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LIBRARY

AT

CORNELL UNIVERSITY

 

JAMES E. RICE
MEMORIAL
POULTRY LIBRARY

 
Pl

192
The University of Chicago

“ FOUNDED BY JOHN BD, ROCKEFELLER

THE FERTILIZATION AND EARLY DEVELOPMENT
OF THE PIGEONS EGG |

he DISSERTATION

SUBMITTED: TO ‘THE FACULTIES OF THE GRADUATE. SCHOOLS oF ARTS,
. LITERATURE, AND SOIENCE, IN CANDIDACY. FOR THE |.
‘DEGREE, OF DOCTOR OF PHILOSOPHY ‘

- ' ZOOLOGY

4 BY a
_ EUGENE HOWARD HARPER

‘

CHICAGO
1902
The University of Chicago

FOUNDED BY JOHN D, ROCKEFELLER

THE FERTILIZATION AND EARLY DEVELOPMENT
OF THE PIGEONS EGG

A DISSERTATION

SUBMITTED TO THE FACULTIES OF THE GRADUATE SCHOOLS OF ARTS,
LITERATURE, AND SCIENCE, IN CANDIDACY FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY

ZOOLOGY

BY
EUGENE HOWARD HARPER

CHICAGO
1902

D
EIS5312

CQL
al
WA4
THE FERTILIZATION AND EARLY DEVELOPMENT OF THE
PIGHON’S EGG.

BY
EUGENE HOWARD HARPER, Pu.D.

With 4 DousLe PLates AnD 6 DIAGRAMS IN THE TEXT.

TABLE OF CONTENTS.

Introduction.
Methods.
Some observations on the breeding habits of the common pigeon.
The fertilization of the egg and its passage through the oviduct.
Some late stages of the ovarian egg.
Nucleoli in the ovarian egg.
The fertilized egg:
Maturation divisions.
Fertilization stages.
Early cleavage stages. ;
Development of the accessory nuclei (diagrams 1-6).
The yolk nuclei of later cleavage stages.
Polyspermy in other eggs.
Some features of mitosis in the pigeon’s egg.
Conclusions.
Bibliography.
Description of plates.
Plates.

The investigation, the results of which are here described, was pro-
posed to me by Dr. C. O. Whitman, and the work has been carried on
under his direction, being designed as part of a more general work
upon the Natural History of Pigeons. My thanks are due to Prof.
Whitman for his encouragement and suggestions and his assistance in
obtaining the material.

In this paper the aim has been to get a view of that period of develop-
ment of the bird’s egg which has hitherto been scarcely touched upon,
including thé maturation, fertilization and early cleavage. Material was
obtained from only one species, the common pigeon, Columba livia
domestica. On account of the prolonged breeding season of pigeons
and the ease with which they may be kept in confinement, they are
2 Fertilization and Early Development of Pigeon’s Egg

certainly better adapted to furnish material for studies of this sort
than any other bird.

About the early development of the large meroblastic eggs compara-
tively little is known. This has remained true in spite of the thorough-
ness with which the embryonic stages of selachian and chick have been
studied. As a result of the work of a number of investigators, chiefly
Riickert, there is now a fairly complete general survey of the fertilization
and early stages of the selachian egg. Observations upon the early
development of the bird’s egg are very few. Some of the early cleavage
stages of the chick were figured by Coste, and Balfour contributed some
observations. The internal phenomena of the egg during maturation,
fertilization and early cleavage have remained an open field for investi-
gation. .

Upon the ovarian history of the bird’s egg observations have been
quite numerous. The paper of Holl, go, upon the hen’s egg may be
mentioned as one of the most important.

The development of the large meroblastic eggs obviously presents
numerous problems. In this paper the stages of the egg obtained are
scattered over a considerable period, and present glimpses of various
phases of maturation, fertilization and early cleavage. A few stages
of the ovarian egg have also been introduced.

METHODS.

The method followed has been to fix the whole egg before attempting
to remove the germinal area. The oviduct is removed, the position of
the egg being carefully noted, as this enables one to judge the approxi-
mate stage in development and determines the subsequent treatment in
staining. The portion of the oviduct containing the egg is then cut off,
immersed in the fixing fluid and slit open underneath the liquid. In
case of an egg which is free in the body cavity, with some caution the
body may be inverted over the fixing fluid, allowing the egg to drop
out. The large ovarian egg may be fixed long enough to allow the fluid
to penetrate the disc, then hardened in alcohol and the germinal area
subsequently dissected out.

The choice of fixing fluids is somewhat limited, since many of them
leave the disc too brittle to stand the subsequent treatment, and washing
in water is undesirable. The picro-acetic mixtures have been chiefly
used. Long fixation is not necessary or desirable, owing to the swelling
of the yolk, which is apt to distort the disc.

It is well to cut out a considerable portion of the surrounding yolk
with the disc and then to float this piece into a shallow watch-glass and
a

Eugene Howard Harper 3

allow it to remain with the convex surface down in the watch-glass
through the washing and hardening treatment. Lying on a flat surface
tends to warp and often crack the disc. It should be trimmed evenly all
around to overcome the tendency to curl in one direction. Occasionally
the egg membrane will come off easily before or even after fixation.
If not, the sharpness of the knife must be depended on to overcome this
difficulty. Of course the knife should strike the inner side of the disc
in its descent.

The abundance of the yolk and its obscuration of other structures
would seem to make it desirable to use a stain which should mask the
yolk as much as possible. In all but the fertilization stages the nuclei
are surrounded by areas tolerably free from granules, and this is especi-
ally true of the sperm nuclei in their later divisons, which are sur-
rounded by very large granule-free areas. For this reason the iron-alum
hematoxylin stain is workable, and possesses besides an advantage in
differentiating certain areas in the cytoplasm during its amceboid changes,
which are less conspicuous with a stain which masks the yolk. The. dif-
ferent degrees of extraction of the stain in the different areas of the
cytoplasm is a highly desirable feature.

SOME OBSERVATIONS ON THE BREEDING Hasits or THE ComMMON
PIGEON.

The fact that the pigeon breeds so readily in confinement makes
possible a close observation of its breeding habits. As is well known,
the special instincts displayed in connection with reproduction are more
highly developed in the pigeon than in the common fowl. These com-
plex instincts are associated with monogamy, which reaches a type of
development in the pigeon which is very high among birds. For
example, the feeding of the young with “pigeon milk” may be men-
tioned. It is only with the earlier manifestations of the reproductive
instincts prior to egg-laying that we are here concerned.

It might be supposed that in the case of a domesticated bird breeding
readily in confinement, such as the pigeon, some approach might be
made toward an exact method for determining the time of fertilization
of the egg. The time of egg-laying is approximately definite, as all
breeders know. The common pigeon ordinarily lays two eggs at a sit-
ting, occasionally only one. The first egg is regularly laid late in the
afternoon. The second egg will be laid early in the afternoon of the
second day following.

It is evident that the determination of the time of fertilization of the
second egg of the pair and the length of time taken in its passage
4 Fertilization and Early Development of Pigeon’s Egg

through the oviduct would be a simpler matter than to determine from
external signs when the first egg is fertilized. It has been found that
after the first egg is laid, in the course of a very few hours the second
egg becomes detached from the ovary, is fertilized, and passes into the
oviduct.

As stated above, the first egg is laid late in the afternoon. Larly in
the evening the second egg becomes free from its capsule in the ovary
and enters the oviduct. In all cases observed this has taken place
between seven and nine o’clock. The time taken in passing down
‘the oviduct is relatively short, the far larger part of the time which
elapses before the egg is laid being spent in the lower portion, known as
the uterus, or shell-gland. It is evident that the second egg of a pair
may be obtained at approximately any stage desired, beginning with a
period a few hours before its fertilization.

The question arises whether there may be any criteria found for judg-
ing the time of fertilization of the first egg. It might be thought from
analogy with the mammalia that the time of copulation would furnish
such a criterion. It is quite plain from the regularity of the history
of the second egg, as given above, that the exact period when the egg is
freed from its capsule is dependent upon the female organization, and
would be likely to occur at some definite period, probably at night. A
moment’s thought would, however, make it plain that it is highly
improbable that a periodical receptivity, or period of heat, should be
displayed by the female at this time. Experience of the writer has
shown that any violent movement of the animal at this time is likely
to result in a broken egg. Of course, such an egg as the bird’s cannot
be retained in the oviduct to await fertilization. Sperms are stored
in advance, and the critical passage of the egg, after leaving its tough
capsule in the ovary, through the oviduct till it acquires its coating of
albumen and a shell, occurs at night when there are no movements
of the animal to endanger its safety. The period of receptivity of the
female is prior to this series of events. Copulation is repeated so often
that no definiteness could be attached to it as a criterion. The question
then arises whether the period of receptivity of the female has any
definite duration, so as to indicate in this way when the maturation of
the egg is taking place. From analogy with the mammal and with
many birds, such as the common fowl, we commonly think of ovulation
as exclusively a female function, going on regardless of whether the
eggs produced are fertilized or not. Thus the common fowl produces
unfertilized eggs regularly in the absence of a male. In the pigeon,
however, ovulation is delayed until mating. When a mature pair ready
Eugene Howard Harper 5

for mating are put together, egg-laying ordinarily ensues at the end
of a rather definite period, at the least eight days. The female functions
are held in abeyance till the proper stimulus is received from a mate.
The maturing of the egg is so exclusively a female function that it
seems odd at first thought that an apparent exception should occur to
the rule. Of course, we know that the final maturation of the egg, or
the giving off of the polar bodies, awaits in most animals the act of
fertilization. But here the effect is. produced upon the egg by the
entrance of sperms. How mating itself and the act of copulation could
influence the ripening of the egg in the ovary is another problem. In
this connection the curious fact must be mentioned that two female
pigeons placed in confinement together may both take to laying eggs.
The function of ovulation is in a state of tension, so to speak, that
requires only a slight stimulus, “mental” apparently in this case, to
set the mechanism to working. At any rate, it is impossible to regard
the presence of sperm in the oviduct as an essential element of the
stimulus to ovulation, although it may have an important influence in
the normal case. Our attention is directed to the various and complex
instincts of the male which come under the head of courtship, both
before and after mating is effected, as furnishing a part of the stimulus
to the female reproductive organs.

Phylogenetic considerations would lead us to consider the peculiar
habits of the pigeon as recently acquired. The retention of ova in
the unmated female, is in particular not very firmly fixed, as the facts
stated show. The habits of the common fowl are certainly more primi-
tive. In monogamous birds it might be expected that the function of
ovulation would be adjusted so as to take place only after mating, inas-
much as it is probable that in a state of nature mating may be delayed
for various causes, and the production of an unfertilized egg is no trifling
loss, as in the mammal. In polygamous birds mating is sure to occur, and
the female functions may be adjusted for continuous ovulation, with
the practical certainty that in nature no unfertilized egg will be pro-
duced.

The complex reproductive instincts of the pigeon, displayed in their
highest form in the male, are matters of common observation among
those who have observed pigeons, and need not be dwelt upon at great
length. :

As is well known, the strutting of male pigeons is not simply a fea-
ture of courtship and rivalry among males. It is continued until egg-
laying begins, and is accompanied by a less active similar manifestation
by the female. It is in fact an accompaniment of the whole period
6 Fertilization and Early Development of Pigeon’s Egg

from mating to egg-laying, during which copulation is of frequent
occurrence. .

There is an act which regularly precedes copulation, in which there
is an apparent regurgitation of some secretion by the male which is
taken from his throat by the bill of the female, in somewhat the same
manner as the young birds take their food. It is a less violent mani-
festation than the feeding of the young, however. It is easy to see
that here may be one of the sources of indirect stimulation to the female
reproductive organs.

The male has the habit of frequently taking to the nest and calling
the female by emitting a low growling noise and gently vibrating his
wings. It is evident from a consideration of the complexity of these
and other instincts, such as nest-building, that the initiation of repro-
ductive activity in the female can ordinarily only be dated from the
time of mating, or from the resumption of activity by an already mated
pair. The female pigeon is either a very dull bird or a very exacting
one, requiring constant attention and flattery to rouse her to her proper
functional activity, or else the male must be accused of greatly magni-
fying his office.

There is a possibility that the nesting habits of the female could be
used as a clue to the time of egg-laying. The female has the habit of
sitting on the nest occasionally for some time before the first egg is laid,
but in practice this has not been found to give sufficiently definite data.

No certain method has been found for determining the time of ferti-
lization of the first egg. By making use of the second egg, any stage
after or shortly before fertilization may be obtained. This method has
the disadvantage of yielding only one early stage of the egg from each
bird. The first egg when laid has reached the ‘close of the segmentation
period. The second egg would remain in the oviduct nearly forty-eight
hours after the first was laid. To obtain a series of the late ovarian eggs
is more a matter of chance.

In elasmobranchs and reptiles a considerable number of eggs are
found in the oviduct and all in nearly the same stage of development.
The greater certainty with which the pigeon’s egg may be obtained is a
compensatory feature, when we are considering the relative difficulty of
obtaining material for a study of these forms.

THE FERTILIZATION OF THE Ecc AnD Its PassaGE THROUGH THE
Ovipuct.

The passage of the egg through the oviduct until it acquires a thin
shell within the lower portion, or shell-gland, is a nocturnal function.
Eugene Howard Harper v

That, as such, it is adapted to secure the safety of the egg is evident
from the thinness of the egg membrane when it leaves its tough ovarian
capsule and its consequent liability to be ruptured at this critical period.
Careful handling is necessary to secure the egg at this time. The cap-
sule of the egg splits along the pole opposite to its attachment in the
ovary. A gradual thinning out of the capsular wall occurs along the line
of splitting, causing a pale streak across the egg. The vessels of the
capsular wall are at this time highly charged with blood. Two such
pale streaks across the egg have been seen at right angles. During the
rupturing of the capsule, the egg bulges out in various places, producing
an irregular appearance with several protuberances. Inasmuch as the
wall grows quite thin during the process, it is quite possible that the
spermatozoa may be able to penetrate and reach the germinal vesicle
before the egg leaves its capsule: When the egg escapes, it is found well
surrounded by a thin albuminous liquid with which the body cavity at
this time is charged. It is like the albuminous secretion of the oviduct,
except that it is much thinner. This liquid serves both as a medium
for the spermatozoa, as stated by Balfour, and as a support to the egg
at this critical juncture, when it is invested by only a very thin membrane.

The egg membrane or yolk membrane is about 3.54 in thickness.
The outer margin of the cytoplasm is somewhat denser and also takes on
something of the character of a membrane. In some preparations this
is found actually separated for a little way from the underlying cyto-
plasm. But for the most part it appears like a very thin non-separable
layer.

The egg membrane appears structureless. It seems to increase in
tenacity, since, when the egg is first set free from its capsule, it is very
easily ruptured. The flattening of the yolk from its own weight in the
fixing fluid is enough to cause the rupturing of the membrane. The
increase in tenacity later may be the effect of the deposition of closely
adhering layers of albumen.

The egg is clasped by the funnel-like mouth of the oviduct, which at
this time has been observed to display active peristaltic contractions, as
if in the act of swallowing the egg. The contractions were confined to the
funnel portion of the oviduct. The fact as stated rests upon a single ob-
servation. As the transition from the funnel to the glandular portion
is abrupt, it would seem that the egg must be engulfed by muscular con-
traction, but after it is within the glandular portion of the oviduct it is
driven simply by ciliary action along its spiral course through the ovi-
duct, as has been stated by Cushny, 02, in regard to the hen’s egg. The
peristaltic motions were sufficiently active to be unmistakable. The
8 Fertilization and Early Development of Pigeon’s Egg

desire to obtain the egg interfered with the continued observation of the
movements, and it is not known how long they might continue. Morgan,
97, states that the old view that the frog’s egg is swallowed by peristaltic
motions of the infundibulum of the oviduct is probably mistaken, and
that the egg is doubtless driven along its entire course by ciliary action.
The oviduct of the pigeon is usually from twelve to fifteen inches in
length, but sometimes over twenty inches. The funnel portion or infun-
dibulum is less than one-fourth of the entire length; the glandular por-
tion which secretes the albumen is a little less than one-half of the
ordinary length. The remaining portion, the uterus or shell-gland,
is separated from the preceding part by a definite constriction.

The entrance of spermatozoa is previous to the time when the egg
is clasped by the funnel of the oviduct. An egg at this stage contains
numerous sperm nuclei which have undergone considerable transforma-
tion and others in various early stages of transformation. Hence
the entrance of spermatozoa must take place as soon as the germinal disc
is exposed by the rupture of the follicular wall. This may be while the
egg is still attached to the ovary, but the point has not been definitely
ascertained.

The stage of development reached by the egg at any time is indicated
approximately by its position in the oviduct. Thus the polar bodies are
given off within the proximal part of the glandular portion, and cleav-
age begins just about as the egg enters the shell-gland. The passages
through the upper portion of the oviduct in which the albumen is secreted
is relatively rapid. The following table gives some data for an estimate
of the time.

Beginning of first maturation division............. 7:40- 9:00 P.M.
- “ second “ SS Sheaanigeaihw es 7:45-10:15 P.M.
ee “ first cleavage SO Sospta Wee Gren aiars 10 :30-12 :30 P.M.
ee “ second “ APY hae ales as Eac mesg 12:30- 1:00 A.M.

From such data only a rough estimate can be made as to the time
elapsing between the impregnation of the egg and the first cleavage.
Balfour, 85, states that in the fowl cleavage of the egg begins just
before it enters the shell-gland. There is consequently a close simi-
larity between the pigeon and the fowl in this respect. Since the abso-
lute length of the oviduct varies in different birds, it would hardly be
expected that the same relative position in the oviduct would generally
be reached by the egg at the same stage of development. As a matter of
fact the observed cases so far have been so close to the average as to
furnish no evidence as to variation in this respect.
Eugene Howard Harper 9

Some Lars Straces ofr THE Ovarian Ecc.

The growth of the ovarian egg may be roughly divided from one point
of view into two periods. The first is a long one of very slow growth,
the second is a short period in which the main increase in size of the
egg is effected. Two eggs mature at a time, occasionally only one. The
second pair in order of development are usually quite small, the larger
being ordinarily several millimeters in diameter when the first pair are
mature, but it may be half-grown occasionally at this time.

The full-sized egg in its capsule is nearly an inch in diameter. In
Fig. 1 is shown the nucleus of an egg 1.4 mm. in diameter. It measures
238u in diameter, and, being nearly spherical, is greater in volume than
the nucleus of an egg which was in the midst of its rapid growth and
15 mm. in diameter. The latter nucleus is lens-shaped, flattened against
.the follicular envelope, and its diameter is 378, (Fig. 3). In a smaller
egg 12 mm. in diameter the nucleus is shown in horizontal section
(Fig. 2b). Its greater diameter is 3294. The ground substance of the
nucleus or germinal vesicle is of a finely alveolar character, appearing
under a low power to contain only a few scattering deutoplasmic granules.
Under a higher power, however, it is seen to be thickly studded with
microsomes of the same character as the larger granules. The chromo-
somes are in a group in a somewhat eccentric position, surrounded by a
system of radiations. Apparently pairs of dumbbell-shaped dyads are
lying side by side. They are unequal in size, three of the pairs being
considerably larger. Two of the pairs are crossed, lying very close
together (Fig. 17). There are numerous glistening refractive bodies
scattered among the chromosomes, some small and some in vesicular
masses. At the center of the germinal vesicle is a considerable amount
of chromatic staining material in the form of short threads, and also a
group of rounded bodies like nucleoli lying underneath them. The
nucleoli in some cases have short remnants of chromatic threads clinging
to them.

A later stage is shown in Figs. 4a and b. In Fig. 4a the whole ger-
minal area of this egg is shown, the germinal vesicle enlarged in Fig..
4b. This egg was the older of a pair, the nucleus of the younger of
which is shown in Fig. 3. A comparison of the size of the nuclei shows
a great diminution. The wall of the nucleus is seen to be breaking
down. Its contour is no longer regular, but it has shriveled up and
retreated from its manifestly former position. The disintegrating wall
is surrounded by a zone of the nuclear ground substance. The diameter
to the outer limits of this zone is 2104, showing an invasion of the yolk
10 Fertilization and Early Development of Pigeon’s Egg

into the area formerly occupied by the germinal vesicle. The retreat
of the nuclear wall is unlike ordinary plasmolysis from fixing agents.
There is no vacant space left from the shrinking of contents. Such
plasmolysis is evident in the case of the younger egg of the pair, Fig. 3,
indicating a more watery condition of the nucleus in the younger and
still rapidly growing egg. In this egg there are no remnants of threads,
except for slight indications upon the more or less rounded nucleoli.
The refractive bodies previously mentioned are present. The chromo-
somes are shortened as compared with the former instance and are irregu-
larly placed. The deutoplasmic microsomes in the nuclear ground sub-
stance are less prominent owing to a greater extraction of the stain.

Underneath the germinal vesicle is a core of lighter staining material,
extending inward to the bed of white yolk. The whole germinal area
under a low power appears very finely granular compared with the coarse
underlying yolk.

The next stage obtained is that of an egg which in the ordinary course
of events would have become free from its capsule and passed into the
oviduct in the course of several hours. To be more definite in this
instance, the egg was taken from the ovary at 7:00 P.M., the first egg
having been laid in the afternoon.

The cross section (Fig. 5) shows the equatorial band of chromosomes
lying obliquely to the surface of the egg at the margin of the deutoplasmic
area. There is an accumulation of a liquid substance at this point
between the follicular wall and the granules, which, as in other eggs, may
be called the perivitelline liquid.

The areca occupied by the germinal vesicle is obliterated. There is
very little if any difference in the appearance of the germinal area at this
point, except for the perivitelline liquid above mentioned, and a much
greater accumulation of a substance having seemingly the same character
directly underneath the finely granular layer. This body of more liquid
protoplasm appears in structure and staining properties like the con-
tents of the former germinal vesicle. It fills a wider area, however,
and is bounded beneath by the coarsely granular yolk. Two eggs were
obtained at this stage, both showing the same appearance. It does not
appear like an artefact, and though peculiar to this stage of the egg,
it seerns to be definitely related with later changes in the fertilized egg.

No stages were obtained between those shown in Figs. 4b and 5. Fig.
5 shows the acme of development of the ovarian egg. If the accumu-
lation of granule-free protoplasm underneath the granular layer of the
disc is derived from the contents of the germinal vesicle, as its appear-
ance would indicate, it. seems as if a centripetal movement of this sub-
Eugene Howard Harper 11

stance had taken place simultaneously with a lateral invasion of the
granular protoplasm. Possibly the path of this centripetal movement
is indicated directly beneath the contracting germinal vesicle in Fig. 4b.
Fig. 5 represents the stage supposedly when activity in the egg has
reached a minimum. When activity is resumed in the maturation stages
attention will be called to the fact that the underlying bed of granule-
free protoplasm has disappeared and in its place is a cone-shaped active
area (Fig. 8) extending clear to the periphery of the egg with the spindle
at its apex. This would seem to indicate a centrifugal movement of the
granule-free area at the time of giving off of the polar bodies.

The spindle is fully formed in this egg, although its oblique position
makes it difficult to recognize the achromatic structures. The equa-
torial band of chromosomes shown in Fig. 18 is from the other of the
two eggs above mentioned, and the section was almost parallel with the
equatorial plate. The chromosomes appear as tetrads of unequal size.
There is an appearance peculiar to the first polar spindle to which atten-
tion is called. There are within the circle of chromosomes and lying
in the same plane, a number of deeply staining granules at the nodes
of the linin network. They plainly differ from the deutoplasmic granules
outside, having the staining properties of chromosomes or centrosomes.
There are four or five especially large ones at the center.

Nucleoli in the Ovarian Hgg.—The nucleoli which have been described
in this later part of the ovarian history are, as has been stated, evidently
derived from a chromatic network which becomes aggregated into rounded
masses, and these soon undergo dissolution in the form of refractive
bodies. Lebrun, 02, describes nucleoles derived from the granular chrom-
atin which are present at the first appearance of maturation in the egg
of Diemyctilus and speaks of their propensity to fuse together.

In the amphibian egg, King, 01, mentions the occurrence of such
refractive bodies in the germinal vesicles. They are described as “ vel-
lowish green refractive bodies,” which result from the disintegration of
nucleoli. It seems likely that the small refractive bodies among the
chromosomes are remains of nucleoli which are nearly disintegrated.
The larger aggregations of refractive bodies and nucleoli found else-
where in the germinal vesicle are in an earlier stage of the disintegrating
process (b and ¢ in Fig. 4b).

The nucleoli which change to refractive bodies and disappear have a
different fate from the nucleoli in the previous history of the germinal
vesicle. According to Carnoy and Le Brun, in the amphibian egg the
nucleoli are aggregations of the chromatin network, which at definite
periods break down and give rise once more to a chromatic thread. They
12 Fertilization and Early Development of Pigeon’s Egg

are a resting stage of the chromatin. According to the opinion of Wil-
son, 00, such nucleoli are to be regarded as chromatin masses distinct
in nature from true nucleoli or plasmosomes. In the smaller pigeon
ova such net-knots of chromatin are frequently seen, looking like the
beginning of the formation of a nucleolus or the contrary, the unrolling
of one to form a thread (Figs. 1a and b). But better evidence on the
nature of these chromatin nucleoli may be obtained from other material
than the pigeon. Without going too far afield from the purpose of this
paper, it may be mentioned that the ova of the sparrow in the winter
condition give an excellent example of chromatin aggregated into the
form of nucleoli. There is an almost entire disappearance of the chro-
matin network and a large and variable number of nucleoli having the
staining reaction of chromatin. This phenomenon is accompanied by
a watery condition of the nucleus as shown by the great plasmolyzation
from fixing agents. The eggs may be fixed so as to show no distortion,
but the nuclei are invariably plasmolyzed.

This condition disappears when the growing season recommences in
March. The chromatin threads reappear and the nucleus is no longer
so easily plasmolyzed. The difference between the pigeon and the spar-
row ova is accounted for by the fact that the ovary of the sparrow is
in a resting state in the winter, while the breeding season of the pigeon,
in comfortable quarters, is continuous except for a slight cessation in
the fall, during moulting. If the view of Carnoy and Le Brun is correct,
the evidence for the continuity of the chromosomes through the ovarian
development alleged by Born, 94, must be mistaken.

A frequent appearance found in the pigeon ova was that of pale,
broken-down nucleoli, looking rather like the “shells” of nucleoli, either
inside the nucleus or outside close to the membrane. This appearance is
entirely different from that of the previously described refractive bodies.
Such bodies are described by Carnoy and Le Brun in the amphibian egg.

Tue FeErtiuizep Eaa.

Maturation Dwisions—The earliest stage of the fertilized egg obtained
is shown in horizontal section in Figs. 6a and b. The surface appear-
ance of the disc of such an egg is shown in Fig. 6. The slightly oval
disc has a greater diameter of 3.5 mm. It is divided into two zones
quite clearly distinguished in opacity, the outer zone being due to the
abrupt thinning out of the fine granular matter of the disc. With a
hand-lens the region of the nucleus may be made out in the living egg
as a spot surrounded by a lighter ring or halo, the “ fovea.” The whole
affected area surrounding the nucleus is shown in Fig. 6a. The nucleus
Eugene Howard Harper 13

is in the center of a granular area which is surrounded by a hyaloplasmic
zone. The inner ring of the zone of hyaloplasm is quite free from gran-
ules and here the sperm nuclei are imbedded. Outside of the clear ring
the cytoplasm is less densely granular and there is an appearance of
watery rays or channels passing out. The diameter of the affected area
is about .5 mm. One side is more vacuolated and hyaloplasmic than the
other, which fact will be recalled in connection with later appearances
during development. There is a rather clearly marked ring which is
not like the hyaloplasmic ring in appearance, but is filled with a ground
substance having a more finely alveolar structure. This ring, which
may be called the “polar ring,” will be better described in connection
with vertical sections of the disc. The sperm nuclei shown in this
section are in an advanced stage of transformation, and their identity
with entering sperms must be discussed further later on.

The egg nucleus is in the equatorial plate stage (Fig. 19). There are
eight apparent tetrads (pairs of dyads) in a ring, the diameter of which
is greater than in the mature ovarian egg. The chromosomes also are
larger, and are of unequal sizes as before. The central spindle granules
are present, lying in the plane of the chromosomes, at the nodes of the
linin network. The central group of larger ones is conspicuous, as in
the previous instance. It may be again stated that these granules have
not been found in any other spindles than the first polar, and, as already
mentioned, they present a similar appearance in the four observed cases.
They would seem to be concerned in some way with the formation of the
first polar spindle, and may indeed be condensations of the linin network
at the foci of the system of radiations surrounding the group of chro-
mosomes before the formation of the spindle (see Fig. 17), though this
may sound like a rash suggestion, since the stages in the formation of
the spindle are yet to be observed. If they are “accessory” chromatin
material, they evidently do not undergo dissolution like the chromatin
nucleoli.

The nuclei embedded in the hyaline zone are all of similar structure
and staining properties. They vary in size from four to seven ». They
are of irregular shapes and do not have any bounding membrane. No
asters have been found, but want of material has prevented the use of vari-
ous staining methods. Those which have penetrated deeper are somewhat
larger. Stages in the transformation of the sperms are shown in Figs.
Ya-h. The entrance of sperms seems to take place anywhere within the
affected area, but those which enter the hyaline zone seem to undergo
a more rapid development. The fate of the great majority of sperms
can best be inferred in lack of direct evidence from the numbers found
14 Fertilization and Early Development of Pigeon’s Egg

in the fertilization stages, where in the cases obtained the number was
from 12 to 25. Apparently those not entering at the right time and
place meet with some unfavorable influence hindering their development.

In the perivitelline liquid are found numerous large cells from the
follicular membrane of the ovarian egg (Fig. 7i). These cells are with-
out walls, but the nucleus and accompanying body of cytoplasm bears an
unmistakable resemblance to the follicular cells. Associated with them
are found also blood corpuscles. From their size and appearance they
may be distinguished from the sperm nuclei, since they are of a much
greater order of magnitude. There is much nuclear debris which is evi-
dently derived from these cells also present in the perivitelline space,
showing that their fate is to degenerate. Riickert, 99, has found the same
“inwandering follicular cells” in the selachian egg.

A vertical section of the germinal disc during the maturation stage is
shown in Fig. 8. The egg was taken from near the beginning of the
oviduct. The section contains the second polar spindle, and first polar
body, situated at a slight depression in the surface of the disc. The
vertical section of the germinal disc shows that the central or affected area
has the shape of a cone with the spindle at the apex. The distinguishing
characteristic of the affected area which mark it off from the surrounding
homogeneous appearing disc, is its lighter staining property. This seems
due both to the relative fewness of the granules and the greater extraction
of the stain from those present. The protoplasmic ground-work is ap-
parently more watery, and vacuoles are very numerous. The finely gran-
ular material of the disc surrounding the affected area retains the stain
with great tenacity. The deeply-staining layer is quite sharply marked
off from the underlying yolk, which loses its stain completely. The
deeper yolk composed of very large granules, retains the stain, and thus
there is a lighter area sandwiched between two dark staining regions.
This may be seen in the section of the ovarian egg (Fig. 4a). The “ polar
ring” mentioned previously is seen to be shallow, appearing in cross-
section as two lighter staining V-shaped areas outside of the apex of the
affected area. The distinctness and conspicuous character of this ring
make it evidently something more than an accidental feature. It can be
traced through adjoining sections and shown to be a complete ring. Simi-
lar appearances in other eggs will be recalled, as, e.g., in that of the
leech.

Turning to the nuclear phenomena at this stage, we see that the spindle
lies close to the egg membrane. Centrosomes are inconspicuous, but a ra-
diating arrangement of the alveoli may be made out at the poles. The
spindle is in the equatorial plate stage (Fig. 22).
Eugene Howard Harper 15

In another egg we find the chromosomes just separating (Fig. 23).
There are eight pairs, and they are quite unequal in size, as was shown
in the first polar spindle. They show a marked increase in size compared
with those of the first division. The spindle here lies in a decidedly
lighter staining area. The polar globule fills a bowl-like depression in
the disc instead of a position at the side of the depression, as in the
previous case shown. The eight dyads in the polar body are not fused
together, and some retain a slightly dumb-bell like shape. The wall of
the polar body is well marked. The polar ring was present in this, as in
all of the other maturation stages obtained after impregnation of the
egg.

The second maturation spindle is shown in Fig. 21 in a slightly earlier
stage. The spindle is not yet completely reformed. One of the chromo-
somes lies between the equatorial plate and the egg membrane not yet be-
ing drawn into position. It has the appearance of a tetrad, in which case
it would be a chromosome of the first division which failed to divide.
It is, however, probably a dyad in the first stages of splitting. The
chromosomes as shown above are originally pairs of dyads lying closely
side by side. The polar globule is in this case more rounded than in
the last, which is undoubtedly due to its not yet having had time to
become flattened by the pressure of the egg membrane

One other stage of the second maturation spindle is shown in Figs. 9
and 24. In this egg the central core of the affected area directly under-
neath the spindle is different from the other cases obtained, being entirely
free from deutoplasmic granules.

The egg nucleus is shown in Fig. 25 with the second polar globule just
given off. The chromosomes are fused together into a mass with a some-
what crenate contour. The polar globule is still connected with the
egg by a cytoplasmic neck, and its wall is not formed. The egg nucleus
has penetrated the egg farther than is found to be the case in some later
stages, but this may be explained by the exceptional fewness of yolk
granules in the affected area which ordinarily might hinder its freedom
of movement.

The further reconstructed egg nucleus is shown in Fig. 26. Both polar
bodies are shown, being present in adjacent sections. The inner sphere
and centrosome (?) is in this case recognizable and appears larger than
during division. The egg nucleus does not have a distinct membrane.

Fertilization stages—In a still later stage the egg nucleus is seen to be
completely reconstructed and has a distinct membrane (Fig. 27). Yolk
granules crowd about it so as to hide any other structures. The polar
bodies were both found in the same section. The one which is farther
16 Fertilization and Early Development of Pigeon’s Egg

from the egg nucleus has the chromosomes nearly all fused together. The
nearer one is a fused mass of chromatin with a ragged outline showing a
decided tendency to form a network. In displaying this tendency it
resembles its sister nucleus in the egg. It tends to enter a metabolic
phase. The first polar body, on the other hand, retains the kinetic ten-
dency like the second maturition spindle, although it fails to divide.

The polar ring is present at this stage, but has not been found later than
this, being apparently obliterated by cytoplasmic movements occurring
within the affected area.

The sperm nuclei which are found in this egg are rather faintly staining
bodies and no one of them is especially near to the egg nucleus.

The next stage obtained shows the pronuclei very near together, one
being slightly smaller and deeper in the egg (the male?) There is a
hyaline area adjoining, but no distinct astral appearance (Fig. 10).

In a later stage the pronuclei, about the same size as in the last in-
stance, are seen in contact (Fig. 11). A diagram (Diagram 1), shows
the other sperm nuclei present and their distribution. Only one, and it
a quite large one, is in the affected area, the rest being scattered away from
the center in all directions.

At a later stage the conjugating nuclei are flattened against each other
(Fig.12). The cytoplasmic surroundings of the disc are shown in the
drawing. The peculiar orientation of the lighter staining central area
in the form of a cone which was seen in the maturation stages is not any
longer manifested in any of the fertilization stages. There is a con-
siderably vacuolated area at one side of the pronuclei and on the other
side a curious apparently normal appearance like a hyaline channel ex-
tending from the vicinity of the nuclei toward the egg surface. Astral
appearances if present are hidden by the yolk. The number of sperm
nuclei shown in the diagram (Diagram 2), is only twelve, which is
probably too small, as the series of sections was imperfect.

When the segmentation nucleus is formed the accompanying cytoplas-
mic changes in the germinal disc are so striking as to indicate plainly the
approach of division (Fig. 13). The affected area is spread out laterally
and shows a differentiation into a more hyaloplasmic margin and a gran-
ular interior. This fact is, however, still more clearly perceived by refer-
ence to a surface section as shown in the subsequent stage (Fig. 14).
The segmentation nucleus (Fig, 13) lies near the center of the affected
area, rather closely surrounded by yolk granules. It has moved nearer
to the egg surface than at the time of copulation. There are no sperm
nuclei remaining within the affected area, but a pair are to be seen a
little distance outside of its margin. The segmentation nucleus may be
Eugene Howard Harper 17

identified by its size and the appearance of the chromatin, as well as by
its surroundings. It has a well-developed double contoured membrane.
The contents are slightly plasmolyzed, a feature which has not been ob-
served to occur at any earlier stage. The chromatin is beginning to be
gathered into long threads.

The First Division —A later stage containing the prophase of the first
cleavage spindle is shown in Fig. 14, in horizontal section. The spindle
lies at the center of-a small area free from granules (Fig. 28). Centro-
somes and asters are very indistinct, as would be expected at this stage
of division. The centrosome is not a deeply staining granule. The cyto-
plasm shows only indistinct radiations. The spindle is rounded at the
ends and rather broad. The chromosomes, sixteen in number, are partly
in the equatorial plate. None of them are yet splitting. The prevailing
shape of the chromosomes.is that of a broad V. In the surrounding
protoplasm may be seen the same appearances as in the stage of the
segmentation nucleus (Fig. 14). There is an area of protoplasm whose
outer border is hyaline and the center surrounding the nucleus is more
granular. The area is elongated in the direction of nuclear division. Its
margin shows an appearance like that of outpushings. These are large
lobes, as if indicating an amceboid movement of the whole mass. Curi-
ously enough, the granular interior conforms to the same outline, showing
lobes corresponding to the outer margin. Balfour, 85, states that “In
elasmobranchs before segmentation commences, the germinal disc ex-
hibits amceboid movements.” Here these amceboid movements, if so the
appearances described are to be interpreted, are seen to be confined to a
region at the center of the germinal area whose diameter is about 0.5 mm.,
or about one-sixth the diameter of the inner area of the disc. The area
of active protoplasm is differentiated into a more granular and a more hy-
aline pole. There are indications of a constriction which, if carried out,
would. thus divide the cytoplasm of the area qualitatively.

A stage of the first division is shown in Fig. 15, in which the nuclei
are separated a considerable distance. They are of quite small size. The
affected area of protoplasm shows a dumb-bell shaped figure and
the nuclei lie at about the centers of the two ends. The hyaline
outer border and the inner granular condition is still preserved.
The first furrow is being formed at the constriction, but is shallow and
does not appear in the section containing the nuclei. In the surface sec-
tion it is seen as a broad, shallow depression filled with cytoplasm of a
finely alveolar structure. Around the affected area lighter streaks may
be seen extending out into the surrounding protoplasm. One blastomere
is seen to be more hyaloplasmic than the other.
18 Fertilization and Early Development of Pigeon’s Egg

The nuclei and first furrow have been found at a stage when the nuclei
are very much larger. They had moved apart relatively little compared
with the former stage, while they had greatly increased in size. If the
small size at the former stage indicated that little time had elapsed since
division, then their movements at first must have been more rapid. Per-
haps the cytoplasmic constriction would be the cause of the early, rapid
separation of the nuclei. Whatever may be the link connecting nuclear
with cell division, it would seem that the constriction of the cytoplasm
must play a part in the separation of the nuclei. At this stage, the
completion of the first division, there is no differentiated area about the
nuclei recognizable. Apparently the amceboid changes cease during the
resting stage of the nuclei.

The Second Division —In Fig. 16 is shown the beginning of the second
division. The first furrow is longer. The nuclei are about equidistant
from it. The spindles are formed and are in the prophase, approaching
the equatorial plate stage. They lie in small areas free from granules.
The differentiation in the cytoplasm is like that surrounding the first
cleavage spindle, except for greater complexity. There is a clearly
marked polarity. One blastomere is more granular, the other more
hyaloplasmic. In the latter there is a complex affected area surrounded
by homogeneous protoplasm. The hyaline border of the active area is
even more distinct than at the first division. But this blastomere must
apparently be identified with the more hyaloplasmic pole at the first
division. The hyaline border shows a sinuous contour. The whole area
is elongated, as before, in the plane of the next nuclear division. The
prominences or outpushings are more complex. They correspond some-
what at the two poles, but are more developed at one pole (the left).
There is an evident beginning of constriction, and division at this point
would separate again a more hyaloplasmic blastomere from a more granu-
lar one. In the other blastomere the affected area has an even contour.
One other egg was obtained at this stage and shows the same general fea-
tures, but with minor differences in the apparent ameeboid changes. It
would hardly be expected that amoeboid movements of this character
would give rise to identical appearances in different eggs. The observa-
tion of Whitman, 87, on cytokinetic phenomena in general may be quoted
in this connection: “They are diversiform in the.extreme, rarely pre- -
senting regular form series, and thus stand in marked contrast with
nuclear metamorphoses, which everywhere, both in plant and animal
cells, exhibit a most remarkable uniformity.”

Comparison of Cleavage and Maturation Diwvisions.—The appearances
here described in connection with cleavage may be compared with the
Eugene Howard Harper 19

maturation divisions. The body of active protoplasm is differently
oriented in the two cases, as has been pointed out. During maturation
the active protoplasm underlies the spindle and extends radially in the
egg, widening centripetally, so that its appearance is roughly that of a
cone from the apex of which the polar bodies are pinched off. At cleav-
age, the area assumes a horizontal position with reference to the surface,
and a constriction occurs at its middle. At the first maturation division
(Fig. 6a), the spindle lies at the center of a granular area encircled by

a hyaline zone. From analogy with the cleavage divisions, it would ‘ap-
pear that this hyaline zone is a normal cytoplasmic feature of the
maturation division. Within it lie many sperm nuclei and it’is the
favorable zone of entrance for the male elements. It would thus appear
to have a double function, the relations of which are not, however, neces-
sarily close, since the entrance of spermatozoa is not confined to this
zone. In the second maturation division the hyaline zone around the
spindle seems less conspicuous than at the first.

Kupffer, 75 and go, pointed out that the primary differentiation of
protoplasm seen in‘the unicellular organism, into an outer hyaline and an
inner granular protoplasm surrounding the nucleus, is also found in the
animal tissue cell and egg. In the egg of petromyzon, as shown by
Bohm, to which Kupffer, 90, made especial reference, this differentiation is
clearly shown. The hyaloplasm, which during fecundation appears as a
cap upon the egg, later moves back into the yolk and undergoes further
amceboid changes, elongating in the direction of nuclear division. The
behavior of the sphere substance in the egg of unio, as described by
Lillie, or, may be compared with that of the active protoplasm in the
pigeon. The sphere substance results from the growth of the egg centro-
-some- and sphere, and extends across nearly the whole diameter of the
egg, elongating in the plane of nuclear division. In the pigeon’s egg one
stage was found, of the partially reconstructed egg nucleus, where the
centrosome and sphere appeared greatly enlarged, as described by Lillie
(Fig. 26). Conklin’s demonstration of protoplasmic currents in the egg
of crepidula may be mentioned in this connection. As mentioned above
Balfour states that in the selachian egg amceboid movements occur
before cleavage begins. Whitman, 87, insisted that the cytoplasm could not
be regarded merely as a passive nutritive substance, although, as he said,
the majority of writers are inclined to seek the primum mobile in the
nucleus and to make the nucleus responsible for the kinetic phenomena
displayed in the cytoplasm.”

Loeb, 95, relying upon Quincke’s experiments and certain experiments
of his own upon the echinoderm egg, ascribes cell-division to diffusion
5)

 

-

DIAGRAMS 1-6.

NUMBER AND DISTRIBUTION OF ACCESSORY NUCLEI IN EARLY STAGES OF
PIGEON’S EGG.
Eugene Howard Harper 21

EXPLANATION OF DIAGRAMS 1-6.

DrackamM 1. Diagram showing number and relative position of accessory
nuclei in the egg. The male and female pronuclei are in the center, lying in
contact. Two sperm nuclei nearer than the rest. Same egg as in Fig. 11.
X 10.

DiacRam 2. Pronuclei in closer contact. Same egg as shown in Fig. 12.
Owing to the incompleteness of the series the number of sperm nuclei is too
small. x 10.

DracraM 3. Stage of segmentation nucleus. Accessory nuclei are mostly in
pairs, indicating previous division. Same egg as Fig. 13. > 10.

Disceam 4. Stage of first cleavage spindle. Same egg as Fig. 14. > 10.

Diackam 5. First furrow is beginning to appear at surface (f). The two
cleavage nuclei equidistant. Same egg as in Fig. 15.  » 10.

Dracram 6. Beginning of second division. The accessory nuclei appear
nearer the center of the disc than in the previous stage. x 10.
22 Fertilization and Early Development of Pigeon’s Egg

phenomena and amoeboid movements occurring on the surface of the egg
along the circle whose plane separates the two astral systems.

The Early Cleavages—In Figs. 40-45 are shown the 2-4-8-16 cell stages,
drawn from a surface view. The accessory cleavage is also shown, except
in the sixteen-cell stage. The first furrow crosses the disc along its
shorter diameter. It is slightly eccentric in position. In the four-cell
stage is seen the so-called “ cross furrow ” connecting the second furrows.
In the eight-cell stage, considerable variety exists in the position of the
furrows. A more regular type is shown in Fig. 43, and an irregular one
in Fig. 42.

The more regular type shows meridional furrows at quite corresponding
positions in the four quadrants. In the sixteen-cell stage shown the
accessory cleavage is not represented (Fig. 45). The nuclei in the ana-
phase of division are shown as they appeared in a surface view in a whole
mount of the blastoderm, and give an idea of the relative size of nuclei
and blastomeres.

In the sixteen-cell stage there is a clearly marked polarity of the
egg due to the small size of the blastomeres on one side. This asymmetry
of cleavage was pointed out by Kolliker in the case of the chick as pro-
ducing an evident polarity during the early cleavages, whose relation,
however, to the polarity of the embryo is undetermined.

DEVELOPMENT OF THE Accessory NUCLEI.

The accessory nuclei, whose appearance soon after the time of entrance
was described in connection with Fig. 6b, have been found in later stages,
varying considerably in number. In fertilization stages from twelve to
twenty-five have been counted. After division sets in among them their
number in some cases becomes very great, and no attempt has been made to
count them. The diagrams (Diagrams 1-6) show the number and dis-
tribution of these nuclei in a series of stages. The general fact is dis-
closed that they migrate away from the point of entrance and soon become
outside of the vicinity of the pronuclei. During the earlier stages of copu-
lation one or more of the accessory nuclei may remain in the vicinity
within the affected area of the germinal disc. But this is not true of the
later stages, as is indicated by the absence of accessory nuclei in
Figs. 14-16.

In Fig. 13 a single pair are found at one side, near the affected area.
This pair are in close apposition, as if conjugating. Moreover, twenty-
five pairs of such nuclei are found in this egg together with some earlier
division stages. Some of the pairs are in apposition, but most of them
are a slight distance apart, some being in a stage very soon after division.
Eugene Howard Harper 23

A difficulty is presented in finding the division stages, on account of the
surrounding yolk. It must be remembered that these nuclei are not in
the favorable region for observation in the center of the disc where the
yolk granules are fewer, but they are migrating through the deeply stain-
ing surrounding region. Hence during the division stages when at their
minimum size they are to be seen only under the most favorable circum-
stances. Several clumps of chromatin threads in the spireme and later
stages were seen, enough to indicate the probable presence of other divi-
sion stages. The resting nuclei are, of course, larger and easily seen.
For the above reason the details of the first division of the nuclei have
not been made out in the very limited material at hand. This difficulty,
however, later disappears, and the mitotic division of these nuclei may be
made out with perfect ease.

If the sperm nuclei divide before the cleavage nucleus, then the rate of
division of the latter is a resultant of a slower and a faster rate. The rate
of division of the unfertilized egg nucleus may be considered as approach-
ing zero. There have been contradictory observations as to whether the
unfertilized blastodise of the chick may segment parthenogenetically.
Appearances have been observed which at any rate suggest this. Barfurth,
94, offers a different explanation of these phenomena holding that there
is no true cleavage in the unfertilized eggs. Assuming that possibly it
may occasionally happen that the unfertilized egg nucleus may divide for
several generations of cells, it is in accord with the accepted view as to
the nature of the sperm protoplasm that the sperm nucleus should show
a faster rate of division than the egg nucleus, and that the fusion nucleus
should have a somewhat slower rate than that of the sperm nuclei. But
Riickert found in the selachian egg that the sperm nuclei divide syn-
chronously or nearly so, with the cleavage nucleus. Oppel, 92, found in the
reptilia, on the other hand, that the accessory nuclei divided more slowly
or not at all in many cases. It is thus seen that special adaptations have
arisen in different groups. Environment seems to have more to do with
the division of the sperm nuclei than the nature of their own protoplasm.

In the course of their further migration the nuclei reach the coarser
yolk surrounding the inner zone of the germinal disc. Here, either be-
cause of a difference in the chemical nature of the materials surrounding
them, or because their progress is impeded by the coarser yolk granules,
the nuclei remain and their division is followed by a cleavage of the sur-
rounding cytoplasm. The first indications of this accessory cleavage on
the surface of the egg are seen when the first furrow is established be-
tween the cleavage nuclei (Fig. 40). The continuation of this cleavage
and division of the surface into small cell-like areas is indicated in the
24 Fertilization and Early Development of Pigeon’s Egg

two-, four- and eight-cell stages. In the later stages obtained the ac-
cessory cleavage is not shown in the figures. This accessory cleavage is
then set up after the second mitotic division of the sperm nuclei. They
are found at the time of this division surrounded by wide cytoplasmic
areas free from yolk granules. The resting nuclei after the first division
are accompanied by small areas of a “sphere substance” which entirely
resembles this material. This sphere substance rather than being re-
garded as an organ accompanying the nucleus would seem to be an accum-
ulation of the products of the nuclear activity. During the migration
of the nuclei, the amount accumulated next to a nucleus appears small
(Fig. 36), but after they are settled down the substance soon gathers
in large quantities. Of course this statement is not meant to imply
that the sphere substance may not at certain times take on a definite
form, like a permanent organ, as in the young ova, for instance. Accord-
ing to the view of Van Bambeke, 97, the sphere substance is the center
of formation of plastic and of nutritive elements.

To explain the peripheral migration of the sperm nuclei, Riickert has
developed a theory of mutual repulsion which applies to all nuclei of a

like character, and is exerted by and. through the means of the sphere’ *

substance. The sperm nuclei show a mutual repulsion for each other,
which prevents their conjugation with one another. The cleavage nuclei
have a superior power of repulsion, and so drive the sperm nuclei from
the cleavage area into the yolk. The egg nucleus having no centrosome
and sphere, or only a slightly developed one, is on the contrary attracted
to the male pronucleus. The early and rapid migration of the sperm
nuclei out of the cleavage area is, however, a fact which does not seem
to fall within this explanation. The sperm nuclei for some reason mi-
grate to the periphery and give rise to an accessory cleavage there, while
the egg is still in the two-cell stage. Only a few straggling nuclei are
at this time remaining within the inner area of the disc. There seems
to be a tendency on the part of the sperm nuclei to migrate, only one
of them being caught at the early stage by the attraction of the female
nucleus; and this conclusion is certainly not inconsistent with the
motility of the sperms during the stage of their free existence. As an
active cause for the migration of the sperm nuclei, it might be assumed
that the activity is but the continued expression of the labile nature of
the protoplasm which gives the sperm its motile character during the
period of its independent existence. An indication of the rapid move-
ment of the sperm nuclei has already been pointed out, namely, that
the accumulation of altered protoplasm or “ sphere substance” about
the nuclei while migrating in the germinal disc is very small. As soon,
Eugene Howard Harper 25

however, as they reach the marginal yolk they become surrounded by
wide areas of protoplasm free from granules of yolk, owing, according
to the assumption, to the cessation of their rapid movements and their
delimitation to fixed areas, which results in the'accumulation about them
of the products of their metabolism, instead of its diffusion into the
surrounding protoplasm.

Mitosis in the Sperm Nuclet—The mitosis of the accessory nuclei
appears to be normal in the early divisions, at least in the sense that it
results in an equal division of the chromosomes. The details of mitosis
have not been compared with that of the cleavage nuclei, although such
a study might indeed be valuable. The determination of the number of
chromosomes in the spindle has a bearing upon the origin of the nuclei,
of course. It is not asserted, since it has not been definitely proved, that
mitosis is always perfectly normal, even at this stage, since abnormalities
do appear later which lead eventually to amitosis. No pluripolar
spindles have, however, as yet been observed. Regular equatorial plate
stages are found. The reduced number of chromosomes is present, which
is eight. In the metabolic nuclei the chromatin network is somewhat finer
than that of the cleavage nuclei. This difference extends to the fully
formed chromosomes, which are narrower and somewhat more elongated
than those of the cleavage nuclei. In the prophases very long, slender
chromosomes are formed which become shorter and thicker as they
approach the equatorial plate stage. A typical longitudinal splitting of
V-shaped chromosomes takes place (Fig. 33), and as the daughter
chromosomes pass to the poles, one end of each becomes thicker. Gradu-
ally the chromatin accumulates at this end (Fig. 34) until in the late
anaphase the chromosomes appear as short oval bodies approaching a
spherical shape (Fig. 85). The achromatic structures are well defined.
The centrosome is a sharply defined, deeply staining spherical granule,
not so large as in the late cleavage nuclei, but decidedly more conspicuous
than the centrosome of the maturation and early cleavage stages. The
spindle fibers are distinct and the spindles are very regular in form.
These characteristics of mitosis and their similarity to that found in
later cleavage stages and dissimilarity with that found in the early
cleavage seems clearly correlated with the nature of the substance
by which ‘the nuclei are surrounded, which is in the one case a
highly plastic cytoplasm, the immediate product of the nuclear activity,
and in the other is the largely unmodified egg cytoplasm, which from
its coarse alveolar structure reacts differently to the mitotic forces and
gives less evidence of their operation by a change in form than does
the more plastic medium.
26 Fertilization and Early Development of Pigeon’s Egg

In regard to the synchronousness of division in the sperm
nuclei, there appears to be a considerable difference. In one egg
of the two-celled stage, over one hundred sperm nuclei were present,
The accessory cleavage began at one side and here the nuclei had
nearly all passed into a resting stage. On the opposite side of the
disc, the nuclei were nearly all in some phase of division from spireme
to late anaphase. It did not seem that concentric zones could be distin-
guished, as Riickert has found to be the case in the selachian, in which
the gradations in phase of division could be found in successive zones,
Rather in this egg there was a difference in phase on opposite sides, or
what may be called a polar difference. The evidence of this is seen also
in the beginning of the accessory cleavage, as shown in Fig. 40.

Tur YoLtk NucieI or Later CLEAVAGE STAGES.

With the advance of the cleavage nuclei, the sperm nuclei are driven
into the surrounding yolk. In a stage about fifteen hours after fertili-
zation, the sperm nuclei were found dividing amitotically. The inter-
vening stages have not been filled in. The identity of the yolk nuclei
at this stage with the earlier sperm nuclei is undoubted in the light of
the selachian egg, whose phenomena can be duplicated, at least as to chief
details, in the pigeon.

Balfour has described the yolk nuclei in the chick as lying at the
margins of the blastoderm, and under the peripheral cells, but not
under the center. The nuclei are found very largely in nests or clusters,
the members of which are very unequal in size. Sometimes 6-8 nuclei
may be found thus clustered together (Figs. 39a, b, etc.). They are
surrounded by wide areas of protoplasm free from granules of yolk.
Besides the nuclear nests, many are found singly and these very fre-
quently at the margin of the blastoderm near the surface. These often
show a distinct difference in staining capacity, retaining the stain with
more tenacity than the underlying ones. This difference would seem
to be correlated with the environment, since these are found in the
coarse, deeply-staining, yellow yolk and the underlying ones in the white
yolk. There are also “ giant” resting nuclei as large as the entire proto-
plasmic area which surrounds one of the nuclear clusters (Fig. 37%b).

Transitional stages from mitotic to amitotic division may be found
at this period. In some of the protoplasmic areas are found nests of
daughter nuclei not yet reconstructed (Fig. 38). The separate chromo-
somes or chromatin vesicles are distinct or partially fused together. In
some of the groups of chromatin vesicles approximately eight could be
counted, although the exact number could not be identified on account
Eugene Howard Harper a”

of fusion, and also apparent disintegration of some. ‘These appearances
may arise from pluripolar spindles, which have not yet been found in
the pigeon, but which are undoubtedly to be found here as in the
selachian. Other evidences of attempts at mitosis are to be found. Often
a spireme is found of irregular appearance, indirect division proceeding
no farther. There were no accessory cleavage furrows recognized at this
stage.

Are the nuclei incorporated into the cleavage area? ‘There is no
affirmative evidence on this point. On the contrary, there is a distinet
separation between cleavage cells and yolk underneath the blastoderm,
the marginal cells having complete cell boundaries. No nuclei at all
resembling the yolk nuclei have been found within the cleavage area.
The cleavage nuclei are distinct in appearance and are not easily to be
confused with the nuclei in the yolk.

The only evidence obtained having a possible bearing on the fate of
the yolk nuclei is the occurrence of great numbers of peculiar refractive
bodies closely associated with the nuclei in the large nuclear nests. These
bodies resemble the disintegrating nucleoli of the late ovarian egg. Ina
nest such as shown in Fig. 38 there are large masses of this refractive
substance made up of clusters of vesicles. There are also isolated rod-like
bodies of the same material. May these not indicate that there is a
constant reduction in the amount of chromatin material due to “ karyo-
litie” action? ‘The yolk nuclei are very numerous at this stage, and
form a fringe around the blastoderm, but do not go far out into the
yolk. Their numbers at the start, if augmented by division followed by
migration, ought soon to fill the yolk, as it would seem. On the con-
trary, the margin of the blastoderm seems to be the only region occupied
by them. The liquefied products of this karyolitic action are doubtless
absorbed by the embryonic cells. The refractive bodies are probably
material in process of dissolution, as is apparent in the case of the
nucleoli of the late ovarian egg.

POLYSPERMY IN OTHER Eaes.

The term physiological polyspermy has been applied to all cases where
more than one spermatozoon normally enters the egg. The fate of the
supernumerary sperms is by no means the same in the different groups
in which the phenomena occur. Hitherto, in the elasmobranchs alone
has their persistence to form yolk nuclei or “ merocytes” been observed.
The nearest approach to this condition was found by Oppel, 92, in the
reptilia, where the sperm nuclei though present in large numbers, divided
slowly and karyolitically, and soon degenerated. The evidence in the
case of the reptilia is, however, fragmentary.
28 Fertilization and Early Development of Pigeon’s Egg

If the cause assigned by Riickert for the occurrence of physiological
polyspermy be correct, namely, the absence of protection against it on
account of the thinness of the egg covering in these internally fertilized
eggs, it might well be expected to occur in the bird’s egg also. Balfour,
85, stated in regard to the chick, that “In the bed of white yolk nuclei
are present which are of the same character and have the same general
fate as in Elasmobranchs. They are generally more numerous in the
neighborhood of the thickened periphery of the blastoderm than else-
where.”

Among the amphibia, polyspermy has been found in the urodela. Thus
Jordan, 93, found it to be universal in the newt. He states that “ there
is every reason for regarding such physiological polyspermy in the newt
as a natural, normal and in fact usual occurrence.” The extra nuclei
degenerated shortly after the fusion of the pronuclei. Fick, 92-93, found
polyspermy occurring in axolotl, but inconstant. Braus, 95, in triton
found the sperm nuclei dividing amitotically from the start, a fact which
Riickert correlates with the entrance of the spermatozoa through the
yolk, since in elasmobranchs the sperms enter through the germinal dise

and change from the mitotic to the amitotic method of division after they -

have migrated into the yolk. The anura, on the other hand, are mono-

spermic according to the evidence of Hertwig, Born, 86, Roux, 81, and

King, ox, although opposing observations were recorded by Kupffer, 82,
in the case of Bufo.

In the elasmobranchs the yolk nuclei have been the subject for much
controversy and speculation. Balfour, 74, recognized the existence of
such nuclei in the late cleavage stages of the selachian blastoderm. He
surmised that they arose spontaneously in the yolk. Schultze, 77, dis-
agreed with such an assumption as to their origin and took it for granted
that they arose from the cleavage nuclei. Riickert, 85, traced these free
nuclei as far back in development as the eight-cell stage, which was the
earliest stage he found. He argued that they arose from an equatorial
cleavage of the nuclei of the four-cell stage, and that their position in

the yolk indicated that they were the homologues of the nuclei of the

vegetative pole in the frog’s egg. Their peripheral position was taken
as strongly favoring such an homology. Riickert termed them “ mero-
cytes,” indicating thereby that they were parts of cells, namely, the
nucleus with some surrounding protoplasm, which after division migrated
away from the cellular region into the yolk. Kastschenko, 88, found
these merocytes in the stage of the formation of the first furrow. He
proposed a theory that the first cleavage nucleus gave rise to a multi-
nucleate plasmodium before the first division of the egg. Riickert, 90-92,
Eugene Howard Harper 29

pursued the investigations still further, and by the discovery of fertili-
zation stages, was enabled to announce the origin of the much-discussed
yolk nuclei from spermatozoa. He thus not only accounted for the
origin of the merocytes, but established the fact of physiological poly-
spermy for the selachian group. He traced a continuous series of stages
from the entering sperm head to the fully developed merocyte. He
obtained finally the conclusive evidence of their origin from spermatozoa
by determining that the dividing nuclei contained only one-half the
somatic number of chromosomes. Riickert’s results for selachians were
confirmed by Samassa, 95, Beard, 96, Sobotta, 96. His own complete
account appeared in 1899.

The present state of the controversy which involves the ultimate fate
of the merocytes is outside of the province of this paper. The question
whether there may be another generation of yolk nuclei arising in late
cleavage stages, homologous with the periblast of teleosts, has been the
subject of controversy chiefly between His and Riickert.

The announcement of Riickert of the origin of merocytes from sperma-
tozoa necessitated the modification of the prevalent assumption as to the
universally pathological nature of polyspermy and opened up a field of
inquiry as to the causes of normal polyspermy; its adaptiveness; its dif-
ference from the so-called pathological type; the influences which pre-
vent multiple conjugation with the egg nucleus; the cause of the migra-
tion of the supernumerary sperms into the yolk; their change from
mitotic to amitotic division, etc. The identification of these sperm nuclei
with the long known “yolk nuclei,” to which had been assigned by
common assumption the function of yolk digestion for the embryo, both
in selachians and teleosts, raised the question whether in reality in the
selachians the sperm nuclei have a normal or physiological role in
embryonic development.

As mentioned above, the presence of nuclei in the yolk during the early
cleavage stages, forming a syncytium supposedly derived from the cleay-
age nuclei, had been used as an argument to support the theory of the
homology of the yolk of the selachian egg with the lower pole cells of
the frog’s egg.

Riickert holds that the cause of polyspermy in the selachian egg is
simply the absence of protection against it, due to the thinness of the
egg membrane. The phenomena of conjugation of sperm nuclei with
each other and their multiple conjugation with the egg nucleus, seen
in the case of nicotinized eggs (Hertwig, 87), he holds to be due not to
polyspermy, per se, but to changes brought about by nicotinization. Such
phenomena are absent in polyspermatic eggs, and he proposes a theory that
30 Fertilization and Early Development of Pigeon’s Egg

the sperm nuclei exhibit normally a repulsion for each other due to the
presence and activity in some way of their accompanying sphere sub-
stance. The absence or feebler development of the sphere substance in
the egg nucleus accounts for the mutual attraction displayed by the male
and female pronuclei. Moreover, the mutual repulsion of sperm nuclei
disappears with the change of their environment from the germinal disc
to the yolk as it disappears in the abnormal environment of nicotin-
ized eggs. Hence conjugation of nuclei occurs in the yolk, giving rise
to giant nuclei, pluripolar spindles and progressively increasing irregu-
larity of division ending in amitosis.

As to the adaptation of polyspermy to the large meroblastic egg,
Boveri suggested that polyspermy was necessary in the case of the large
egg to insure certainty of fertilization. Riickert maintains that the
force of this argument is weakened by the fact that the region where
the sperms may enter is very limited and is in close proximity to the
egg nucleus. Sobotta accepted the above view of Boveri and also argued
that the size of the egg was the factor which prevented multiple conju-
gation with the egg nucleus, since the sperms could never in so large
an egg enter at exactly the same time. Hence they would always be
unequal in development, and the largest would become the male pro-
nucleus. Riickert points out the inconsistency of maintaining that the
size of the egg requires the entrance of many sperms to insure fertili-
zation, and that the size of the egg is also what prevents multiple fertili-
zation. Riickert believes that the mutual repulsion of sperm nuclei is
at least to be regarded as a fact in the selachian egg, if not proved true
for all monospermic eggs. He thinks it improbable that the sperm-
nuclei have a normal function in the embryonic development, and leaves
open the question of a possible second generation of nuclei arising from
the cleavage cells in later stages homologous with the periblast of teleosts.

As to the cause of migration of the accessory nuclei into the yolk,
Riickert adduces his theory of repulsion, holding that the sperm nuclei
are driven from the germinal disc by the advancing cleavage nuclei,
owing to their superior power of repulsion. If the observations upon
the pigeon in this regard prove anything, it is that the sperm nuclei
migrate so early to the periphery of the germinal disc that it is difficult
to believe they do this under the influence of the cleavage nuclei. As
pointed out, they are found in the accessory cleavage region, far removed
trom the affected area in the center of the disc, which seems to be the
sphere of influence of the cleavage nuclei, as early as the formation of the
first cleavage furrow. Moreover, they are nearly absent from the inter-
mediate region at this time. This seems to point to the independent
Eugene Howard Harper 31

activity of the sperm nuclei, rather than any mechanical driving of them
from the inner region. What chemotactic influences there may be pres-
ent we of course have no means of knowing.

Some FraturEs oF Mirosis IN THE PIGEON’s Eac.

Centrosomes and asters are structures which are frequently asserted
to be absent from eggs heavily laden with yolk. For example, in the
maturation stages of some amphibians they are said to be wanting.
Speaking of the egg of unio, Lillie says that “rays form more readily
in protoplasm free from yolk granules.” The bird’s egg is certainly
as heavily laden with deutoplasmic granules as any, and these granules
are of relatively large size.

This question cannot be considered properly without taking into view
more than the earliest phases of development. If we take these into
view in connection with the later cleavage, we find that there is a pro-
gressive increase in the distinctness of achromatic structures as devel-
opment proceeds. A typical mitosis from a rather late cleavage stage is
shown in Fig. 29. Here the centrosome is a very large, well-defined
granule, and spindle and astral fibers are distinct.

In inquiring into the reason for the feeble development of astral fibers
in the maturation stages of the pigeon’s egg, it does not seem that the
interference of yolk granules in all cases accounts for the fact. For
occasionally yolk granules are not especially near to the spindle, and the
structures in question are not exceptionally well developed in these cases.
In the maturation stages in the pigeon’s egg, the spindle is sometimes
in an area free from granules, but the achromatic structures are essen-
tially similar in all cases. Centrosomes and asters are inconspicuous,
but the alveolar structure about the poles of the spindle, when copied
with the camera, shows a somewhat regular radiate arrangement. There
is no well defined centrosome, more than perhaps a cluster of minute
granules difficult to make out.

Some light is thrown on this matter of asters and centrosomes by
mitosis in the sperm nuclei. As has been pointed out, these nuclei when
they reach the periphery of the disc in their migrations, come to rest
and become surrounded by large areas of cytoplasm, which is identical
in appearance with the sphere substance associated with the nuclei
earlier. As has been suggested, this cytoplasm is apparently the product
of the activity of these nuclei, and is evidently of a highly plastic nature,
giving rise in division to very regular mitotic figures, and well defined
centrosomes. 5

In the later cleavage stages of the blastoderm, the same is true. The
32 Fertilization and Early Development of Pigeon’s Egg

nuclei then are surrounded by entirely similar areas. They are at this
stage limited in their movements by the cell boundaries, and so con-
fined to very small areas. Consequently they become surrounded by
cytoplasm, which is the product of their own activity in altering the con-
stituents of the yolk. This material is highly plastic and responds to the
forces operating in mitosis so as to produce regular figures.

Compare with the limited movements of these nuclei the wide migra-
tions of the nuclei resulting from the first cleavage, and we see that the
latter have no chance to become surrounded by an altered material, since
all products of nuclear activity must rapidly become diffused into the
surrounding cytoplasm. The reason for the different appearance in
mitosis is seen when we compare the alveolar structure of the unaltered
egg cytoplasm with that surrounding the later cleavage nuclei. The
criginal egg cytoplasm is coarser, i. e., the alveoli are larger, and takes
the cytoplasmic stains much less deeply. This is not very apparent in
the drawings. In the maturation stages the absence of a metabolic
phase of the nucleus for so long a period makes the surroundings of the
nucleus least favorable of all apparently for the production of typically
regular figures. It has been noted by some observers that the second
maturation division differs from. the first in the poorer development of
asters, a phenomenon which might be due to the altered character of the
surrounding cytoplasm. It would seem in the case of the pigeon’s egg
that the hyaline zone surrounding the first polar spindle (Fig. 6a) is
not reformed so conspicuously at the second division, there being only
scattered vacuoles present at this period in the region surrounding the
nucleus. The suggestion that the deutoplasmic granules surrounding
the nucleus in these early stages inhibit the formation of achromatic
structures is perhaps an incomplete explanation, since the nature of the
cytoplasmic groundwork may be a more fundamental cause.

CoNCLUSIONS.

1. As a result of the monogamous habit of pigeons, ovulation is nor-
mally held in abeyance till aroused by the stimulus received from the
male. The passivity of the female is compensated by the highly devel-
oped and complex instincts of the male bird. The determination of the
time of fertilization and egg-laying must date from the time of mating.
The second egg of a pair is set free from the ovary and enters the
oviduct within a few hours‘after the first is laid. The egg is impreg-
nated before entering the oviduct.

2. Polyspermy is normal. The most favorable region for entrance of
sperms is the “fovea,” in a zone surrounding the egg nucleus. Never
Eugene Howard Harper 33

more than one male nucleus has been found in very close proximity to
the egg nucleus.

3. The stage of development of the egg may be approximately inferred
from its position in the oviduct. The first polar spindle is formed in the
ovarian egg. The first cleavage occurs about the time the egg is entering
the shell-gland. The time elapsing between impregnation and the first
cleavage is apparently between two and three hours.

4. The polar bodies lie within the egg membrane, in a depression in
the cytoplasm. The second disintegrates before the first, showing a
tendency to form a network and become metabolic like the egg nucleus.

5. There is an area of active protoplasm surrounding the nucleus
which during the maturation stages is oriented as a cone with the spindle
at its apex, from which the polar bodies are pinched off. In preparation
for cleavage, this area becomes oriented horizontally in the germinal
disc. It undergoes amceboid changes and displays a differentiation into
an outer hyaloplasmic and an inner granular area. It elongates in the
direction of nuclear division, and divides with the division of the nucleus,
The appearance of amceboid movements dies out during the resting period
of the nucleus, and reappears at the second division. One blastomere is
more hyaloplasmic than the other, and shows more complex ameboid
changes.

6. The supernumerary sperms which enter the egg pass from the
point of entrance toward the periphery of the disc. The accessory nuclei
undergo division earlier than the cleavage nucleus. At the margin of
the inner disc they come to rest within the coarser granular material, and
give rise to an accessory cleavage on the surface of the disc. They divide
niitotically without abnormalities, so far as discovered at this stage.
They contain the reduced number of chromosomes, which is eight. The
chromosomes differ in shape from those of the cleavage nuclei and the
maturation spindles, being more slender. In late cleavage these nuclei
are found outside the blastoderm, at or near the margins, and dividing
amitotically. Some traces of abnormal mitosis were found.

”. Asters and centrosomes were found in the maturation stages, though
not conspicuously developed. There is a progressive increase in the
‘distinctness of these structures as the nuclei become limited to narrower
areas by cell division, so as to become surrounded by the more plastic
cytoplasm resulting from their activity in altering the yolk. The less
fronounced development of these structures in the maturation and early
‘cleavage stages seems due to the nature of the cytoplasmic groundwork,
.as well as to the casual interference of yolk granules. The sperm nuclei
likewise do not display well developed achromatic structures till they are
54 Fertilization and Early Development of Pigeon’s Egg

delimited within narrow boundaries in the accessory cleavage area, when
they become surrounded by large cytoplasmic areas free from yolk gran-
ules and display well developed and regular mitotic figures.

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Engene Howard Harper 35

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36 Fertilization and Early Development of Pigeon’s Egg

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EXPLANATION OF PLATES.

PLaTE I.

Fic. 1. Ovarian egg 1.4 mm. in diameter, showing nucleus. X 120.

Fics. 1a and b. Nuclei of ovarian eggs about 1 mm. in diameter. x 312.

Fig. 2a. Nucleus of ovarian egg 4 mm. in diameter. 120.

Fig. 2b. Horizontal section through germinal disc of ovarian egg, 1% inch
in diameter, showing nucleus; a, group of chromosomes; b, chromatin network.
x 120.

Fic. 3. Nucleus of an ovarian egg % of an inch in diameter. 120.

Fig. 4a. Vertical section through germinal disc and nucleus of ovarian egg,
% of an inch in diameter. X 50.

Fic. 4b. Nucleus of same, enlarged; a, group of chromosomes; b, group of
nucleoli; c, refractive substance; d, wall of nucleus; e, follicular envelope of
egg, outer layers of capsular wall not shown. Combination of two sections.
x 885.

Fie. 5. Vertical section through disc of mature ovarian egg, taken from
ovary after laying of first egg. Time, 7.30 P.M.; a, spindle; B, perivitelline
layer; c, layer of substance free from deutoplasmic granules. >» 200.

4

/~ Prater IL.
Fic. 6. Surface appearance ee disc about time of fertilization; a,

inner area; b, outer zone; c, fovea. x 10.

oa

Fic. 6a. Horizontal section of germinal disc of egg loosed from ovary and
not yet entered oviduct. Time, 9.00 P.M.; a, egg nucleus; b, sperm nuclei;
c, polar ring. < 125.

Fig. 6b. Same enlarged, showing zone in which the sperm nuclei lie.
x 1000.

aS 7a-h. Stages in transformation of entering sperms. ) 2000.

_“¥ie. Ti. Inwandering follicular cell. >< 2000.

Fie. 8. Vertical section through dise showing second polar spindle, polar
body and surroundings. Combination of two sections. Position in upper end
of oviduct. Time, 7.45 P.M. > 200.

Fic. 9. Vertical section, showing second polar spindle, polar body and
surroundings. Time, 10.15 P.M. » 333.
Eugene Howard Harper 37

Fie. 10. Vertical section. Pronuclei and surroundings. ¢ pron. at left.
From two sections. Time, 11.50 P.M. > 400.

Fic. 11. Vert. section, showing pronuclei. Time, 10.15 P. M. x 400.

Fig. 12. Vert. section, showing pronuclei and surroundings. Time, 10.40
P.M. > 200.

Fig. 18. Vert. section, showing segmentation nucleus and surroundings.
Pair of sperm nuclei at left. Time, 12 P.M. »x 200.

Fie. 14. Horizontal section, showing first cleavage spindle and surround-
ings. Position in oviduct at constriction between upper portion and shell
gland. Time, 10.30 P.M. x 200.

Fig. 15. Horizontal section, showing first pair of cleavage nuclei and sur-
roundings. Combination of two sections. Time,12 P.M. x 80.

Fic. 16. Horizontal section showing first pair of cleavage nuclei at begin-
ning of second division and surroundings. n, nucleus; f, first furrow. Posi-
tion in shell gland of oviduct. Time, 1.00 A.M. » 80.

Prats III.

Fie. 17. Horizontal section through nucleus of ovarian egg (Fig. zb),
showing group of chromosomes. a, pair of dyads; 0, refractive substance in
vesicular mass. x 2000.

Fie. 18. Group of chromosomes in equatorial plate from a mature ovarian
egg. Same stage as Fig. 5, but from a different egg. x 2000.

Fic. 19. Group of chromosomes in equatorial plate of first polar spindle,
showing central spindle granules. From same egg as Fig. 6a. X 2000.

Fie. 20. Vertical section, showing first polar spindle. Central spindle gran-
ules present as in 19, but not drawn. Egg clasped by funnel of oviduct. Time,
8.50 P.M. > 2000.

Fie. 21. Vert. section, showing second polar spindle, not completely formed
and first polar body. One chromosome not in equatorial plate. Combination
of two sections. Time, 8.55 P.M. x 2000.

Fig. 22. Vert. section, showing second polar spindle and first polar body.
See Fig. 8. x 2000.

Fig. 23. Vert. section showing second polar spindle and first polar body.
Chromosomes are separating. Combination of two sections. Time 8.15 P. M.,
x 2000. :

Fie. 24. Second polar spindle. See Fig. 9.

Fig. 25. Vert. section, showing polar body, and egg nucleus as a fused mass
of chromosomes. Combination of two sections. Time, 10.45 P.M. > 2000.

Fig. 26. Vert. section, showing polar bodies, egg nucleus without definite
membrane, inner sphere enlarged. Combination of two sections. Time, 8.55
P.M. > 2000.

Fie. 27. Vert. section, showing completely reconstructed egg nucleus and
polar bodies. Time, 8.30 P.M. > 2000. ,

Fig. 28. Horizontal section showing first cleavage spindle in prophase.
Chromosomes not drawn into equatorial plate. See Fig. 14. > 2000.

Fig. 29. Spindle from cell of blastoderm about fifteen hours after fertili-
zation. Chromosomes not all shown. x 2000.

Fie. 30, Sperm nucleus from 2-cell stage of the egg. In prophase of divi-
sion. xX 2000.
38 Fertilization and Early Development of Pigeon’s Egg

Puate IV.

Fig. 31. Pair of sperm daughter nuclei. See Fig. 18 at left. x 1000.

Fic. 82. Sperm nucleus in prophase of division. x 2000.

Fig. 33. Sperm nucleus in division; shows chromosomes splitting. A ves-
icular refractive body and a yolk granule at right. X 2000.

Fig. 34. Anaphase of division of sperm nucleus in polar view, lighter
gioup of chromosomes are in different plane. X 2000.

Fig. 85. Late anaphase of division of sperm nucleus. Figures 30-85 are
all from the same egg. xX 2000.

Fic. 36. Vert. section through blastoderm about fifteen hours after fertili-
zation. Shows nuclei free in the yolk at the edge of the blastoderm. One
nuclear nest is shown. X 120.

Fig. 37. Similar to above. A “giant nucleus” is present in yolk. Xx 120.

Fic. 88. From same egg as above. Shows an area in which two groups
of chromatic staining bodies lie. a, chromatin vesicle; b, refractive body; c,
yolk. > 2000. :

Fies. 39 a, b, c, d, e. Nuclei in yolk showing amitosis. xX 1000.

Fie. 40. Surface view of germinal disc showing first furrow and accessory
cleavage beginning at one side. Time, 12.20 A.M. 10.

Fia. 41. Surface view of four-cell stage, showing accessory cleavage. Time,
3.15 A.M. 10.

Fig. 42. Surface view of 8-cell stage with accessory cleavage. Time, 2.10
A.M. x 10.

Fie. 48. Surface view of eight-cell stage. Accessory cleavage not shown.
Time, 3.55 A.M. >» 10.

Fic. 44. Surface view of sixteen-cell stage. Accessory cleavage not drawn.
x 10.

Fie. 45. Surface view of sixteen-cell stage. Daughter nuclei are visible
in a whole mount of the disc and are shown in the drawing. Accessory
cleavage not drawn. The boundary represents the margin of the inner zone
of the germinal disc. x 20.
FERTILIZATION AND EARLY DEVELOPMENT OF PIGEON’S EGG.

«. H, HARPER.

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FERTILIZATION AND EARLY DEVELOPMENT OF PIGEON’S EGG.

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