meridional plane as- the first or entrance part of the sperm-path (Fig. 46). Hence it does not necessarily lie in the same meridional plane as that which includes the line of union of the pronuclei.

We shall see in the next chapter that the meridional plane of the first division always includes the line of union of the pronuclei, and hence does not always coincide with the meridional plane of the grey crescent.


It is clear that, whereas the unfertilized egg was radially symmetrical about its axis, it can now be divided into similar halves by only one plane, that which includes the axis and the middle point of the grey crescent. About this plane it is bi-laterally symmetrical. The greatest interest attaches to this alteration of symmetry, since the side of the grey crescent will become the dorsal side of the embryo, the side on which the sperm entered its ventral side. Since the animal and vegetative poles mark respectively the future anterior and posterior ends (approximately), it follows that the plane of symmetry of the fertiUzed but unsegmented egg coincides with the median longitudmal or sagittal plane of the future embryo. The whole bilateral symmetry of the embryo is now predetermined in the cytoplasmic structure of the egg.



Fig. 45. - Formation of the grey crescent in the frog's egg (R. ternporaria). a, b from the side ; c, d from the vegetative pole. In A, c there is no crescent, in b, d a part of the border of the pigmented area has become grey.


That the blastodisc has a bilateral structure in Birds and Elasmobranch fishes also seems to foUow from the fact that the cells in both these cases are larger at one end of the blastoderm than at the other. Further, this structure is definitely related to that of the embryo since the large-celled end becomes anterior.

Whether the change from the original radial to the definitive bilateral symmetry is in these cases also brought about by the spermatozoon, future researches must show.

In the Teleostei the concentration of the superficial cytoplasm (periblast) to form the blastodisc is an effect of fertihzation.



Fig. Diagrams to show the relation between the first and second parts of the sperm-paths. The paths are projected on a plane perpendicular to the axis. In a the two parts are in the same meridional plane, in B m different meridiona.1 planes. 1, First part of the sperm-path ; 2. Second part; o^, male pronucleus ; ?, female pronucleus ; ^.c, grey crescent : on the opposite side (side of entrance of the sperm) the superficial pigment Z rS; ' «,<^^ has divided in a plane perpendicular to thf a^ at right angles to the second part of the path. '


In conclusion we may attempt to estimate the parts played by the cytoplasm and the nucleus of the germ-cells in inheritance.


That some at least of all the inheritable characters of the species- and not only specific but varietal and individual characters as well- can be inherited from the father as readily as from the mother is obvious. Since the nucleus, beside the centrosome which is merely an organ of cell-division, and the acrosome which merely provides for the entrance, is the only part of the male cell which is always incorporated in the fertilized ovum for the tail may be left outside, we are obliged to regard the nucleus, that is, the chromosomes, as the vehicles by which tliese characters are transmitted.


The chromosomes of the nuclei of the germ-cells - which, as we have already pointed out, are different from one another - are in some sense the determinants of inheritance in the offspring : on their presence depends the ultimate appearance in the offspring of certain characters, and, in respect of their capacity for transmitting these characters, the two germ-cells are similar : each possesses a full set of the necessary chromosomes. In ordmary sexual reproduction the offspring receives two such sets, but one will suffice, as in parthenogenesis and merogony.

It does not, however, follow that the determinants for the whole of the inheritance are located in the nucleus.

As we have just seen, the material for the different parts of the body of the embryo is present in the cytoplasm of the fertilized but unsegmented egg ; to that structure the spermatozoon has . contributed nothing, beyond the rearrangement of material, the substitution of a bilateral for a radial symmetry. Experiment teaches us that the various parts of this structure are so many organ-forming substances, causally related to the development of certain organs, and therefore determinants of a part of the whole inheritance ; and recent researches on heterogeneous hybridization show clearly what this part is. The ovum of a sea-urchin, if the proper precautions are taken, may be fertilized by the sperm of a starfish, a feather-star (both of which of course are, like the urchin, Echinoderms), or even of a Mollusc or Worm. The result is always the same. A typical sea-urchin larva is developed. Even an enucleate egg-fragment will develop a little way when so fertilized, and exhibits the maternal characters alone. The spermatozoon employed does nothing but convey to the egg a stimulus, which sets the process in action ; its chromosomes sometimes persist, sometimes do not.


Hence the characters, the determinants of which reside in the cytoplasm, are the large characters which put the animal in its proper phylum, class and order, which make it an Echinoderm and not a Mollusc, a Sea-urchin and not a Starfish ; and these large characters are transmitted through the cytoplasm and therefore through the female alone. The smaller characters - generic, specific, varietal, individual - are equally transmissible by both germ-cells, and the determinants of these are in the chromosomes of their nuclei.


And yet the cytoplasm of the egg-cell is indebted very largely for its structure to the activity of the nucleus. As we have seen, the nucleus makes two contributions to the cytoplasm, first, the so-called ' yolk-nucleus ', the substances concerned in the deposition of the yolk, and second, the contents of the germinal vesicles dispersed when the latter breaks down at matm'ation. These processes are perhaps independent of the chromosomes. Further, they find no parallel in the male sex.

Even if, therefore, the cytoplasmic determinants are ultimately to be assigned to the nucleus, the share taken by the female in the transmission of the whole heritage is greater than the part played by the male.


Literature

W. E. Agab. The spermatogenesis of Lepidosiren paradoxa. Quart. Journ. Micr. Set. Ivii, 1911.

G. Belikens. Die Reifung und Befruchtung des Forelleneies. Aiiat. Hefte, x; 1898.

J. B. Caenoy et H. Lebeun. La v6sicule germinative et les globules polaires chez les Batraciens, La Cellule, xii, xiv, 1897, 1898.

J. B. Farmer and J. E. S. Moore. On the meiotic phase in animals and plants. Quart. Journ. Micr. Sci. xlviii, 1905.

K. Herfort. Die Reifung und Befruchtung des Eies von Pelromyzon fluviatilis. Arch. miJcr. Anat. Ivii, 1901.

'J. W. Jenkinson. Observations on the maturation and fertilization of the egg of the Axolotl. Quart. Journ. Micr. Sci. xlviii, 1904.

E. KoRSCHELT u. K. Heider. Vergleichende Entwicklungsgeschichte der wirbellosen Tiere. Allg. Th., Lief. 2, Jena, 1903.

F. Meves. Ueber die Entwicklung der mannlichen Geschlechtszellen von Salamandra mactdosa. Arch. mikr. Anat. xlviii, 1896.

F. Meves. Es gibt keine parallele Conjugation der Chromosomen ! Arch. Zellforsch. i, 1908.

T. A. Montgomery. The heterotypic maturation mitosis in Amphibia and its general significance. Biol. Bull, iv, 1903.

A. Oppel. Die Befruchtung des Reptihencies. Arch. mikr. Altai, xxxix,

J. RijOKEET. Zur Bofruchtung des Selacliioroies. Ami. Am. vi, 189L A. u. K. E, SoHEEiNER. Uio Rcif ung der maiinlichen Geschlechtszelleu

von Salamandra maculosa, Spinax niger und Myxine gluHnosa. Arch, de Biol, xxii, 1906.

J. SoBOTTA. Dio Befruchtung und Furohung des Eies der Maus. Arch, mikr. Anat. xlv, 1895.

E. B. Wilson. The cell in development and inheritance. New York, 1902.

H. VON WiNrwABTER. Rechcrches sur I'ovogentee et I'organogcneso de I'ovaire des Mammif^rea. Arch, de Biol, xvii, 1901.



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Jenkinson JW. Vertebrate Embryology. (1913) Oxford University Press, London.

Vertebrate Embryology 1913: 1 Introduction | 2 Growth | 3 The Germ-Cells, their Origin and Structure | 4 The Germ- Cells, their Maturation and Fertilization | 5 Segmentation | 6 The Germinal Layers | 7 The Early Stages in the Development of the Embryo | 8 The Foetal Membranes of the Mammalia | 9 The Placenta | Figures

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