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THE
MATURATION OF THE EGG OF THE MOUSE

J. A. LONG and E. L. MARK

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WASHINGTON, D. C.
Published by the Carnegie Institution of Washington

191 1

Carnegie Institution of Washington, Publication No. 142

Contributions from Zoological Laboratory of the Museum of Comparative
Zoology at Harvard College. E. L. Mark, Director. No. 216.

Copies of this Book
were first issued

APR 3 1911

PRESS OF J. B. LIPPINCOTT COMPANY
PHILADELPHIA

CONTENTS.

Page

I. Introduction i

II. Literature 2

III. Material and methods 6

IV. Time relations of parturition, maturation, ovulation, insemination, and

semination 15

V. Ovulation 22

VI. Size of egg 24

VII. Observations on the maturation processes 25

A. OScyte I 25

1 . General description of stages 25

Stage I. — Germinative vesicle 25

Stage II. — Formation of first maturation spindle 26

Stages III-V. — Development and division of first ma-
turation spindle 26

Stage VI. — Telophase of first spindle, and the first polar

cell 27

2. Chromatin parts of first maturation spindle 27

3. Achromatin parts of first maturation spindle 31

4. Centrosomes, circumpolar bodies, and clear region 32

5. Position and orientation of first maturation spindle 33

6. Abstriction of first polar cell 34

B. Oocyte II 35

1 . General description of stages 35

Stage VII. — Formation of second maturation spindle. . . 35
Stage VIII. — " Equatorial plate " of second maturation

spindle 35

Stage IX. — Division of second maturation spindle 36

Stage X. — Telophase of second spindle and second polar

cell 36

2. Chromatin parts of second maturation spindle 36

3. Achromatin parts of second maturation spindle 38

4. Centrosomes, circumpolar bodies, and clear region 39

5. Position and orientation of second maturation spindle. ... 40

6. Abstriction of second polar cell 40

C Ripe egg 41

Stage XI. — The pronuclei 41

D. Polar cells 41

First polar cell 41

Second polar cell 44

VIII. Criticisms and conclusions 45

.4. Material 45

B. Methods 45

C. Time relations 46

D. Ovulation 49

E. Size of egg 50

iii

CONTENTS.

VIII. Criticisms and conclusions — Continued. Page

F. Maturation processes 51

1 . Germinative vesicle 51

2 . First spindle 51

Chromatin 51

Achromatin 55

Centrosomes, circumpolar bodies, and clear region 56

Position and orientation 57

Division of first spindle and abstriction of first polar cell 59

3 . Second spindle 60

Chromatin. 60

Achromatin 62

Centrosomes, circumpolar bodies, and clear region 63

Position and orientation 63

Division of second spindle and abstriction of second

polar cell 64

4. Polar cells 64

5. Reduction 66

IX. Summary of the principal results in the study of the egg of the mouse. ... 67

Bibliography 69

Explanation of plates 71

THE MATURATION OF THE EGG OF THE MOUSE.

By J. A. Long and E. L. Mark.

I. INTRODUCTION.

Researches into the maturation phenomena of both plants and
animals have been extended greatly in recent years, and, although they
have given rise to numerous different and sometimes conflicting theories,
they point on the whole toward a striking uniformity of processes for
all of the forms of life studied. Among the metazoa investigations
have covered not only the maturation of eggs, but also the production
of spermatozoa. These investigations have shown the general rule to
be that by means of two mitoses, not separated from each other by a
resting nuclear stage, there are formed in the one sex a ripe egg and two
(or three) polar cells and in the other sex four spermatids. In many
cases the origin, structure, and divisions of the chromosomes involved
in these mitoses have received particular attention.

The greater number of works on the maturation divisions of eggs
have been carried out on invertebrates, which furnish the most easily
obtainable material. Work on vertebrates has been largely devoted to
the study of amphibians and mammals. In the case of mammals, which
perhaps present the most interesting field for the study of oogenesis,
the investigation is especially difficult, since the kinds of mammals
lending themselves to such researches are for several reasons relatively
few; among these reasons are the large size of the more common domestic
forms, the difficulty of breeding wild animals in captivity, and the
infrequency of the breeding periods. Of the mammals most carefully
studied (bat, rabbit, guinea-pig, and mouse) the last has been believed
to be the only exception to the general rule that two polar cells are formed
in the maturation of the egg.

According to the excellent works of Tafani and Sobotta, the egg
of the mouse forms two polar cells in only a small proportion of cases;
in the greater proportion of instances it produces only one polar cell. It
was because of this apparent exception to the general law of maturation
in metazoan eggs that the present piece of work was undertaken. It was
begun in 1903 with the hope of finding some explanation for the sup-
posed two classes of eggs.

It soon became clear that it would be necessary to go over the whole
subject in a systematic way on the basis of the changes taking place in
the chromosomes. To do this thoroughly has involved so much time
that it has not been possible to give special attention to the cytoplasm.

2 THE MATURATION OF THE EGG OF THE MOUSE.

Since the summer of 1906 papers on this subject have been published
by Gerlach, Coe and Kirkham, Kirkham, Lams et Doorme, and lastly
by Sobotta. It is a satisfaction to confirm some of the results of these
investigators. There are, however, a number of points in which we do
not agree with any of our predecessors ; some of these are due to differ-
ences of interpretation, some to differences of technique, and others to
the insufficiency of material at the command of some of those who have
preceded us.

A considerable part of the expense incurred in maintaining and
caring for the mice has been covered by a grant from the Carnegie
Institution of Washington, and a part of the same grant has been used
in procuring the assistance of an aid to do part of the less important
technical portion of the preparation of slides.

II. LITERATURE.

It is not our intention to give here a summary of the subject of
the maturation of the egg of either invertebrates or vertebrates. The
reader is referred to Boveri (1892), Rlickert (1894), Hacker (1899),
Korschelt und Heider (1903), and Gregoire (1905) for excellent general
reviews of the literature of the whole field or special portions of it; to
R. Hertwig (1903) for similar information relative to vertebrates; and
to Sobotta (1895) and Kirkham (19076) for surveys of the papers on
mammals. The following brief account of the several works on the
mouse will serve as an introduction to the results set forth in this paper.
More detailed references will be made wherever necessary.

The first to study the egg of the mouse was Bellonci (1885). He
described in ovarian eggs the spindle and the chromosomes arranged at
its equator and considered them as being similar to those of some inver-
tebrates. According to his account the first polar cell and the second
spindle are formed while the egg is still in the ovary. The polar cell he
considered a true cell with a membrane.

Tafani (1889) studied both living and preserved eggs. He believed
that the chromosomes of the first spindle, numbering twenty, were
formed from the nucleolus while the egg was in the ovary, but that the
division of the first spindle and the formation of the first polar cell took
place after ovulation. He thought that in one-fifth of all cases the
chromosomes left in the egg after the formation of the first polar cell
produced a second spindle, while in the remaining four-fifths they were
directly transformed into the female pronucleus. Thus, in his opinion,
in about one-fifth of the eggs two polar cells were produced, while in
four-fifths there was only one, the second polar cell being in the latter
suppressed. No explanation of the cause of this difference was offered.
He said that each of the polar cells contained either a nucleus or granules,

LITERATURE. 3

and that the first polar cell, though it could change its shape and also
vary in size, remained at the spot where it was formed.

Holl's paper (1893) dealt with the formation of chromosomes from
the nucleolus. He made the number eighteen. Unfortunately, his ma-
terial was so poorly preserved that his results are unreliable.

Sobotta (1895), who studied a large number of eggs (1402), stated
that only one polar cell was formed in about nine-tenths of the eggs —
a larger proportion than maintained by Tafani — while in the remaining
one-tenth two were formed. Those eggs which abstrict only one polar
cell are set free from the ovary in the stage of the germinative vesicle
or of the early prophase of the first maturation spindle. This spindle
is formed from the germinative vesicle after the egg reaches the ovi-
duct. Just before the polar cell is cut off the spindle becomes radial in
position.

In the other tenth of the eggs (those forming two polar cells) a first
spindle is formed in the ovary 24 hours before ovulation. He does not
say how it is formed, but emphasizes the fact that it lies deep in the
egg and is twice as large as the spindle of eggs which produce but one
polar cell. The chromosomes also are different from those of the single
spindle. The division of the spindle which accompanies the abstriction
of the polar cell in the ovary is only rarely seen. Then ovulation occurs,
and, while the ovum is in the oviduct, the second spindle arises from the
chromosomes remaining in the egg. This spindle is exactly like the
single spindle of eggs forming but one polar cell. Consequently, in those
eggs which produce a single polar cell, it is the first spindle and polar
cell that are suppressed, the polar cell that is formed being the equivalent
of the second polar cell of eggs that form two. In all spindles the chromo-
somes number twelve and divide transversely. There are no centrosomes
nor polar radiations.

In a later paper Sobotta (1899) describes and figures the division
of the first spindle. He emphasizes its large size and deep position in the
egg and the infrequency of this stage. He believes that the spindle axis
turns from a tangential position, and, just before the cutting off of the
polar cell, becomes radial, with one pole lying in the protuberance which
will become the polar cell. He further says, in correction of his earlier
statement, that the second spindle may be formed immediately before
ovulation.

Gerlach (1906) agrees with Tafani and Sobotta that some eggs
produce one polar cell, others two; but in his opinion the proportions
are as three to one. He describes the origin of the first spindle, the
chromosomes (twelve in number), and the formation of the first polar
cell. This cell and the second spindle may be formed either in the ovary
or in the oviduct. Consequently ovulation may occur at any time from
the stage of the first spindle to that of the second. According to his
view, eggs in the oviduct with no polar cell must have the first spindle.

4 THE MATURATION OF THE EGG OF THE MOUSE.

Although, he says, only 25 per cent of all the eggs form two polar
cells, all form two spindles, both of which divide; however, in those
eggs which have only one polar cell, it is the second polar cell which is
suppressed. This failure of the second polar cell to be formed is brought
about by a rapid division of the spindle. As a result the chromosomes
which would have been in the polar cell are retained in the egg cyto-
plasm, where they degenerate. The rapid division is, in turn, a conse-
quence of late semination.

Gerlach finds that the two polar cells are separated by a varying
distance. This he explains as the result of the migration of the second
spindle from the point at which the first polar cell was formed. Semina-
tion interrupts the migration and causes the spindle to divide in the
position it may have reached when it was stopped, whatever that position
may be. He believes that in both divisions the chromosomes are divided
crosswise, but he thinks that, for theoretical reasons, one of the divisions
should be considered longitudinal {i.e., an equation division). The chro-
mosomes of the first spindle are tetrads, those of the second, dyads.
In one case he found what he considered a centrosome. The first polar
cell is larger than the second.

Lams et Doorme (1907) deal chiefly with the cytoplasm. They,
however, describe both spindles. The second spindle is slightly smaller
than the first, but it can be identified only by the presence of the first
polar cell. They believe that both spindles divide and that two polar
cells are cut off in all cases. The abstriction of the first polar cell and the
formation of the second spindle from the chromosomes left in the egg
take place in the ovary. Ovulation occurs, then, during the stage of the
second spindle. The second polar cell is formed in the oviduct after
semination. They maintain that the second polar cell is larger than the
first, also that the first degenerates. Each spindle has twelve chromo-
somes; centrosomes may exist, though they are not regularly present.

Kirkham (1907) believes that in all eggs two polar cells are formed,
the first always being produced while the ovum is in the ovary. In his
opinion the first and second spindles differ in the nature of their chromo-
somes, those of the first being tetrads, the second, dyads. The number
of chromosomes is twelve. Centrosomes occur at the poles of both spindles.
The first polar cell is larger than the second and different in chromatin
content. He assumes that in most eggs the first polar cell is forced
through the zona pellucida and is lost.

Melissinos (1907), in his paper on the development of the mouse
makes, in passing, a few remarks on maturation. He thinks that 25 per
cent of the eggs form two polar cells, and he places the number of chromo-
somes at eight. But his figures are so diagrammatic and indicate such
poor fixation of his material that not much weight can be given to them.

Since 1895 Sobotta (1907) has considerably changed his former views.
He now maintains that one-fifth (instead of one-tenth) of the eggs form

LITERATURE. 5

two polar cells, and that not only this one-fifth, but all of the eggs,
produce two spindles. However, he still thinks that in 4 out of every 5
eggs the first spindle does not divide, but is metamorphosed directly
into the monaster of the second spindle, and that half of its chromosomes
must degenerate in the egg. Thus, in his opinion, the first polar cell in
four-fifths of the eggs is suppressed by the failure of the first spindle to
divide. He thinks that this conclusion is supported by the fact that
the metakinesis of the first spindle is only rarely seen. When the first
polar cell is formed it is cut off while the egg is in the ovary, and the
second spindle, too, arises before ovulation. He adds somewhat to his
previous description of the chromosomes, the spindles and their divisions.
His view has changed also in regard to the number of chromosomes in
both spindles. He now counts sixteen instead of twelve. Sobotta reviews
and criticizes the work of Gerlach, and touches on the papers of Kirk-
ham and Lams et Doorme.

Sobotta (1908), in his latest paper, gives a clear summary of the
present state of investigation on the maturation processes, and points
out that he believes the mouse to be an exception to the general rule.
He then briefly outlines his own results and reviews and criticizes the
recent papers of Gerlach, Melissinos, Kirkham, and Lams et Doorme.

6 THE MATURATION OF THE EGG OF THE MOUSE.

III. MATERIAL AND METHODS.

The mice used at the beginning of this work were received from the
lot reared by Professor Castle and Dr. G. M. Allen in connection with
Dr. Allen's work on the Heredity of Coat Color in Mice. Some were
white and some were hybrids obtained by crossing wild gray mice (Mus
musculus) with the white variety bought of dealers. There were a few
white and hybrid individuals of less simple ancestry; also black, choco-
late, and golden agouti (Allen, 1904). These served as a beginning for
the subsequent stock of 400 to 500 kept on hand for material during the
greater part of the past five years.

As the vigor and fertility of the stock became lessened by inbreeding,
new white mice procured from several dealers in different parts of the
country and a few gray mice caught wild were introduced with bene-
ficial results. Thus the animals furnishing eggs for study were of cosmo-
politan ancestry. Besides the introduction of new blood, pains were
taken to mate as distantly related animals as possible in order to keep
up the standard of the stock. With the idea at first of finding out whether
there was any possible relation between the number of polar cells and the
coat-color inheritance, whites and hybrids were mated (giving whites and
hybrids in equal proportions) ; but on finding no such relation, hybrids
and whites were paired only for convenience in distinguishing sex.

As a supplement to the account of the care of mice by Dr. Allen
(1904), whose methods the writer has in general used, the following may
be of value to those working with mice and rats. Fig. A (plate A) shows
a modification of the cage originally used in the Harvard Zoological
Laboratory. The improvement consists in making the lids a few inches
shorter and putting the hinges, not at the highest part of the cage, but
further down on the inclined surface. This arrangement greatly decreases
the danger of pinching under the lid frightened mice which have run up
the sides to the top, and, finding an opening, are trying to get out; it
also facilitates catching the mice in the upper corners.

Since water left in open dishes soon becomes fouled, use was made of
the supply bottle shown near the corner of the left-hand cover in fig. A
and in section at 5, fig. D (p. 9). One of these was put on each cage.
It consists of a 3 -ounce, wide-mouth bottle fitted with a rubber stopper
pierced by a bent glass tube of about 6 mm. inside diameter. The tube
has its lower end bent just enough to prevent the escape of water when
undisturbed and is at the same time large enough and open enough to
allow air bubbles to ascend as the water is lapped out of the free end by
the mouse. This device, arranged as shown in fig. A, with the tube
projecting through the wire mesh into the cage, insures an easily acces-
sible supply of clean, fresh water.

Mice thrive well on rich bread-and-milk, oats, and sunflower seed.
They find an occasional bit of lettuce a welcome addition.

LONG and MARK- Maturation of Egg of Mouse

Plate a

n»*H

a b e d e f

II I' i j /•• /

A. Mouse Cage. (For description see p. 6.)

B, C. Suspended mouse cages, with self-recording apparatus to indicate approximately the

time of parturition of a gravid female. (See pp. 7-10.)
G. Chromosomes of first maturation spindle. (See pp. 28-30.)

MATERIAL AND METHODS. 7

For distinguishing individuals the system of holes and notches
punched in the ears, used by Professor Castle and Dr. Allen, was em-
ployed (Allen, 1904). In addition to a book for serial numbers, sex,
parentage, color, and date of birth arranged according to the serial
numbers, it was found convenient to have another book in which there
was devoted to each cage a separate sheet, whereon were set down the
serial number, sex, and color of each of the mice in the corresponding
cage. When mice were transferred from one cage to another, corre-
sponding records were made on each sheet, making it possible, when
necessary, to trace a mouse from one cage to another, and to determine
its matings.

Individual records were kept of the breeding females only. These
records, the record of litters, etc., were made on paper of uniform size
perforated for file-binding. To lessen the possibility of error, the same
sheets also served for all subsequent records of insemination, killing,
fixing, etc. Finally, a new serial number, corresponding with the numbei
on the slides prepared from the killed individuals, was also recorded on
these sheets.

Sobotta (1895) states that under natural conditions mice breed
most actively during two periods in the year, one in the spring (April and
May), the other in late summer and early autumn (from the middle of
August to the end of^September) ; but that if kept warm they breed all
winter. Since the mice used in this work have been kept warm and
well fed at all times of the year, the conditions have not been favorable
for determining the natural breeding seasons.

As previous investigators have shown, female mice are in heat and
ovulate soon after parturition. The eggs for the present work have
been obtained, with one exception, from the ovaries and oviducts during
the first 40 hours after parturition.

It has been the custom of the junior writer to look over the stock
of breeding mice every 5 days (5 days being the average time before
parturition when pregnancy is first easily recognizable) to note pregnant
females and to remove from males such as were to be observed and killed.
Apparently it has been the habit of former investigators to leave the
two sexes together and not to determine with exactness either the time
of parturition or of fertilization. It was felt from the first that a fair
degree of accuracy in the determination of the times of parturition and
insemination would be of great advantage; and, since it was found that
parturition may occur at any time during the 24 hours of a day, it was
necessary to make frequent observations.

In order to increase accuracy in observation and to save much time
during both day and night, the apparatus illustrated in figs. B, C
(plate A), and D was planned and made by the junior writer. Its pur-
pose was to serve in recording automatically the approximate time of the
birth of litters. In this apparatus advantage has been taken of the

8 THE MATURATION OF THE EGG OF THE MOUSE.

fairly constant habit of mice to take food or water at frequent intervals.
The food is placed on a stationary shelf in the cage, whereas the nest
and the floor of the cage are made independent of the rest of the cage
and of each other. By making the movements of the delicately poised
nest and floor self-recording, the activities of the mouse can be deter-
mined. The change in the record after parturition is due to the increase
in the weight of the nest depending on the presence of the young mice
in the nest even when the mother is away. The apparatus is constructed
on the principle of a simple balance, the movements of which are re-
corded on a chronograph drum revolving once in 12 hours. The parts
shown in fig. D at 1 and 2 constitute a unit and accommodate one mouse.
The apparatus as finally perfected, fig. C, has a capacity of four mice, all
the records being made simultaneously on the same chronographic drum.
The essentials of each unit are shown in fig. D; 1 is a diagrammatic
side view, and 2 is an end view. Each unit consists of a box, fixed in
position, but having a movable floor composed of two parts, each of
which is suspended independently of the other and may move in a
vertical direction. The box (B), about 15X12X10 inches, rests upon
supports as seen in figs. B and C. Each box has either the top or side
made of wire netting having quarter-inch meshes and is provided with
a door (D) at one end or on the top (see lower box on right side and
upper box on left side, fig. C). The floor is of thin, light wood made in
two separate parts — a central part, the nest-floor (fig. D, 1 and 2, NF),
supporting the nest (N), and a marginal part, the main floor (MF).
The two parts of the floor are suspended from the ends of two levers or
balance arms (NL, FL), the opposite ends of which terminate in pointers
(NP, FP) in contact with the revolving drum of a chronograph (CR).
The levers are supported on pivot fulcrums at O O, and the pointers are
made of very thin spring-brass so pointed and bent as to scratch the
smoked paper enveloping the drum. The suspension of the floors is
effected by means of thin strips of wood the upper ends of which are
attached to the ends of cross-beams. Each cross-beam in turn rests on
the end of its lever by means of a glass-and-steel bearing. To the under
side of the middle of each cross-beam is attached a piece of glass (G, fig.
D, 1, 2, 3, 4), which rests on a steel knife-edge (E) secured to the end of
the lever (NL or FL) . Slipping of the glass on the steel edge is prevented
by making a slot (fig. D, 3 and 4, SL) in each of the two pieces of sheet
zinc (Z) with which the glass (G) is held in place on the under side of
the cross-beam, the knife-edge (E) occupying the slot. To all the edges
of each floor are fastened strips of light tin (T). These prevent the
mouse from easily gnawing out and also keep in place the nest (N, which
is an inverted strawberry basket) and the sawdust with which the main
floor is sprinkled. To the floors are further attached light wood strips
(S S, provided with metal ends for reducing friction), which keep the
floors from touching each other or the box. The floors and attached

MATERIAL AND METHODS. 9

parts are counterbalanced by the weights (W W) , which may be so
adjusted that the floors move up and down at a very light touch. The
extent and place of the excursion of the levers are controlled by check
blocks, shown in plate A, figs. B and C, attached to the outside of the
chronograph box. The feed dish (FD) is on a little shelf attached to the
inside of the box, and thus independent of the movable parts, as is also
the water bottle.

Fig. D.

In use, the weights are so adjusted that the empty nest (N) with its
floor (NF) is raised to its upper limit, but may be depressed by a weight
of only 2 to 3 grams; the main floor (MF), on the other hand, requires
about io grams to depress it.

When, therefore, there is no mouse present, both parts of the floor
(NF and MF) are up, and the pointers (NP FP, fig. E) are down. If,
under these conditions, the chronograph drum is set in motion, the two

THE MATURATION OF THE EGG OF THE MOUSE.

pointers will inscribe lines in the position of the lines between a and b,
fig. E. A pregnant female placed on the floor (MF) causes its pointer
(FP) to go up, as at b. As long as she remains on the floor the record
is like that between b and c. When she enters the nest, the nest pointer
(NP) goes up and the floor pointer (FP) down, the record being that
between c and d. When she leaves the nest and goes directly to the
food, the record becomes that between d and e, as at first. The record
at e shows that she again enters the nest, but on her way jumps to the
main floor (vertical mark on line FP). If, before again making an exit
(as she must for water and food), she gives birth to a litter of little ones,
on the one hand her weight will still be sufficient to depress the floor
(MF) , as at /, and on the other, the young will be heavy enough to keep
the nest down, so, that no matter how often she goes in and out, the nest
pointer (NP) will make an unbroken line, the floor pointer alone making
vertical marks.

FP-

NP-

Fig. E.

f 9

Knowing the time when the record began or ended, it is an easy
matter to ascertain the limits of a period of time, of day or night, within
which parturition occurred. The length of the period — depending on
the frequency of the excursions which the mouse makes — may vary
from about 1 5 minutes to 6 hours, but is usually from \ hour to 2 hours.
Table 1, based on the observation of 147 mice, is interesting as showing
the degree of precision of these observations.

Table i. — Observations.

Length of period. individuals. Percentage.

Over 6 hours

45
42

37
20

3°-6- 1 „ 1
28.6- /S9-2 g4 4_

25-2- J
13-6 +
2.0 +

From this it is seen that in nearly one-third of the cases the period
is not over 1 hour; in nearly two-thirds (about 60 per cent) it is not
over 2 hours, and in nearly 85 per cent it does not exceed 3 hours. In
all subsequent calculations the middle point of the period is adopted
in each case, so that the greatest inaccuracy as to time can not exceed
half the length of the period, and assumably will be on the average
much less.

MATERIAL AND METHODS. II

At this point it is convenient to define two terms which will be fre-
quently used in the following pages, viz, "insemination" and "semina-
tion." The first refers only to the introduction of the male sexual
elements into the genital tracts of the female by the act of coitus or
otherwise. The second, which means the same in this connection as the
German "Besamung," applies to the access of the spermatozoa to the
eggs in the oviduct, the coming into contact of the male and female
reproductive cells. Both terms are distinct from "penetration" and
"fertilization."

Fig. F. — Glass syringe and speculum, about three-fourths actual size.

In order to control the time of semination, artificial insemination
has been used in nearly all the cases where fertilized eggs have been
desired. The method is a simple one, and with a little experience the
operation becomes easy. It may be performed quickly and without
the use of ether, and apparently produces neither pain nor injury to
the mouse. The spermatozoa are obtained from the vasa deferentia of
a male killed for the purpose and are put into a small amount of tepid
physiological salt solution (0.75 per cent ordinary table salt), in which
they will live for several hours. The spermatozoa from one male are suffi-
cient for several females. The mass of spermatozoa thus diluted is
drawn into the long, narrow part of a glass syringe (S, fig. F), made
for this purpose. If the spermatozoa become disseminated in the salt
solution — a fact easily recognized in the syringe because of the increas-
ingly milky appearance of its contents and a diminution of the solid
mass of spermatozoa— they are active and capable of fertilizing. In
practicing artificial insemination, the mouse is held under the left hand,
being confined between two pieces of cotton-batting, one above and one
below. The base of the tail is grasped between the first joint of the left
thumb and the metacarpal of the left forefinger.

By means of a glass speculum (SP, fig. F) introduced into the vagina
and held between the left thumb and the tip of the left forefinger, it is easy
to see the somewhat constricted orifice of the neck of the uterus, and to
introduce into the uterus by means of the syringe operated by the right
hand a very few drops of the fluid containing spermatozoa. The specu-
lum and syringe are best kept at body temperature by immersing them
in hot water, taking care not to injure the spermatozoa. Only sperma-
tozoa from freshly killed males were used.

There can be no doubt that the eggs fertilized by means of artificial
insemination are perfectly normal. Artificial insemination is a common
practice in the breeding of horses and dogs, the offspring produced by
such methods being quite sound and perfect (Heape, 1897; Iwanoff,

12 THE MATURATION OF THE EGG OF THE MOUSE.

1903). Moreover, in cooperation with Professor Castle, the junior writer
obtained in 1904 by the above-described method a litter of three rats,
which have been used for breeding purposes in Dr. Castle's experiments.
Similar breeding experiments with mice are too few to be of any value ;
but eggs of mice artificially inseminated when compared with those of
mice naturally impregnated appear normal in every respect.1

In all, 149 mice have been artificially inseminated, but as only 85
have been studied in detail the rest unfortunately can not be included
here. 31 of the 85 have furnished eggs which contained spermatozoa
or pronuclei. A further discussion will be found on page 20.

Only sound mice, white, hybrid, and black, have been used for
study. They have been killed at all hours of the day and night during
the first 40 hours after parturition. While at first chloroform was used,
it was found to be quite as humane and quicker to stun them and then
break their necks by pinching them quickly with the thumb and fore-
finger just behind the head.

The ovaries with the oviducts attached were immediately removed
and fixed for from 20 to 60 minutes in the following modification of
Zenker's fluid : 2 per cent corrosive sublimate, 2 per cent potassium
bichromate, 10 per cent glacial acetic acid. The fluid was made up in
two separate solutions: (A) 4 per cent bichromate, (B) 4 per cent (aque-
ous sol.) sublimate and 20 per cent acetic acid. When desired for use,
equal portions of A and B were mixed. After fixation the ovaries and
oviducts were washed in several changes of warm water until fairly
white, i.e., from 12 to 24 hours; then left in 70 per cent alcohol contain-
ing iodine for from 12 to 24 hours; quickly dehydrated, cleared in xylol,
and embedded in paraffin. This process gives clear fixation of ovarian
eggs without shrinkage of eggs or nuclei and without destroying the
finer st.ucture. Various other mixtures, with and without osmic acid,
have not given satisfactory results.

The whole ovary and oviduct were cut into sections 8 micra thick, as
thinner sections divide the nuclei and spindles into too many parts.
The sections were affixed to slides with albumen, being spread by the
water method, and were stained by one or the other of these three methods :

(1) with iron hematoxylin followed by either Congo red or orange G,

(2) with Bohmer's hematoxylin and Congo red, or (3) with Mallory's
(1905) phosphotungstic-acid hematoxylin. The first gives clear out-
lines, but does not show the structure of chromosomes well. Bohmer's
dye when used for 24 hours or more gives excellent results. Mallory's,
when used in just the right way, is the best of any of the stains tried.
The method employed for Mallory's stain was as follows: From water
the sections were put into a constantly agitated solution of 0.25 per cent

1 Since the above was written the junior writer has obtained two litters of
perfectly healthy mice by artificial insemination performed about 24 and 30 hours,
respectively, after parturition.

MATERIAL AND METHODS. 13

potassium permanganate for 10 minutes; rinsed in water; transferred
to a 5 per cent solution of oxalic acid for 20 minutes; washed thoroughly
in water; and left in the stain for from 18 to 36 hours. The process was
completed by a final rinsing, rapid dehydration, and mounting in balsam.
The results given in the following pages are based on 1,000 eggs
obtained from 147 mice. Only clearly normal eggs have been used,
those in the ovaries being in all cases in large ripe or nearly ripe fol-
licles, never in small or manifestly degenerating ones. Each egg was
carefully studied with a Zeiss 2 mm., homogeneous immersion, apo-
chromatic objective and a No. 12 compensating ocular. Sketches and
measurements were made for each egg on separate sheets of paper of
uniform size (see page 7), which could be subsequently arranged as
desired. Tables 2 and 3, which will be referred to again, show in a
comparative way the number of eggs in various stages, and also other
data to be discussed later.

THE MATURATION OF THE EGG OF THE MOUSE.

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TIME RELATIONS OF PARTURITION, MATURATION, ETC. 1 5

IV. TIME RELATIONS OF PARTURITION, MATURATION, OVULA-
TION, INSEMINATION, AND SEMINATION.

Parturition may occur at any hour of the day or night; although,
as table 3 shows, it takes place more frequently in the early morning.

Table 3. — Number of cases of parturition during each of 6 four-hour periods of a day.

Period.

No. of cases.

Period.

6 a.m. to 10 a.m.
10 a.m. to 2 p.m.

2 p.m. to 6 p.m.

6 p.m. to 10 p.m.
10 p.m. to 2 a.m.

2 a.m. to 6 a.m.

24
2 1
18

23
21
40

1
147

30
24
22
21
18
32

147

4 a.m. to 8 a.m.

8 a.m. to 12 m.
12 m. to 4 p.m.

4 p.m. to 8 p.m.

8 p.m. to 12 night.
1 2 night to 4 a.m.

The distribution is nearly the same whether the periods begin at
4 a.m. or at 6 a.m.

The eggs which mature at each ovulation average nearly seven, and
are in general fairly evenly divided between the two ovaries. In the
maturation processes of the eggs of each individual there is a synchro-
nism which appears tolerably exact when the adopted stages cover fairly
long periods; more specifically, in most cases all the eggs of a given mouse
are in one or the other of the following stages: with (1) the germinative
vesicle, or (2) the first maturation spindle, or (3) the first polar cell and
second spindle. It rarely occurs that the eggs from one ovary are very
much in advance of those produced by the other; in fact, a marked
difference was observed in only two mice, and in these the most widely
separated stages exhibited, on the one hand, the germinative vesicle,
and on the other, the first polar cell and second spindle. Between eggs
from the same ovary there is still less difference.

If, however, the processes of maturation are divided into shorter
periods, as in table 2 (p. 14), the synchronism appears less perfect.
Neglecting, for the time, mice with eggs in the stage of the second spindle
(VIII, table 2) — a stage which may persist for 24 hours or more — and
considering only those (50 in number) which show eggs in stages between
the beginning of the formation of the first spindle and the abstriction of
the first polar cell, inclusive (Stages II to VI inclusive), it was found
that in a few less than half the mice (22) each individual had all its
eggs in only one stage (either Stage I, III, IV, or VI), while the other
28 mice had eggs which fell within some two or three consecutive stages
from Stage I to Stage VII. In no individual were the eggs confined to
either of the single Stages II, V, VII. In other words, one or the other
of two conditions prevails; either, first, all the eggs from a given mouse
may be in one or the other of the four following stages: (I) the ger-
minative vesicle, (III) the first spindle with the chromosomes not yet

l6 THE MATURATION OF THE EGG OF THE MOUSE.

drawn into the equatorial plate, (IV) the first spindle in the equatorial-
plate stage with or without circumpolar bodies (see p. 33), (VI) the
telophase of the first spindle and the first polar cell just cut off; or,
secondly, some of the eggs may be in one stage, some in another. If,
under the latter condition, some eggs show either (II) the beginning of
the first spindle within the germinative vesicle, or (V) the separation of
the daughter chromosomes of the first spindle, or (VII) the formation of
the second spindle, others are sure to be in one or more of the adjoining
stages.

The conclusions to be drawn from these observations are, first,
that some stages occupy less time than others, since, owing to the some-
what imperfect synchronism, in some cases all the eggs fall into one
stage, whereas in other cases some fall into one stage, others into another
stage; and, secondly, that the stages passed comparatively quickly are
those of the formation of the first spindle (II), of the dividing of the
first spindle and the cutting off of the first polar cell (V), and of the
formation of the second spindle (VII). Furthermore, the small numbers
of eggs in these three stages bear out these conclusions. In a similar
way it can be shown that the division of the second spindle takes place
in a relatively very short time.

In the foregoing considerations Stages IVa and 1Mb can not with
fairness be separated, since there is much less difference between them
than between any other two stages. Also, neither of them is rare. Of
the two, IV6 is more often associated with other stages.

There is considerable variation among mice in regard to the time
relation between the stage of the egg and the interval between partu-
rition and killing. This variation may be so great that mice killed for
eggs in the oviduct are found to have them still in the ovary, and vice
versa. Nevertheless, a detailed study of this relation shows a uniformity
sufficient to enable one to say about when certain stages occur, and to
determine approximately the time of ovulation. Moreover, in connec-
tion with a knowledge of the relative length of the stages, it is possible
to form something of an idea of the rapidity of the whole process and of
its parts.

How long before ovulation the germinative vesicle presents the
conditions shown at parturition is not known. It may be weeks or even
months. But it is quite certain that for several days, perhaps weeks,
before ovulation it has the structure which is found during the first
12 hours after parturition. Usually within 15 or 16 hours after parturi-
tion the vesicle has given place to the first maturation spindle. More
seldom it persists longer, even up to 20 \ hours after parturition.

The earliest first maturation spindle that we have observed was
formed 13! hours p.p.1; and the latest was in existence at 28^ hours p.p.
Since the formation of the spindle is very rapid, it is probable that the

1 For sake of brevity we have employed for post partum the abbreviation p.p.

TIME RELATIONS OF PARTURITION, MATURATION, ETC. 1 7

first spindle may arise as late as about 28 hours p.p. According to these
observations, then, the first spindle divides and gives rise to the first
polar cell not earlier than 13! hours p.p., nor later than 28^ hours p.p.
This conclusion is rendered the more probable by the observations that

13J4 HX 15Ji 16M 11H 18X 1W 20X 2 IX 22M 23X 24X 25M 26X 27X 28 K 29V£
14 15 16 17 18 19 20 21 22 23 24 25 20 27 28 20 30

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FIRST

CLASS

the youngest egg in which the first polar cell was completely cut off
was taken from a female killed 14 hours p.p., and that the oldest egg in
which the formation of the first polar cell was barely completed was from
an individual killed 27 hours p.p.

Very frequently the second spindle has been found completely
formed as early as 16 hours, and occasionally as early as 14^, p.p. The

1 8 THE MATURATION OF THE EGG OF THE MOUSE.

latest epoch at which it may originate depends on the time when the
first polar cell is formed, which, as above stated, may be as late as 28^
hours p.p. It can be found in unfertilized eggs in a normal condition up to
at least 40 hours p.p.

The accompanying "curves" (page 17) are given to illustrate the
ratio between two classes of eggs: the "first class" embraces eggs con-
taining the germinative vesicle, or the first spindle in any of its stages
up to the recently formed first polar cell (Stages I to VI, table 2); the
"second class," eggs of all succeeding stages (Stages VII to XI). A con-
sideration of these curves reveals some facts, or at least probabilities,
concerning the amount of time required for certain parts of the matura-
tion process. In the upper diagram the unbroken line represents the
number of eggs in the first class, obtained at various indicated epochs
(hours) after parturition, and the dotted line the number of eggs in the
second class at corresponding epochs. The sum of the two curves,
shown by the dot-and-dash line, includes all the eggs obtained up to
30 hours p.p. In the lower diagram the unbroken and dotted lines give,
respectively, in percentages the ratios of the number of eggs in the first
and second classes to the total number of eggs; they are, of course,
reciprocals of each other.

The general trend of the percentage curve shows from the 1 4-hour to
the 16-hour epochs p.p. a rapid decrease in the proportion of eggs of
the first class during the earl 5^ periods. The great fluctuations in the
periods between 16 and 23 hours and between 26 and 28 hours are
probably due to the small numbers of eggs obtained in those periods
(compare upper diagram), and very likely would disappear to a large
extent with more abundant material.

The whole process of maturation can be conveniently divided into
two parts, the first embracing those stages which are included in the
first class of eggs and the second part those in the second class. It
should be borne in mind that eggs of the first class are constantly in a
state of activity and are steadily advancing toward the formation of the
first polar cell; whereas eggs of the second class, if not seminated, re-
main for 24 hours or more in a quiescent condition in the stage of the
second spindle (Stage VIII). Consequently, the length of the period
in which the eggs of the first class fall would be an approximate measure
of the time required for that part of the process; but a similar period
for the second class would not be a measure of the amount of time nec-
essary for the completion of the second part of the process. The time
required for the latter is calculated by other means. Since in eggs above
the twenty-third hour p.p. the proportion of the first class is very small,
it can be said that usually the first part of maturation is completed
within the period between 14 and 23 hours p.p. When it is noted,
further, that the curve representing the percentage of eggs in the first
class drops very rapidly from the 14th to the 18th hour, it is fair to

TIME RELATIONS OF PARTURITION, MATURATION, ETC. 19

assume that the first part of maturation in a large majority of cases
occurs between the 14-hour and the 18-hour epochs p.p. While it is
quite possible that the first part of maturation requires fully 4 hours
(as for example from 14 to 18 hours p.p.), it seems highly probable that
it may be accomplished within 2 hours, for the reason that at the
16-hour epoch as many eggs have reached the second part of matura-
tion as are still in the first part. If that assumption is true, the process
beginning at 14 hours p.p. would be finished at 16 hours, that starting at
16 hours would end at 18 hours, and so on.

The second part of the maturation process — the formation of the
second spindle, the division of the spindle, and the formation of the
second polar cell — probably requires only a very short time (perhaps
only a few minutes). But the period when this takes place depends,
as Tafani and Sobotta have pointed out, on the time of semination,
this part of the maturation process being apparently dependent on the
stimulation due to the presence of the spermatozoon in the egg.

Now, the earliest stage of an egg containing a spermatozoon that
we have observed came from a mouse killed 20 J hours p.p., but most of
the fertilized eggs were obtained from animals killed between 23 and
31 hours p.p. Thus generally the second part of the process occurs at a
period which begins somewhere between 2\ (20^ minus 18) and 17 (31
minus 1 4) hours after the completion of the first. Consequently the whole
process of maturation probably requires not less than 4 hours. However,
as we have seen (p. 1 7) , the first part of maturation may occur quite late —
as late as 28^ hours p.p. In such case it is entirely conceivable that
spermatozoa might reach the oviduct simultaneously with the eggs, and,
as a result, the second part of maturation might not be delayed but begin
immediately on the completion of the first.

It must be concluded, then, that the process of maturation {i.e.,
from the disappearance of the germinative vesicle to the completion of
the second polar cell) may be accomplished within about 2 hours, but
probably requires more, from 4 to 15 hours, the longer period (above
4 hours) being due to delay in the time of semination.

The time of ovulation is not rigidly fixed with regard either to par-
turition or to the maturation of the egg. Table 2 shows the location
(ovary, oviduct, etc.) of eggs in the several stages, and table 4 the in-
tervals p.p. when eggs in Stages III, IV6, VI, VII, and VIII were obtained.
Also, table 4 does not include all the mice whose eggs fall in the above
stages, but only those bearing on ovulation. Two mice (Stage IV6,
table 2 and table 4), one killed 14! and the other 18^ hours p.p., showed
in the periovarial space two eggs and one egg respectively. Each of the
three eggs had the first spindle in the "equatorial-plate" stage with
circumpolar bodies. The ovaries contained other eggs of the same
stage in ripe follicles. Referring again to tables 2 and 4, the eggs in Stage
VI were all found in the ovary except four (from two mice killed 16J and

THE MATURATION OF THE EGG OF THE MOUSE.

24 hours p.p., respectively), which were in the oviduct along with other
eggs in Stages VII and VIII. The eight eggs of Stage VII which were
from the oviduct came from six mice killed at from 15 to 17 hours p.p.,
all being associated with eggs in Stage VIII. Of the three eggs of Stage
VII which were still in the ovary, one was from a mouse killed 22f
hours p.p., and two were from two mice killed 15^ and 16 hours p.p.,
respectively. Of the eggs in Stage VIII, many were in the oviduct even
as early as 14! hours p.p. Among a few (7) mice, however, having all
their eggs in Stage VIII, in three (killed 14^, 19J, and 22^ hours p.p.,
respectively) eggs occurred in the ovary, in the periovarial space, and in
the oviduct; in two (killed 14! and 28 hours p.p., respectively) eggs were
found in both ovary and oviduct; in one (killed 14I hours p.p.) eggs were
discovered in both ovary and periovarial space; and in another (killed
16 hours p.p.) the periovarial space and oviduct contained eggs. In
Stage III some eggs were observed in the ovary 28^ hours p.p. It fol-
lows, therefore, that ovulation occurs from 14^ to 28^ hours p.p., and
that eggs when discharged may be in any stage from the end of the
"equatorial-plate" stage of the first spindle (Stage IV b) to that of the
second spindle (Stage VIII).

Table 4. — Mice killed during ovulation, showing location of eggs and
hours p.p. when they were obtained.

Location.

Stage.

Individual No.
of mouse.

Hours p.p.
when killed.

Ovary.

Periovarial
space.

Oviduct.

Ill

183

28*

IVb

220

I4f

140

i8i

l6J

126

VII

'ioi

io3

"Si

107

"5*

I6£

144

100

"Si

142

22 1

VIII

Several

I4l

I4§

"4i

*9i

118

22^

187

28"

*Eggs in Stage VII in only one oviduct. Eggs in Stage I were also
found, but in the ovary of the opposite side of the body.

As already mentioned (p. 12), out of 85 mice artificially inseminated
31 produced fertilized eggs. No attempt is made here to analyze ex-

TIME RELATIONS OF PARTURITION, MATURATION, ETC. 21

haustively the reasons for failure in so many (54) cases, but some of the
apparent causes will be given for the benefit of those who may wish to
use the method for breeding purposes, or for a continuation of the study
of the phenomena of fertilization in mammals.

The number of hours after parturition when mice were inseminated
varied from g% to 28^; and the time between insemination and killing
varied from 3^ to 17^ hours, as many time combinations as possible
being made. Before considering the two classes of eggs from the insem-
inated individuals — the fertilized and the unfertilized — 14 cases can at
once be deducted from the latter, because the eggs in those 14 mice were
found in the ovaries, where semination is of course not to be expected.

In the first class (cases resulting in fertilized eggs) the times of
insemination were pretty evenly distributed between i6£ and 28^ hours
p.p. ; two, however, lay outside these limits, being at g% and 14^ hours p.p.,
respectively. All these mice were killed at from 4 to 13^ hours after
insemination, the one inseminated at q4 being killed 13^ hours later
(23 hours p.p.). an(i the one at 14^, 6 hours later (20^ hours p.p.). In
the second class (resulting in unfertilized eggs) most of the insemina-
tions were made between 11 and 18^ hours p.p.; a few, however, were
evenly distributed between 20^ and 28^ hours p.p. The animals were
killed at from 3^ to 17^ hours after insemination.

A comparison of the two classes brings out the fact that the insemi-
nations in both extend over almost exactly the same period of time, but
with a somewhat different distribution; and a more detailed examina-
tion of the data (not recorded here) shows that as the inseminations
were delayed more and more after parturition the proportion of suc-
cessful ones increased. Accordingly, the optimum time for insemination
lies between 18 and 30 hours p.p.

The most obvious causes of failure are (1) too early insemination,
in which case possibly the conditions of the uterus are sometimes un-
favorable for the continued existence of the spermatozoa, (2) killing too
soon after insemination to allow the spermatozoa time to reach the eggs,
(3) late ovulation, and (4) combinations of two or all of these factors.

The time required for the spermatozoa, after introduction into the
uterus, to reach the eggs in the first part of the oviduct nearest the
ovary varies from 4 to 7 hours in mice inseminated about the same
number of hours p.p. Of these eggs some contained the heads of sper-
matozoa, some both pronuclei. Assuming, as is reasonable, that all the
eggs, because they lie very near one another, are seminated at nearly
the same time, one must conclude that the time required for a sperma-
tozoon to develop into a pronucleus is very short indeed. According
to the same reasoning pronuclei must grow very rapidly. Since the first
spindle never persists until the egg reaches the oviduct, semination occurs
only during the stage of the second maturation spindle. An account of
the effect of semination on maturation is given on p. 35.

2 2 THE MATURATION OF THE EGG OF THE MOUSE.

V. OVULATION.

The time when ovulation occurs in relation to parturition and the
maturation of the egg has already been given (p. 19). No attempt has
been made to determine how often ovulation occurs, nor the regularity
of such occurrence. It is perhaps worthy of record, however, that a
female kept isolated and killed 6 weeks after parturition gave eggs in
the oviduct in the stage of the second spindle, a fact which does not
conflict with Sobotta's statement that ovulation occurs at intervals of 3
weeks. On the other hand, careful records of the births of litters show
that the 3 -weeks periods are not constant, in fact, that they vary by
several days. As far as known to us, no careful examination into the
causes of these variations in mice has been made.1

Although the irregularity in the occurrence of ovulations — which
may be as great as days or even weeks — may possibly be caused by
coitus, it is certain that the first ovulation after parturition is entirely
independent of such external condition, because females removed from
males before they give birth to young always furnish eggs in the oviduct
if killed at the proper time.

Just as there is a lack of perfect synchronism in the maturation
processes, so here some eggs pass from the ovary early enough to have
already reached the oviduct, while in the same individual others are in
the periovarial space, and still others are in the ovary. Since in each of
seven cases eggs were found in two, or sometimes three, of these places,
it is highly probable that in few or no mice do the eggs leave the follicles

Cxactly the same time.
In the ovaries of a mouse killed 22^ hours p.p., there occurred three
cles (plate 6, figs. 38, 39, 40) showing in a rare way three stages in
the process of ovulation. First, the completely ripe follicle about to
rupture (fig. 38) ; secondly, the ruptured follicle before the escape of the
egg (fig. 39) ; and, thirdly, the flowing out of the contents of the follicle
carrying the egg with them (fig. 40). They are all later conditions than
those figured by Sobotta (1907), and are an interesting supplement to
his observations. In fig. 38 the granulosa cells which form the sides
and fundus of the follicle are so numerous that they form a thick wall
several (four or more) cells deep, as Sobotta has pointed out; but the
side of the follicle next the surface of the ovary has already become
attenuated to such an extent that at its middle the nuclei of granulosa
cells are entirely wanting. The theca folliculi having also disappeared
in that region, the fluid contents of the follicle come into direct contact
with the germinal epithelium, which is stretched out into a thin mem-

1 Postscript. — During the year 1910 Dr. J. Frank Daniel has independently
found the variation in the gestation of mice to be even greater than we have stated.
He has worked this out in considerable detail, as may be seen in his forthcoming
paper in the Journal of Experimental Zoology, Vol. 9, No. 4.

OVULATION. 23

brane with widely scattered nuclei. The discus proligerus is already
separated from the rest of the granulosa, and its cells, except those con-
stituting the corona radiata, which still show the radial arrangement
about the egg, are becoming detached from one another. As in the other
two follicles, the first polar cell has been produced, and the second
spindle (not shown in the drawing) is fully formed. There is a small
space between the zona pellucida and the vitellus.

In fig. 39 (plate 6) the contents of the follicle have begun to flow
out into the periovarial space through an opening at the surface of the
ovary. The opening does not have the appearance one would expect
to result from a rupture due to pressure from within, but rather from a
condition produced by the migration of cells away from the rupturing
region. The viscidity of the fluid is indicated by the sinuous, more or
less parallel, line-like markings of the escaping contents (see also Sobotta,
1 895), and the plasticity of the discus cells is shown by the partial oblitera-
tion of the radial arrangement of the corona cells around the egg. The
distance between zona and vitellus is so much increased on the deep
side of the egg that the polar cell lies in the space thus formed quite free
from contact with either.

In the last stage (fig. 40) the egg lies in the periovarial space, the
follicle having collapsed. Here, too, there is the same lack of evidence
of a violent tearing of the follicle wall. The contents of the follicle still
have the appearance of a viscous substance. The flattening of the egg,
probably caused by unequal pressure— perhaps due to the narrowness
of the space between the ovarian capsule and the wall of the ovary —
suggests considerable plasticity. This condition can also be seen sub-
sequently in eggs which lie between ridges of the oviduct. The zona is
separated from the vitellus, as in the preceding stage, and the polar
cell is detached from the egg, though not shown in fig. 40.

The corona cells surround the egg in its passage to the oviduct and
persist for a varying number of hours.

24 THE MATURATION OF THE EGG OF THE MOUSE.

VI. SIZE OF EGG.

All measurements made to determine the size of eggs at different
stages of maturation have been made on eggs fixed in the same way and
measured with the same objective and the same eyepiece and micro-
meter. The diameter does not include the zona pellucida. Since the
egg is seldom quite spherical the longest and the shortest diameter of
the middle section of the series into which each egg was cut was meas-
ured. Half the sum of these two measurements was taken as the diam-
eter of the egg.

Table 2 (p. 14) shows clearly the changes in size of the ovum as
it advances in maturation. Under the heading "Diameter of eggs"
the first column gives the number of eggs measured; the second column,
the average diameter of all these eggs; the third and fourth columns, the
diameters of the largest and the smallest eggs of each lot measured.

It will be seen that, with one exception, there is a steady decrease
in size from Stage I to Stage VII. The exception, Stage II, shows only
a slight deviation and is probably due to the fact that the average is
based on so small a number (13) of eggs. Stages IVa and IV&, hitherto
treated by us as Stage IV, show the same progressive decrease. There
is a small reduction in size at the time the first polar cell is formed
(Stage VI), and another in Stages VII and VIII, when the eggs have
left the ovary and have been in the oviduct for only a short time. Pos-
sibly the fact that Stage VII is not intermediate in value between Stages
VI and VIII may be due, as presumably in Stage II, to the small num-
ber (10) of eggs on which the average is based. Eggs that were observed
in the oviduct about 29 hours or more p.p. show a slight increase in
size (see foot-note to table 2). The sizes in the remaining stages can
have no special meaning because the eggs had been in the oviduct vary-
ing lengths of time.

Aside from the change in volume, there is, as the column of maxi-
mum and minimum diameters shows,, considerable individual variation.

OBSERVATIONS ON THE MATURATION PROCESSES. 25

VII. OBSERVATIONS ON THE MATURATION PROCESSES.

In considering the various topics of maturation the processes are
dealt with by stages, the chief characteristics of which have been briefly
suggested in table 2 (p. 14). It seems desirable, however, to give a more
precise definition of these stages before proceeding to a detailed account
of maturation.

It should be borne in mind that these stages, though fairly distinct,
are, nevertheless, only periods in a continuous process of development
and therefore connected with each other by intermediate conditions.

A. OOCYTE I.
l. General Description of Stages.
Stage I. — Germinative Vesicle.

The germinative vesicle, nearly up to the time when it is trans-
formed into the first maturation spindle, presents the following condi-
tions (compare plate 1, fig. 1):

It is somewhat eccentric in position, nearly spherical, and from 19
to 26 (on the average 23) micra in diameter. It has a uniformly thin,
lightly staining, smooth membrane, and is filled with a clear, homo-
geneous substance, the karyoplasm. At one side lies the vesicular
nucleolus, usually in contact with the nuclear membrane. Immediately
inside the membrane, and particularly around the nucleolus (plasmo-
some), are masses of chromatic substance attached to these structures
by achromatic material of irregular, though often threadlike, form.
There are a few strands, remnants of the linin network of an earlier
stage, running through the karyoplasm. Figure 1, plate 1, illustrates
these conditions, except for the condition of the nuclear membrane.

The spheroidal, or sometimes lenticular, nucleolus is about 8.5 micra
in its longest diameter, and has a fairly thick, deeply staining wall of uni-
form thickness. It contains only a clear, homogeneous substance, never
any chromatic bodies such as are attached to its outer surface, either as
distinct bodies or as apparent thickenings of its membrane (fig. 1).

The chromatic masses of the nucleus are usually globular, though
sometimes of an irregular form, and have no correspondence with chro-
mosomes of later stages either in number or in shape. In phospho-
tungstic-acid haematoxylin some of them are stained deep blue, like
chromatin; a few pink, like cytoplasm. There are in addition deeply
stained granules scattered through the nucleus. These are usually
associated with the achromatic substance.

Preparatory to the advent of the first spindle, the germinative
vesicle moves a little nearer the surface of the egg, but the depth at
which it comes to lie is not the same in all cases. It then decreases in
size, and its membrane becomes a little fainter and presents a very
irregular, wrinkled appearance (fig. 1).

26 THE MATURATION OF THE EGG OF THE MOUSE.

Stage II. — -Formation of First Maturation Spindle.

The passage from the preceding stage to this one is rapid. The
germinative vesicle has shrunk still more and is surrounded by a narrow,
clearer region, in which, however, there are cytoplasmic granules (figs.
2 and 3). Its contents are no longer clear and homogeneous, but show
a granular condition, much like that of the immediately surrounding
cytoplasm. A few achromatic threads are still visible. The nucleolus
and chromatic spherules have disappeared (compare fig. 1 with figs. 2
and 3), and instead there is a group of chromosome bodies, which is
usually located at one side, rather than in the middle, of the nucleus.

Figs. 2, 3, 4, and 5 show the first steps in the formation of the first
maturation spindle. The fundaments of the chromosomes differ greatly
in form. Some are masses of irregular shape, which it is hard to dis-
tinguish from the large granules; some are ring-like; a few are elongated
and show a simple or a compound curve; still others show divisions into
two or four parts (figs. 2 and 3). Later (figs. 4 and 5), these all become
completely differentiated and assume more definite and characteristic
forms, some in advance of others. As they assume more precise forms
they become more separated from one another. Their number is at
first uncertain, but by the time they have reached the condition seen
in figs. 4 and 5 it is clearly 20 (see table 2, Stage II, p. 14).

At an early stage in their development the fundaments of the chro-
mosomes lie in a group at one side of a homogeneous portion of the
karyoplasm which is denser than the surrounding nuclear contents
(figs. 3 and 4). This denser portion, at first indefinite in form (fig. 4),
increases in size and develops into the first maturation spindle. As it
grows the chromosomes move apart and all come to lie at its surface.
At length it becomes elliptical in outline (fig. 5), and then shows delicate
fibrillations extending from pole to pole. At the same time the sub-
stance of the spindle becomes less homogeneous, showing granules dis-
tributed through it, so that, except for the fibrillations, it becomes in
appearance more like the rest of the karyoplasm. Meanwhile, the clear
zone around the nuclear membrane disappears (figs. 2 to 5), and at the
same time the general contents of the germinative vesicle assume more
nearly the appearance of the surrounding cytoplasm; the nuclear mem-
brane, which meanwhile has shrunk little, if any, more, is gradually
dissolved (fig. 5), vanishing more quickly in some regions than in others.
Its disappearance may begin in some parts very early (fig. 36).

Stages III to V. — Development and Division of First Maturation Spindle.

Stage III (plate 1, figs. 6, 7, and 7a). — With the complete disap-
pearance of the membrane of the germinative vesicle the spindle is
left free in the midst of the cytoplasm. It is broadly elliptical (fig. 6)
and shows, not only on its surface but in the interior as well, very fine
fibrillations, which conform in direction to its shape. As in Stage II,

OBSERVATIONS ON THE MATURATION PROCESSES. 27

there are granules scattered throughout its substance and the chromo-
somes are still distributed over its surface. Sometimes the surrounding
cytoplasm shows a faint radial structure, which has the axis of the spindle
at its center (fig. 7).

Stage IVa (plate 2, figs. 8, 8a, 86, and 9). — In this stage the chromo-
somes are drawn into the region of the equatorial plane, some lying at
the surface and some nearer the axis of the spindle, where all make up
a cluster having the form of an uneven disk, the so-cal'ed equatorial
plate. The spindle fibers are still very delicate. Occasionally the radial
structure of the surrounding cytoplasm seen in the preceding stage can
still be observed (fig. 8).

Stage IV6 (plates 2 and 3, figs. 12 to 14). — The chromosomes, still
near the plane of the equator of the spindle, are sometimes visibly attached
to the spindle fibers, which are now much more easily seen. However, the
chief characteristics of this stage are the tormation of several circum-
polar bodies at each end of the spindle and the appearance of a clearer
cytoplasmic region surrounding the spindle on all sides. The spindle in
this stage begins to elongate and to become correspondingly narrower.

Stage V (plates 3 and 4, figs. 14 to 17). — This stage is characterized
by the division and separation of the chromosomes (metaphase and ana-
phase of nuclear division). Fig. 14 shows several chromosomes already
divided into halves, while others are in process of separation. Figs. 15,16,
and 1 7 show more advanced stages in the migration of the daughter chro-
mosomes toward the poles of the spindle and also an increasing diminution
in the number and size of the circumpolar bodies and in the extent of the
clear region in the neighboring cytoplasm. The more advanced represent-
atives of this stage (figs. 16 and 17) show thickenings of the interzonal
filaments midway between their ends, and also the beginning of the con-
striction which cuts off the first polar cell.

Stage VI. — Telophase of First Spindle and the First Polar Cell.

(Plate 4, fig. 18.)

In this stage the daughter chromosomes, both in the egg and in
the polar cell, have fused into compact masses, which are still joined
to each other by the interzonal filaments. The middle thickenings of
the filaments have united to form the "cell plate," which is continuous
at its edge with the vitelline membrane where the latter has been con-
stricted to form the neck of the polar cell. The circumpolar bodies have
disappeared and the clear cytoplasmic region is very pale.

2. Chromatin Parts of First Maturation Spindle.
The origin of the fundaments of the chromosomes has already been
described (p. 26). Although we are unable to state how these funda-
ments are formed from the chromatin of the germinative vesicle, the
changes by which they are converted into the characteristic mature
chromosomes can be traced with a fair degree of certainty.

28 THE MATURATION OF THE EGG OF THE MOUSE.

Fully formed chromosomes are shown in plate A, fig. G (e to /) and in
figs. 6 to 14 (plates 1 to 3). In fig. G a typical chromosome is shown at /
in face view {i.e., looking toward the axis of the spindle along that
radius of its equator which passes through the middle of the chromo-
some), and at e in side view {i.e., looking in the direction of the tangent
to the equator of the spindle which cuts the chromosome at its middle
point). To the pointed ends of the chromosome are attached the spindle
fibers; the side of e (fig. G) which is directed to the right is that which
is turned away from the axis of the spindle. The chromosome is com-
posed of four deeply stained parts, which are more or less completely
separated from one another by two deep constrictions, one longitudinal,
the other, less complete, transverse. In a sense the separation is incom-
plete in both directions, because the four deeply stained parts are con-
nected to one another by a less deeply stained substance, in which they
are, as it were, embedded. This substance may possibly be in part non-
chromatic, but probably it contains a certain amount of chromatin. This
diminution in the proportion of chromatin is also evident at the pointed
ends of the chromosomes, where, as already stated, the spindle fibers are
attached (see e and /). The chromosome illustrated by the two views
g and h differs from that seen in e and f chiefly in being more elongated,
the four median, deeply stained regions of h being the upturned adjacent
ends of the four parts resulting from the elongation of the correspond-
ing thicker four parts shown in /. In both these cases the transverse
division is less conspicuous than the longitudinal. In j both divisions
are obscured by the temporary fusion or adhesion of the four parts.
The cross-division is, however, represented by a constriction. To one
or other of these three conditions can be referred all the other forms of
the fully developed chromosomes, the differences being due merely to
various degrees of fusion or separation of the parts. All of these chro-
mosomes ultimately he with their long axes approximately parallel to
that of the spindle.

We return now to the fundaments of the chromosomes and their
development into the forms last described. It is to be noted that in the
early stages (figs. 2 and 3) some fundaments show only a single (longi-
tudinal) division. The transverse division, seen clearly in the left-hand
chromosome of fig. 4a, arises a little later, as may be inferred from the
condition shown in the lower right chromosome of fig. 4a and in the
lower (pale) chromosome of fig. 5 ; this division may perhaps arise much
later. The 4-part condition appears to be a typical one. When it per-
sists as late as the time of the formation of the spindle, the chromo-
some generally lies with its long axis parallel to that of the spindle (fig.
5). Were there no forms intermediate between this and the one shown
in f (fig. G), the four parts of the one might be referred in all cases di-
rectly to the corresponding parts of the other. But the forms b and c
{d answering for the face view of both b and c) are apparently intermedi-

OBSERVATIONS ON THE MATURATION PROCESSES. 29

ate between a and e, for the stages of nuclear metamorphosis illustrated
in figs. 5, 6, 7, and 7a, which exhibit these forms of chromosomes (b and
c) are in other respects intermediate between the conditions shown in
fig. 4 and those of figs. 8 and 9, which present respectively the forms of
chromosomes shown diagrammatically in a and / (plate A, fig. G).

Owing to the lack of exact synchronism in the formation of the
chromosomes, it is impossible to say with certainty which of the forms
b and c precedes the other, or even to assert that they are not independ-
ent of each other. If they do represent successive conditions of one and
the same chromosome, it might be imagined that the condition b had
been brought about by a secondary union of the four parts of such a
chromosome as is shown in a, followed by a bending in the equatorial
region, and that the condition c was afterwards reached simply by a
thickening of the chromosome in the region of the bending; but, on the
other hand, the reverse sequence might have occurred, and it may be
urged in support of this view that c and b represent respectively the
stages e and g, differing from the latter chiefly in the obliteration of the
cross-division, the one corresponding with the equator of the spindle.
As the sequence e g seems the more natural one for those two forms,
so in the former the sequence c b would be a natural inference. The
basis for the conclusion that the forms b and c pass through a stage cor-
responding to a is the apparent absence of those forms (6 and c) in the
earlier stages of nuclear metamorphosis and the prevalence of the a con-
dition. It must, however, be borne in mind that this does not amount
to a demonstration, and that individual variations in eggs or slight differ-
ences in preservation may afford the real explanation of the conditions.

In b and c the transverse division of the earlier stage, a, has, then,
either vanished by fusion, or has not yet appeared, whereas the longi-
tudinal one is quite evident (plate A, fig. G, d, and plate 1, figs. 7 and 7a).
At the ends of the chromosome, where the spindle fibers are attached (d),
the chromatin is less deeply stained, as also in /. The change from the
condition seen in d to that of / is accomplished either by the reappearance
of the transverse division, or, in case it had not existed in the fundament,
by the first appearance of a cross-division. There is no reason, however,
to suppose that the form f might not in some cases arise directly from a,
the transverse division never being obscured. As figs. 4, 5, and 6 (plate 1)
show, some chromosomes develop more rapidly than others.

The -individual chromosomes differ somewhat in size and all seem
to become a little smaller as they approach completion. They are at
first distributed over the surface of the spindle only. After they have
become concentrated in the region of the equatorial plane, some are
still found at the surface, but others are in the interior of the spindle.
Even at the beginning of metakinesis all do not lie exactly in the
equatorial plane (fig. 136). For this reason in cross-sections of spindles
many of the chromosomes are cut in two; polar views of the "equatorial
3

30 THE MATURATION OF THE EGG OF THE MOUSE.

plate "are therefore unsatisfactory for counting chromosomes. It is
an interesting fact that in the spindles drawn in figs. 10 and n (plate
2) the chromosomes lie nearer that end of the spindle which is more
pointed and about which the evidences of cytoplasmic radiations are
more pronounced.

The chromosomes are oriented with their long axes parallel to the
long axis of the spindle. The few exceptions may in some instances be
natural, but in others they certainly are due to displacement by the
knife in cutting (figs. 12, 136, x and x').

The separation to form the daughter chromosomes always takes
place at the middle of the chromosome and at right angles to its long axis
(plate A, fig. G, / to /). While, in general, all the daughter chromosomes
migrate toward the spindle poles at the same time (fig. 15), it sometimes
happens that one or more of the chromosomes divides and the halves
move apart at an early stage before their sister chromosomes show any
signs of migration (two pairs in fig. 9). In the latter case the precocious
daughter chromosomes show no longitudinal division, while in the former
they are clearly split lengthwise (plate A, fig. G, i, I; plate 3, fig. 15).
Fig. 1 5 shows a spindle which is nearly parallel to the surface of the egg ;
in this case each daughter chromosome consists of halves, each of which
is elongated and somewhat tapering, the narrower end being directed
toward the pole of the spindle; the halves are parallel to each other or
slightly converging toward the ends which point to the pole. In another
spindle, of like age but occupying a radial position in the egg, the halves
of each daughter chromosome are in contact at their polar ends, but
widely separated at the equatorial end, thus together forming a distinct
V. In fig. 17 the daughter chromosomes are more compact, and fewer
show the longitudinal division. Some of them are much more elongated
than others. The spindle in plate 3, fig. 16, being cut obliquely, shows the
daughter chromosomes more clearly. The two limbs of each daughter
chromosome are easily distinguishable, each being somewhat dumb-bell
shaped. The two lie side by side, and in some cases by bending assume
the form of flattened rings (fig. 166). Later the chromosomes at each
end of the spindle fuse into a compact, deeply staining, disk-shaped, or
sometimes cup-like, mass (plate 4, fig. 18).

In spite of the differences of opinion which have been expressed
concerning the number of chromosomes, we think there can be no doubt
that typically in the animals we have studied it is 20. A knowledge of
the structure of the chromosomes makes it possible in many cases to
be absolutely sure that this is the number. Table 2 gives the results of
our observations on this subject. The accuracy of the counting depends
on the stage of the spindle and the position which it occupies with respect
to the plane of cutting. When the chromosomes are scattered along the
spindle (figs. 6, 7, and ja), they obscure one another least and frequently
can be counted with perfect accuracy. Upon the formation of the

OBSERVATIONS ON THE MATURATION PROCESSES. 3 1

"equatorial plate," however, they become crowded, and the crowding
increases as division approaches. Figs. 15 and 16 illustrate exception-
ally favorable cases, in which the number can be determined satisfac-
torily, at least at one end of the spindle. It rarely happens that a spindle
lies wholly in one section; it is usually cut into two or three parts. This
is frequently of advantage. (See figs. 7 and ja, 8a and 8b, 10a and 10b,
etc.) When the axis of the spindle is parallel to the plane of cutting,
the chromosomes, which are hardened by the process of preservation,
are seldom cut by the knife, but are pushed to one side. Sometimes
they are dragged out of place (figs. 12,%, 136, x and x') , or even completely
out of the spindle into the cytoplasm (fig. 12), where they lie at the
surface of the section on the side of the spindle toward which the
knife moved. In the spindle shown in fig. 12 the chromosomes (not all
of which are drawn) number 20, including the one lying to the left of
the spindle. This fact, the displacement of chromosomes, doubtless
accounts for some of the cases where there seem to be fewer than 20.
In the spindle shown in figs. 13a and 136, for example, where there are
only 18, displacement is clearly shown in two chromosomes (x and x')
lying at the upper surface of the lower section (136); and it is quite
possible that others have been completely removed.

3. ACHROMATIN PARTS OF FIRST MATURATION SPINDLE.

The origin of the spindle has been described under Stage II. At
first broadly elliptical, it changes its form, becoming slightly sharper
at the poles and, on the average, longer and narrower, especially in the
later stages, as division approaches. The fibers are not limited to the
surface of the spindle, nor to any part of it, but are uniformly distrib-
uted, as can be seen in cross-sections of the spindle. They do not con-
verge as straight lines to a point, but curve inward toward the poles,
without, however, meeting (figs. 8, 9, 11 left end, 12, 13a, 136, 14a).
Consequently they are never parallel, and the spindle poles are more
or less open. However, in two otherwise apparently normal spindles
(figs. 10, 11) the fibers at one pole do meet at a point, from which there
are a few radiations extending into the surrounding cytoplasm.

Besides the change in proportions, there is also, on the average, a
small increase in volume. At Stages III, IVa, and IVb the average
dimensions are, respectively, in micra, 18.7 X 10.4, 19.2 X 10.8, and
22.4 X 9.9. The variations in size in each stage are considerable (see
table 2, p. 14). With metakinesis the spindles elongate considerably
and become narrower. Three such spindles, parallel or nearly parallel
to the surface of the egg (fig. 15), give as an average a length of 26 micra
and a diameter of 8 micra; another, almost exactly radial in position,
gives the corresponding measurements of 23 X n micra.

As the spindle develops, the fibers, at first in the young spindle
evident only as feeble fibrillations, become more distinct. They are

32 THE MATURATION OF THE EGG OF THE MOUSE.

usually smooth in appearance and of uniform size from end to end (figs.
8a, 86, 9, io, u). A little later they often exhibit minute, granular
thickenings at irregular intervals along their lengths (figs, n, 13a, 136,
14a). The polar ends of the fibers become thickened and more or less
confluent in the later stages (IV6 and V; figs. 12, 13a, 136, 14, 15),
frequently to such an extent that the end of the spindle looks homo-
geneous, and the fibers are distinguishable only as faint striations (fig.
15). In some cases the attachment of some of the fibers to chromosomes
is evident (figs. 10, 11, 13a, 136, 14, 14a). In addition to these fibers
there are, however, others, very delicate ones, running from pole to pole
without being attached to any chromosome (figs. 13a, 136). These
probably constitute a part of the interzonal filaments. The latter, when
the daughter chromosomes have separated, are very fine (fig. 15). Later
(figs. 16 and 17) they become thicker, and in the telophase (fig. 18) they
apparently become fused into a pale, nearly homogeneous, faintly stri-
ated bundle, lying between the two deeply stained masses resulting
from the confluence of the chromosomes. The chromosomes, drawn
nearly to the end of the spindle, lie in a somewhat deeply staining matrix
(fig. 17), which is perhaps derived from the spindle fibers.

At the middle of each interzonal filament is a thickening, a "Zwi-
schenkorperchen. ' ' The number of these was not determined. The thick-
enings, at first elongated, become more globular (fig. 1 7) , and at length by
fusion give rise to the "cell plate" (fig. 18), a disk-shaped mass staining
moderately deeply. The further fate of the interzonal filaments and the
" Zwischenkorperchen " will be discussed later (pp. 34 and 43).

4. Centrosomes, Circumpolar Bodies, and Clear Region.

Although recently the existence of centrosomes in connection with
the first maturation spindle in the ovum of the mouse has been asserted,
the evidence, so far as our preparations show, points clearly to the entire
absence of centrosomes. Not even in the two cases illustrated in plate 2 ,
figs. 10, 11, is there any hint of a centrosome at the ends where the fibers
converge to a point, although there are clearly a few fiber-like radiations
in the surrounding cytoplasm. If there were any centrosomes present,
one would expect them to stain as sharply as those in the surrounding
follicle cells during division. In the eggs from which figs. 10 and 11 were
drawn there are no polar radiations except those figured and mentioned
above, nor have any other instances been observed in which there were
polar radiations as marked as these. Occasionally a few fibers may be
observed outside the limits of the spindle (figs. 9, 11, 12, 136) and ex-
tending from the poles obliquely toward the plane of the equator.

The two conditions mentioned as characteristic of Stage IV6 are
the circumpolar bodies and the clear region around the whole spindle.
The two arise at about the same time and likewise disappear together;
they both reach their greatest prominence at the stage when the chro-
mosomes divide — at metakinesis.

OBSERVATIONS ON THE MATURATION PROCESSES. 33

The circumpolar bodies have been so named because they are
grouped around the poles of the spindle (figs. 13, 14). Their origin is
not known beyond the fact that they come into existence gradually at
the spindle poles. They are variously shaped (figs. 13a, 136, 14, 14a,
15), no one form having predominance over others. Some have irreg-
ular forms or are roughly spherical, others are pear-shaped, still others
disk-like. In ordinary plasma stains they are very inconspicuous,
apparently being composed of a homogeneous substance somewhat
denser than the surrounding cytoplasm. In phosphotungstic-acid haema-
toxylin, on the contrary, they become deep blue, like the chromosomes,
from which they are distinguishable only by their forms. They appar-
ently have no connection with the spindle fibers (figs. 13a, 136, 14a),
and after the chromosomes have reached the ends of the spindle they
fade away (plate 3, fig. 16) and disappear altogether (plate 4, fig. 18).

The clear region around the spindle is often visible in sections as a
faint, broad zone before the circumpolar bodies appear (figs. 11 and 12),
and it often persists for a short time after they have vanished (figs. 16
and 18). When most conspicuous it is comparatively narrow. It ap-
pears more homogeneous than the surrounding cytoplasm by reason of
its being less granular; but at ho time is it quite free from granules.

5. Position and Orientation of first Maturation Spindle.

The depth at which the spindles lie is variable. Whether the fully
formed spindle remains at first in the position which was occupied by the
germinative vesicle when its membrane vanished is undecided. At all
events, before the time when the chromosomes divide, the spindles may
be found at different depths. When the polar cell is about to be cut off
the spindle comes to lie near the surface of the egg, assumably in the
region of the animal pole. The axis of the spindle may be parallel,
oblique, or perpendicular to a tangent to the surface of the egg at the
point nearest the spindle. These positions are not characteristic of
particular stages, but may be found at any epoch in the maturation.
The perpendicular position is least often met with, the oblique at vari-
ous angles, and the parallel positions are the most frequent. It seems
quite possible that the spindle maintains its original orientation when
it approaches the surface to divide. At least, it is certainly true that
the perpendicular position is not requisite for the formation of the polar
cell (see p. 34), for of ten spindles in the stages shown in figs. 15, 16, and
17, only one was perpendicular, the others being either parallel or some-
what oblique. The perpendicular one was in a stage corresponding to
that illustrated by fig. 15. In nearly all examples of the stage shown in
fig. 18 the bundle of interzonal filaments is oblique to the radius of the
egg, though sometimes it varies only a little from that position. In
other cases it is very much bent, apparently as a result of a more rapid
ingrowth of the cell wall on one side during abstriction of the polar cell.

34 THE MATURATION OF THE EGG OF THE MOUSE.

6. Abstriction of First Polar Cell.

The process of abstriction begins as soon as the daughter chromo-
somes have come close to the poles of the spindle and the " Zwischen-
korperchen" have attained the condition shown in fig. 17. While the
spindle may sometimes be perpendicular to the surface of the egg, as
already stated, one pole lying in an elevation or protrusion, the condi-
tions indicate that, in most cases at
least, the spindle is either parallel or
oblique to the surface (figs. 15, 16,17).
Jpig.lba I \ -phg p0ie nearer the surface does not

M " u -/^ ... ) at first lie in the middle of the protru-

u ^.__3i/j sion, but at one edge of it (fig. 17).

The constricting process begins on
the side nearest the " Zwischenkor-
perchen," where in the surface of the

(Compare figs^a fo i6d, plate 3.) eSg a deep, sharp groove brings the

vitelline membrane into contact with
the "Zwischenkorperchen" of the side of the spindle nearest the surface.
The same condition exists also in fig. 16, in which the plane of sectioning
is very oblique to the axis of the spindle, as may be seen by comparison
with fig. H, which is a diagrammatic reconstruction of an imaginary
section of the egg in a plane perpendicular to that of the actual sections,
but parallel to the axis of the spindle. (Compare plate 3, figs. 16a
to i6(i.)

No other stage between this and that shown in fig. 18 having been
found, the further steps in the process can only be inferred. However,
it is highly probable that the contact between the vitelline membrane
and the "Zwischenkorperchen," shown in fig. 17, advances until it has
quite encircled the spindle. The result is that the entire periphery of
a disk-like body formed by the fusion of the "Zwischenkorperchen"
is finally in contact with the vitelline membrane (fig. 18), and the orig-
inal protrusion, now become more voluminous and containing the super-
ficial group of chromosomes, is thus separated from the egg. The inter-
zonal filaments, brought into a more nearly radial position during the
constriction, form the bulk of the neck of the polar cell. A little later
the constriction is completed by the ingrowth of the cell membranes of
both egg and polar cell in such a way as to cut off the interzonal fila-
ments and leave the "Zwischenkorperchen" on the outside of the cell
membranes of both polar cell and egg. Thus is formed the first polar
cell and the oocvte of the second order.

OBSERVATIONS ON THE MATURATION PROCESSES. 35

B. OOCYTE II.
1. General Description of Stages.

The chief criterion according to which an egg may be judged to
be an oocyte of the first order or of the second order is the character of
the chromatin contents. As the sequel will show, this is the only relia-
ble standard. It will naturally occur to the reader that the oocyte of
the second order must be accompanied by the first polar cell, and that
this fact would be a satisfactory criterion. But the following facts com-
plicate the situation: first, some fertilized eggs exhibit two polar cells,
some but a single one; secondly, there is dispute as to whether this
single polar cell is homologous with the first or second one of eggs hav-
ing two. In the description of the following stages it will be assumed
that the egg naturally has two polar cells, and the question as to how
many polar cells are actually formed will be treated of in a later chapter.

Stage VII. — Formation of Second Maturation Spindle.

(Plate 4, fig. 19.)
It is fair to infer from the comparatively long duration of the pre-
ceding Stage (VI) that the disk-shaped mass of chromatin which re-
sults from the more or less complete fusion of the chromosomes left
in the egg after the formation of the first polar cell probably remains
for some time without perceptible change of morphological conditions,
and that the persisting half of the interzonal filaments likewise under-
goes little change during this period. With the close of this period of
apparent inactivity Stage VII begins. It embraces only the metamor-
phosis of the chromatin mass and what are probably the achromatic
remnants of the first spindle into the fully formed second maturation
spindle. This process, unlike the one involved in the completion of
the first spindle, is so rapid that it can not be subdivided into stages
and traced step by step.

Stage VIII. — "Equatorial Plate" of Second Maturation Spindle.

(Plates 4, 5, figs. 20 to 27.)

As this stage is unique, in that it depends on the occurrence of
semination for its normal termination, it may have a greater length
than any other part of the whole maturation process, and is therefore
the one most easily obtained. If semination is early, the spindle divides
without undergoing any previous alterations; on the other hand, if the
access of spermatozoa be hindered, the spindle, though remaining com-
paratively inactive, undergoes certain changes as a result.

When newly formed, the second maturation spindle (plates 4 and
5, figs. 22 to 24) is very similar to the first spindle immediately before
its metakinesis, differing from it only in being a little smaller, in the
structure of its chromosomes, and in their more exact arrangement in
the plane of the equator. If semination is prevented, the resulting pro-
longed quiescence of the spindle is characterized by a diminution in

36 THE MATURATION OF THE EGG OF THE MOUSE.

the number of the circumpolar bodies, and often by their complete
disappearance, and by the disappearance of the clear region previously
described as surrounding the first spindle.

Stage IX. — Division of Second Maturation Spindle.
(Plate 5, figs. 28 to 30.)

The separation of the daughter chromosomes takes place, as a rule,
only after a spermatozoon has touched or penetrated the egg. How-
ever, in the case of one animal — a mouse which had not been insemi-
nated— one of the eggs contained the divided chromosomes arranged
in two parallel daughter plates, which were still near the equator of the
spindle ; another egg from the same mouse presented a stage still further
advanced (plate 5, fig. 28), the two groups of daughter chromosomes
in this case having migrated nearer to the poles of the spindle.

Stage X. — Telophase of Second Spindle and Second Polar Cell.

(Plate 5, fig. 30.)

The beginning of the abstriction of the second polar cell resembles
that of the first. This stage, indeed, agrees so closely with the corre-
sponding stage in the formation of the first polar cell (Stage VI, p. 27),
from which it seems to differ only in the presence in the oocyte of the
head of a spermatozoon, that it need not be described here. It may be
said that, of 30 eggs in this stage, only 1 failed to show the head of a
spermatozoon.

2. CHROMATIN PARTS OF SECOND MATURATION SPINDLE.

The chromosomes of the second maturation spindle arise directly
from the chromatin mass which remains in the egg after the abstric-
tion of the first polar cell, i.e., without an intervening vesicular stage
of the nucleus. This mass breaks up into fragments, but whether or
not each of these fragments is the equivalent of a chromosome, either
single or multiple, it is difficult to determine. Whatever their mode of
origin, the fragments are fairly (or even very) irregular in form, incom-
pletely separated from one another, and of uncertain number (plate 4,
fig. 19). Some of them bear a slight resemblance to the daughter chro-
mosomes of the previous division which had nearly reached the poles of
the first spindle (fig. 16) . Sometimes (fig. 19b) they are embedded in a ma-
trix of homogeneous substance denser than the surrounding cytoplasm.
They are never scattered, and soon become arranged in the plane of the
equator of the future spindle, where they may constitute a group having
the form of an imperfect ring. No stages between this and that of the
completely formed chromosomes have been observed.

The chromosomes of the completed second spindle (figs. 23 and 24)
often closely resemble the daughter chromosomes of the first spindle
as they appear when they have nearly finished their poleward migra-
tion (fig. 16), for each mother chromosome of the second spindle is com-
posed of a pair of elements, and these elements vary in form, independ-

OBSERVATIONS ON THE MATURATION PROCESSES. 37

ence, and relative position (figs. 23 to 27, plates 4, 5; fig. /). Fig. /
(m to r) illustrates several of these variations. In the simplest form of
chromosomes, shown at m, each element is a straight rod, either of uni-
form size (see also fig. 25), or slightly constricted in the middle. The
constriction is likewise evident in chromosomes seen when looking
nearly in the direction of the axis of the spindle (figs. 20, 21). Viewed
under these conditions usually one element of the pair is partially cov-
ered by the other. Even after the separation of the daughter chromo-
somes from each other, this constriction or dumb-bell condition of the
daughter chromosome is evident, whether seen endwise (fig. 28) or in
side view. In p (fig. /) the constriction is carried still further, some-
times to such an extent that the mother chromosome appears to be com-
posed of four nearly independent parts (x, fig. 24a). Sometimes the
daughter chromosomes are curved rods (fig. /, r; plate 5, figs. 246, 26),
or are of an irregular crescent shape (0) . Fusion or adhesion of the two
elements at one or more points gives rise to figures like n (see also figs.
20, 23a, x). When the elements are more elongated and curved, rings
(q, also figs. 24a, 25, 26) are formed by the fusion of the corresponding
ends of the two daughter chromosomes. Occasionally, when the fusion
of the ends (as in n) is well advanced and the constriction in the middle
of each is complete, the original
separation between the two ele- ^_^ ^^ air, „|B» JBm §

SS wC o\

ments is obscured and the mother

chromosome then appears to be

composed of two parts, the long m n ° V V r

axes of which are perpendicular to Fig. I.

the plane of the equator. Forms

like those shown in figs. 25 and 2 7, which occur in eggs that have remained

long in the oviduct, are explained by the fact that with age the elements

tend to elongate. In any of these forms of chromosome the parts may

be parallel to each other, or, according to the point at which the spindle

fibers are attached, separated at one end (n, p, r) or at the middle (p, q).

The chromosomes are never arranged at the surface of the spindle,
but from the beginning are uniformly distributed in the plane of its
equator (figs. 20 to 24), and are so oriented that that plane passes be-
tween the two elements of each mother chromosome. This arrangement
of the daughter chromosomes in one plane is preserved even after meta-
kinesis (figs. 28a and 286).

The number of chromosomes is 20; but the proportion of cases in
which the number can be determined with accuracy is smaller than in
the case of the first spindle, because in the second spindle the chromo-
somes are more crowded and their forms are less regular than in the
first spindle. When, in cutting, the chromosomes fall in two sections
the difficulty of counting is usually increased. However, knowing the
structure of the chromosomes, it has been possible in many cases to be

$8 THE MATURATION OF THE EGG OF THE MOUSE.

quite certain that the number is 20 (see table 2, p. 14). In figs. 23a and
236 there are only 19 chromosomes, one probably having been lost in
cutting. Figs. 24a and 24b exhibit together 20, one having been so cut
that a half of it lies in each section. The two sections (24a and 24b)
contain, respectively, 8.5 and 11.5 chromosomes. Polar views of the
"equatorial plate" are usually the most satisfactory ones for counting.
In fig. 20, a polar view, there are clearly 20 chromosomes; one of these
(x), seen in face view, corresponds to fig. /, n. In figs. 28a and 286 (an
anaphase) the number can not be determined with perfect accuracy,
because the long axes of the daughter chromosomes are perpendicular
to the plane of the section. Two of the larger chromosomes (x and x')
may well be double; if so, the number in this case also is 20.

In the division of the chromosomes, the two elements of each
mother chromosome separate and then migrate to the opposite poles
of the spindle. Figs. 28a and 286 (plate 5) are polar views of the two
daughter plates at a stage of migration corresponding to that of fig. 16,
and are drawn from a non-seminated egg. In fig. 29, which represents
a slightly later stage than fig. 28, the individual chromosomes are no
longer distinguishable. They seem quickly to lose their identity and
merge into a single disk-shaped mass (fig. 30), as in the case of the first
spindle.

3. ACHROMATIN PARTS OF SECOND MATURATION SPINDLE.

The interzonal filaments left in the egg after the first polar cell is
cut off persist for a while along with the chromatin mass. About the
time when the chromatin breaks up into fragments, they lose their con-
nection with the cell plate (plate 4, fig. igb). It is probable, but not cer-
tain, that they contribute to the formation of the matrix in which the
chromatin fragments are embedded, and also to the formation of the
fibers of the completed second maturation spindle.

The second spindle begins as a somewhat pear-shaped, apparently
homogeneous body at the time when the chromatin mass divides into
fragments. When completed it is more or less elliptical (fig. 22), like
the first spindle, but it varies more in form than does the first spindle,
being occasionally more slender and having more sharply pointed ends.
However, as observed from the surface of the egg, it often appears very
broad (figs. 23, 24), owing to its being flattened in the direction of the
radius of the egg (fig. 20). Such spindles when seen edgewise appear
very narrow (fig. 22) ; they always lie nearer the surface of the egg than
those which are circular in cross-section.

The fibers of recently formed spindles resemble quite closely those
of the later stages of the first spindle in being smooth, of uniform
diameter — except at their polar ends, where they are thickened — and
curved inward toward the poles (figs. 22 and 24). The thickenings at
the polar ends are not to be seen in fig. 23 , because the spindle was stained
in Bohmer's haematoxylin and Congo red, which are not favorable for

OBSERVATIONS ON THE MATURATION PROCESSES. 39

demonstrating the fibers and circumpolar bodies clearly. As a rule,
the individual fibers and their attachment to the chromosomes are not
easily distinguishable. Although one can not be absolutely certain that
there are fibers which are continuous from pole to pole without being
connected to any of the chromosomes, it is perhaps reasonable to assume
that such is the case, because of the general similarity of the second
spindle to the first one, where such a condition is fairly evident. The
daughter chromosomes, after their migration toward the poles of the
spindle, are connected by interzonal filaments (figs. 29, 30). " Zwi-
schenkorperchen " form midway between the ends of the filaments, as
described for the first spindle (p. 32), and later fuse into a cell plate.

The second spindles do not differ from one another much in size,
nor do their dimensions, on the average, change appreciably with pro-
longed existence due to the absence of semination. This constancy in
size is shown by a comparison of spindles from two groups of eggs : one
group composed of eggs which have been but a short time in the oviduct
(taken not later than 16^ hours after parturition), the other of eggs taken
from the oviduct 29 or more hours after parturition. Because of the unfa-
vorable position of many spindles, measurements of only 30 young and 26
old ones could be used. The average dimensions for the young spindles
are: length 17.9 micra, diameter 7.2 micra; for the old spindles: length
17.5, diameter 7.3 micra. A comparison of these averages with those of
the mature, or nearly mature, first spindles in Stages IVa and IV6 (viz.,
19.2 X 10.8 micra, and 22.4 X 9.9 micra, respectively) proves that the
second maturation spindle is somewhat smaller than the first.

4. Centrosomes. Circumpolar Bodies, and Clear Region.

For the second spindle, as for the first, the existence of typical
centrosomes is highly improbable. However, there are at certain times
structures which to some extent resemble centrosomes.

The circumpolar bodies (figs. 22, 24) correspond exactly in posi-
tion, abundance, and general appearance to those of the first spindle.
When the spindle is first fully formed they are already present, and per-
sist for some time. But with spindles which, in the absence of semina-
tion, persist for a long time they have a tendency to dwindle away,
sometimes, however, leaving a few granules at the poles where centro-
somes might be expected (figs. 25, 26, 27). These statements are based
upon a comparison of the eggs used in calculating the size of the spin-
dle. The eggs in the oviduct (and a few in the periovarial space and
in the ovary) taken from mice killed i6£ hours or less p.p. show in most
instances well-developed circumpolar bodies, whereas most of the eggs
from animals killed 29 or more hours p.p. show very few or none of them.
These bodies also disappear following normal metakinesis induced by
semination after the chromosomes have migrated and become confluent
(figs. 29a, 30), as in the case of the first spindle.

THE MATURATION OF THE EGG OF THE MOUSE.

In some of the eggs from a mouse killed about 33 hours p.p. and
not inseminated, there appear a few cytoplasmic radiations at the spindle
poles, from which the circumpolar bodies have vanished. Also in the
egg illustrated in fig. 29a, the cytoplasmic granules around the inner
end of the spindle are oriented with their long axes in a radial direction ;
but otherwise the evidence of cytoplasmic radiations about the poles
of the second maturation spindle has been lacking.

As already stated regarding the first spindle, the clear region around
the spindle exists at the same time with the circumpolar bodies, except
that it may appear a little before them (fig. 19), and sometimes persists
longer (fig. 30). It can usually be found surrounding the spindles of
eggs which have been only a short time in the oviduct (figs. 20, 21, 22,
23, 24), but in most eggs in which the circumpolar bodies have vanished,
it has likewise disappeared (figs. 25, 26, 27). It becomes quite faint
(figs. 29, 30), or is altogether gone, after the chromosomes have divided.

5. Position and Orientation of second maturation spindle.

The second maturation spindle always lies near the surface of the
egg — in fact, sometimes so near that its flatness (fig. 20) is apparently due
to pressure. There is no satisfactory evidence that it moves through
the cytoplasm, although it is found at different distances from the first
polar cell when that is present. This topic will be taken up later (pp. 44
and 63).

There is less variation in the orientation of the second spindle
than in that of the first. Very rarely, indeed, can it be found perpendic-
ular to the surface; occasionally it is oblique, but in the majority of
cases it is parallel to the surface. It is parallel in all instances in which
the daughter chromosomes have separated and have reached, or nearly
reached, the poles before the abstriction of the second polar cell begins;
but in those in which the abstriction has begun (figs. 29a, 296) it is

oblique ; and in the stage of the telophase
of the formation of the second polar
cell (fig. 30) the interzonal filaments are
usually almost , if not quite , perpendicular.

Fig. 29a

6. Abstriction of second Polar cell.

The process resulting in the forma-
tion of the second polar cell is precisely
like that by which the first polar cell is
Fig. /. produced. The beginning of the process

is illustrated by an egg shown in part
in figs. 29a and 296 and in fig. /. The last is a diagrammatic, imaginary
section of the egg, in a plane parallel with the axis of the spindle, but
perpendicular to the actual sections shown in figs. 29a and 296. The
daughter chromosomes have virtually reached the poles of the spindle
and have lost their identity by being merged together; the " Zwischenkor-

OBSERVATIONS ON THE MATURATION PROCESSES. 41

perchen " now occupy the middle of the interzonal filaments. The spindle
is oblique to the surface of the egg, and one pole is so near the surface
(fig. 29a) that the peripheral mass of chromatin lies close to the edge of
the protrusion which is destined to be cut off to form the polar cell. The
constriction has begun on the side nearest the "Zwischenkorperchen,"
the vitelline membrane being already in contact with the " Zwischenkor-
perchen" nearest the surface of the egg (fig. 296). The rest of the
process, involving the final separation of the polar cell, is as described
on page 34 for the first polar cell.

C. RIPE EGG.

Stage XI. — The Pronuclei.

A discussion of the further development of the ripe egg does not
lie within the scope of the present work. It suffices to say that the chro-
matin mass resulting from the union of the chromosomes remaining
after the formation of the second polar cell is quickly transformed into
the egg nucleus. This usually occurs simultaneously with the develop-
ment of the sperm nucleus. But in two cases the egg nucleus had reached
a diameter of 6 micra, while the head of the spermatozoon had not been
appreciably changed in form or size. In no case has the sperm nucleus
been observed before the chromatin mass has begun to be transformed

into the egg nucleus.

D. POLAR CELLS.

The observations on the polar cells here recorded do not extend to
the cleavage stages of the egg. Therefore, no statement can be made
concerning the further fate of the polar cells, or concerning the changes
which take place in the second polar cell.

First polar Cell.

The first polar cell, originating as described on page 34, is usually
an ellipsoidal or a flattened spheroidal body, the three diameters of which
are nearly always unequal. The average dimensions of 28 polar cells —
each of which had been recently formed (Stage VI), the first spindle
being still in the telophase (plate 4, fig. 18) — were 22.7 X 19.2 X 13.5
micra. These figures indicate the average size at its largest stage. With
age some polar cells diminish very rapidly in size (figs. 18, 32-37, plate 6) ;
others retain nearly their original dimensions. Disregarding for the pres-
ent the very small forms (figs. 35-37), it is found that the first 50 polar
cells (which could be measured most accurately) from 100 of the young-
est eggs which have the complete second spindle give as an average the
following dimensions in micra: 20 X 15.6 X 11.8; and 22 polar cells
(all that could be measured) from 100 of the oldest eggs of the same
stage give the following average dimensions: 16 X 13 X 10.5 micra.
These averages show a considerable decrease in size; and, as a series of
gradually diminishing sizes can be found down to that shown in fig. 37,
and as the smaller sizes are too numerous (55 out of 507 eggs) to be mere

THE MATURATION OF THE EGG OF THE MOUSE.

chance occurrences, it must necessarily follow that the first polar cell

may, in many cases does, dwindle to almost nothing. Indeed, it may

even disappear completely; for out of the 507 eggs with complete second

spindle, 189 have no polar cell. This is made clearer still when the 200

eggs, mentioned above, are examined further. The results are most

conveniently presented in tabular form (table 5). This shows that of

the older eggs, as compared with the younger ones, fewer have the large

polar cells and more have no polar cell. The fewer cases with small

polar cell among the older eggs show that most of the polar cells which

degenerate do so early, being completely wanting in the later epochs.

The same conclusions are borne out by the 162 eggs of Stages IX to

XI (table 2, p. 14), which, as a whole, cover a longer period. Of these

162 eggs, 77 have no polar cell, 22 have a small polar cell, and 63 the

larger sizes of polar cell.

Table 5.

Young.

Old.

E<rgs with larsre first polar cell

72
14
14.

fi7

Eggs with small polar cell (figs. 36, 37) ... .
Eggs with no polar cell

Totals

IOO TOO

The first polar cell contains the peripheral group of chromosomes,
which have become compacted into a single, usually flattened mass
(plate 4, fig. 18). During the formation of the second spindle this mass
divides into irregular parts (fig. 19a), which remain more or less in conti-
nuity with one another. It is only rarely that these parts separate from
one another completely and assume the aspect of dumb-bell shaped bodies.
Their number, however, has no significance, owing to their imperfect
form and individuality. The chromatin may remain for a considerable
period in one, or more than one, loosely formed mass. If it is more finely
divided, the fragments may be distributed with tolerable uniformity
throughout the cytoplasm (plate 5, figs. 30, 31a), or roughly aggregated
into two groups, one at each end of the cell. Not infrequently the chro-
matin bodies exhibit thread-like forms, especially in connection with what
appears otherwise to be anon-mitotic division of the polar cell (figs. 3 2, 3 3).
In no case, however, has it been observed that the chromatin is drawn
to the equator of a well-formed spindle and divided. Often the chromatin
fragments, especially the enlarged ends of the thread-like forms, show
vacuolation (figs. 30, 32, 33). Besides the deeply staining chromosomal
bodies, there are other less deeply staining bodies (figs. 2gb, 32, 33,34,35),
which apparently are modified chromatin; these occur either alone —
especially is this the case in small polar cells (figs. 34, 35) — or associated
with vacuolating parts (figs. 32, 33). These conditions all seem to point
to a degeneration of the chromatin. A nucleus is never formed, unless

OBSERVATIONS ON THE MATURATION PROCESSES. 43

perhaps it arises in divided first polar cells during the cleavage stages of
the egg.

Although the cytoplasm of the polar cell has not been studied care-
fully by us, its general features are as follows. In the newly formed
polar cell the more distal part of the cytoplasm appears very clear (figs.
18, 19). Later, it is of uniform appearance throughout the cell, and in
some cases is apparently like that of the egg ; but more often it is either
more granular or more homogeneous and clear than the egg cytoplasm.
In the smaller polar cells it has the latter structure and it sometimes
shows what appear to be ill-defined vacuoles (fig. 36). The interzonal
filaments within the polar cell are, at first, very evident (figs. 18, 19). In
time they lose their connection with the cell plate (figs. 19 and 31a), which
then quickly disappears. Occasionally there can be observed in the polar
cell fibers which are parallel with one another ; but it is uncertain whether
they are the remains of interzonal filaments or fibers of an abortive
spindle.

It may be inferred from the amitotic (or imperfect mitotic) division
of the chromatin that the whole polar cell is capable of division. Such,
indeed, is the case, for, previous to the formation of the second polar
cell, the first polar cell may be observed in many instances to be divid-
ing into two or more parts, as shown in figs. 32 and 33, or to be simply
constricted (fig. 31a). Less frequently the small polar cell is seen to be
already divided into two parts. This dividing of the polar cell doubt-
less aids in its rapid degeneration by increasing the external surface
exposed to the action of absorption.

The polar cell quickly loses its connection with the egg, because the
interzonal filaments become severed from the cell plate. This separa-
tion is evident as early as the time of ovulation and may be aided by
that process, as described on page 22 and shown in figs. 31a and 316, 38,
39, and 40 (figs. 31a and 316 being enlarged views of sections of the egg
and polar cell of which fig. 40 shows another section) . In the egg illus-
trated in figs. 31a and 316 the polar cell is separated from the egg and
probably from the cell plate, which is seen in fig. 316. (In this case,
however, the existence of the cell plate is a little doubtful.) The evi-
dence leads to the belief that the first polar cell need not remain at the
place where it was formed, but may, according to circumstances, change
its position under the zona, even to such an extent as to come to lie
diametrically opposite the point of its origin. The bearing of these
observations on the question of the relative positions of the first polar
cell and the second spindle will be considered later (p. 63). The first
polar cell usually lies in a depression in the surface of the egg.

44 THE MATURATION OF THE EGG OF THE MOUSE.

SECOND POLAR CELL.

The shape of the second polar cell is similar to that of the first,
though it is perhaps more often uniformly regular in shape. In order
to compare the size of the second polar cell with that of the first, meas-
urements were made of as many newly formed polar cells as possible
(Stage X, fig. 30). Since the condition of the polar cells during cleavage
stages of the egg has not been studied, changes in size are not here con-
sidered. For convenience the sizes of the first polar cells (exclusive of
the small degenerate forms) are repeated in this connection (table 6).

Table 6. — Size of polar cells.
Average dimensions of first polar cell. Mi era.

Newly formed polar cell (first spindle in

telophase) 22.7X19.2X13.5

From eggs but a short time in the oviduct

(complete second spindle) 20 X15.6X11.8

From eggs after 29 hours in the oviduct. .. 16 X13 X10.5

Average dimensions of second polar cell.
Newly formed 19. 3X16. 7X 9.6

When first produced the second polar cell, then, is smaller than the
first polar cell of corresponding age, but is larger than the first polar cell
which has been in existence for 29 hours or more.

At the beginning, the chromatin of the second polar cell is in a
single mass, as in the case of the first polar cell, but it does not long
remain so, for it is quickly transformed into a nucleus.

The cytoplasm in the recently cut off cell (fig. 30) has the clear
appearance noted in the case of the first polar cell, but later it generally
has the aspect of the protoplasm of the egg. The interzonal filaments
persist for a time and can be observed joining the nucleus of the polar
cell with that of the egg, the cell plate remaining as a conspicuous,
deeply stained body outside both egg and polar cell.

The position of the second polar cell with regard to the first (when
the latter is present) is variable, for the two polar cells may lie side
by side or be far apart. The reason is probably to be found in the migra-
tion of the first polar cell, as discussed on page 63. The second polar
cell, like the first, occupies a slight depression in the surface of the egg.

CRITICISMS AND CONCLUSIONS. 45

VIII. CRITICISMS AND CONCLUSIONS.
A. MATERIAL.

This work differs from that of previous investigators in that it has
been done on mice of very mixed ancestry. It is therefore open to the
possible criticism that the material is unlike that on which other papers
have been based. It may be maintained, however, that there is no
essential difference in material for the following reasons: first, the fact
that the white mice of our stock, whether of colored ancestry or not,
breed true, leads one to believe that, in the light of recent work on hered-
ity of coat color, they are as pure as other white mice; secondly, there
is no dissimilarity in the maturation processes of eggs from mice of dif-
ferent coat character; thirdly, there is no real difference in important
points between Mr. Kirkham's preparations and our own.

Sobotta suggests in his paper published in 1907 that some of the
differences between his results and those of Gerlach (1906) may be due,
in part at least, to the fact that he used eggs set free at an ovulation 3
weeks after parturition, whereas Gerlach employed ova obtained during
the first 3 days after parturition. Since Sobotta is the only one who has
made use of eggs derived from an ovulation later than the first one after
the birth of young, his explanation must apply to all other investigations,
including the present one. There seems, however, to be no a priori
reason for supposing a difference between the maturation processes of
eggs maturing and ready for fertilization at different periods after par-
turition; moreover, the dissimilarities in the results of the several in-
vestigators can be accounted for to a large extent on other grounds, as
will appear in the course of the remaining pages.

Considerable significance attaches to the amount of material studied
by other investigators. Tafani, Gerlach, and Kirkham do not state the
number of eggs which they observed, but the number was probably
small. Lams et Doorme based their paper on only 90 ova. Sobotta in
his large work (1895) used 1402 sound eggs; but of this number only 298
(compared with our 877, table 2, Stages I-X), at the most, were of
such age as to show stages in the formation or division of spindles, or
the number of chromosomes, or the abstriction of polar cells. All the
rest (1104) were either in stages showing the pronuclei or still older.

B. METHODS.

It is probable that the value of our results would have been en-
hanced had another set of eggs preserved by other methods been com-
pared at each step with those which have served as the basis for the
present paper. These have all been carefully studied and in part are
described and figured here. However, it should be said that other fix-
ing fluids were tried, and that, in cases where the preservation was good
enough to give reliable pictures, the eggs showed conditions similar to
those obtained with the special preserving fluid described at page 12.

46 THE MATURATION OF THE EGG OF THE MOUSE.

This modification of Zenker's fluid, however, is the only one tried which
shows the finer structure of the chromosomes and does not shrink the
nuclei. Most of the figures by other investigators of the mouse egg
show imperfect preservation, and this has been, in our opinion, a potent
factor in causing the differences in their results.

C. TIME RELATIONS.

The possibility of obtaining a complete series of stages of the proc-
esses of maturation depends on accuracy in determining the epochs
of parturition and insemination. As we have seen (p. 22), there is
probably some individual variation in the length of the periods between
successive ovulations. If eggs from ovulations other than the one which
immediately follows parturition had been used by us, it would have been
extremely difficult, if not impossible, to secure a complete series, both
because of this variability in ovulation, and also because (see p. 17)
the stage of the second spindle may last for many more hours than the
stages which reach from the transformation of the germinative vesicle
to the formation of the first polar cell. It is probably a lack of precision
in this matter which accounts for the failure of others to get those stages
which pass quickly, such, for example, as the origin and metaphase of
the first spindle.

As we have seen (pp. 16 and 19), the first maturation after parturi-
tion may occur during a period extending from about 13 hours to 29
hours p.p. Tafani (1889, p. 20) makes the period extend from 24 hours
to 48 hours, or even (18896, p. 113) 2 or 3 days p.p., a time somewhat
later than that indicated by our observations.

Sobotta (1907, p. 504) says that the prophases of the first spindle
begin at least 24 hours before ovulation; but as he does not say when
ovulation occurs with respect to parturition (which is the only event
that can be determined directly), it is impossible to perceive how he
arrives at this particular number of hours as the minimum time. Appar-
ently Sobotta (p. 507) bases this conclusion on the parallelism which,
he maintains, exists between the histological changes in the wall of the
follicle (its ripening) and the ripening of the egg; but admitting the paral-
lelism, and granting that the prophase begins when the follicle is far
from ripe, we are unable to see any very precise ground for the estimated
time required for the ripening of the follicle.

Kirkham (1907a, p. 259) states that he killed mice at various times
during pregnancy and at intervals from a few minutes after parturition
up to 30 hours after that event. In his later paper (19076, pp. 70, 71)
he adds:

The ovaries of every mouse examined during the height of the breeding season
contained some eggs in which the first polar body had been already extruded and
in which the spindle for the second polar mitosis was fully formed. A majority of
the same ovaries revealed ovarian eggs at the end of the spireme or with the first
polar spindle.

CRITICISMS AND CONCLUSIONS. 47

Also (p. 75):

A large number of eggs in different ovaries have been examined, and in every
instance where the size of the egg, its slightly denser protoplasm, and the large folli-
cle gave evidence of ripeness, the egg was found to be accompanied by the first polar
body. This agrees with the observations of Bellonci (1885), and with Sobotta's
idea regarding 10 per cent of the eggs, which he believed formed two polar bodies.

These two statements appear at first sight either to relate to differ-
ent stages of maturation or else to be difficult to reconcile with each
other; but further consideration leads us to think that the same condi-
tions are intended in both. According to the first quotation, a part of
the more advanced eggs are only just beginning maturation (spireme
or first spindle), while others are further along, showing the first polar
cell and second spindle. In the second quotation only the older eggs,
those with the first polar body, are mentioned; but it is perhaps fair to
infer that here, too (as announced in the first statement quoted), others
were just beginning the process of maturation, though it is explicitly
stated that "in every instance" the first polar body was present. How-
ever that may be, it is clearly stated that in every mouse examined
during the height of the breeding season the ovary contained some eggs
which showed the first polar cell and the second spindle. Since the author
certainly studied and figured (his figs. 12-17) eggs from the Fallopian
tube, it is impossible to avoid the inference that in all females, even in
those in which one set of eggs is in the oviduct, the ovaries contain eggs
with the first polar cell and the second spindle already formed; that is
to say, maturation may begin several weeks before parturition or ovu-
lation. But such a state of affairs is incomprehensible to us, because,
according to our studies, mice killed during pregnancy and at intervals
of 7 and 14 days after parturition furnished ovarian eggs (these have
not been included in the 1,000 eggs recorded in table 2) some of which
were in fairly large follicles. Those in the largest follicles (eggs which
presumably were destined to leave the ovary at the next ovulation)
possessed in all cases the germinative vesicle. Such was also the case in
mice killed during a period extending from 1 to 13 hours after partu-
rition. Eggs with the germinative vesicle, which, as has already been
explained (p. 16), do not acquire the first spindle before about 13 hours
post partum, manifestly could not originate by the transformation of
eggs already possessing a polar cell and second spindle. Moreover,
mice which showed a group of eggs in each oviduct never exhibited any
of the large follicles in the ovary. Lastly, as has already been demon-
strated (p. 15), only two mice furnished eggs in stages as widely separated
as those of the germinative vesicle and of the first polar cell and second
spindle ; and in these two cases the eggs exhibiting the early stage were
in one ovary, while the eggs showing the later stage were in the oviduct
of the other side of the body. At first the only explanation of the differ-
ences between Dr. Kirkham's results and our own which seemed to us
possible was that his mice were of a different breed from ours.

48 THE MATURATION OF THE EGG OF THE MOUSE.

Through the kindness of Professor Coe, of Yale University (Dr.
Kirkham being abroad) , we had the privilege of examining a portion of
Dr. Kirkham's preparations, some 25 slides, on which the position of
eggs with first polar cell and second spindle and that of eggs with a
single spindle had been marked by the author. An examination of these
preparations revealed the fact that nearly all of the ovarian eggs so
marked were in process of degeneration. They were of about normal
size, but occurred in rather small follicles, approximately like the one
shown in Kirkham's (19076) plate V, fig. 11. The zona pellucida was
gone, and the granulosa cells were only rarely in contact with the egg —
sure signs, in our opinion, of degeneration. Such eggs can be found in
nearly all ovaries; but we have always rigidly excluded them, because
they are so obviously different from the normal eggs contained in the
large follicles. Sometimes in these small follicles there can be found
clusters of cells resulting apparently from the abnormal cleavage of de-
generating egg cells. These facts explain, we think, fig. 7 of Kirkham's
second paper (19076), a figure which Sobotta (1908, p. 260) could not
understand, and also fig. 11 of the same paper, which is clearly that
of a degenerating egg. Kirkham (19076, p. 77) says, in explanation of
the absence of the zona from this and all other eggs of the same series
(presumably the same animal), that it is "probably due to the solvent
action of the killing fluid." But it certainly would be remarkable if
the same killing fluid operated so differently on different ovaries. The
explanation which we have suggested — a degenerating condition of the
ova — is rendered still more probable by the fact that "all the ovarian
eggs in this series are likewise naked." Tafani (1889, p. 24) in his criti-
cism of Bellonci expresses the opinion that the latter saw in degenerating
follicles eggs which never would have been set free, but which formed
polar cells. Such eggs are just what Bellonci, having little material,
would probably have seen and misinterpreted, for the reason that they
occur in all ovaries of mature mice at all times, whereas normal eggs con-
taining the first spindle or the first polar cell and second spindle can be
found only during a very limited period. However, it must be borne
in mind that, while Tafani did not misinterpret degenerating eggs, he
did confuse the first and second spindles. He saw the first spindle in
the ovarian egg, but apparently not the formation of the first polar cell,
and seeing a spindle (the second) in eggs in the oviduct without the first
polar cell, he mistook it for the first spindle. That he missed the stage
of the abstriction of the first polar cell is rendered the more probable
by the fact that he placed the period of maturation rather late and studied
so many eggs from the oviduct. Nevertheless, Tafani's criticism of
Bellonci was probably sound.

There are apparently no statements in any of the works on the em-
bryology of mammals which show precisely how much time is required
for any part, or the whole, of the maturation process. Indeed, the

CRITICISMS AND CONCLUSIONS. 49

length of time required in the mouse according to our observations,
namely, from 4 to 15 hours, needs confirmation.

According to the calculations of Tafani (18896, p. 114) the interval
between coitus and the penetration of the spermatozoon is 7 or 8 hours,
of Sobotta (1895, P- 63) and Gerlach (1906, p. 8) 6 to 10 hours. Tafani
and Sobotta think the formation of the pronucleus requires only about
an hour from the time the spermatozoon penetrates the egg; whereas
Gerlach does not believe the pronucleus is formed so quickly. We have
already (p. 21) shown that the interval between coitus and penetration
may be much less, viz, 4 to 7 hours, and that the pronuclei probably
require only a few minutes for their development.

D. OVULATION.

It is desirable to know whether the time of ovulation has any fixed
relation to that of either coitus or parturition.

All investigators except Gerlach (1906, p. 22) agree that in the
mouse ovulation is independent of coitus, although such is not the case
in some other mammals, e.g., the rabbit and the guinea-pig.1 Regarding
the relation of ovulation to parturition, Kirkham (19076, p. 79) is the
only one, so far as we know, who makes any statement. He says that
ovulation takes place in from 1 to 2 hours after parturition; but as he
cites no authority for the statement and furnishes no evidence of his
own, one can not give his conclusion much weight. We have already
given evidence that it occurs at some time during a period extending
from 14^ to 28^ hours after parturition.

There is some difference of opinion concerning the relation of the
time of ovulation to that of maturation, the chief cause of which seems
to us to be the failure to find any critical basis for distinguishing between
the first and the second maturation spindles. Tafani (1889, p. 22) says
ovulation occurs during the stage of the first spindle. While this, in
our opinion, is not true, the statement can be explained on the highly
probable assumption that he confused the first and second spindles.
Sobotta has changed his opinion since writing in 1895, ano^ now (1907,
PP- 5I5> 5X9> 546; 1908, pp. 247, 250) believes that ovulation occurs only
during the monaster stage of the second spindle. He never finds the first
spindle in eggs encountered in the oviduct, but describes, as being found
in the oviduct (1907, p. 524, fig. 8), what he thinks may be a transition
stage between the first and the second spindles. Gerlach (1906, p. 14)
believes that the changes in the wall of the follicle that make ovulation
possible are not directly connected with the maturation changes within
the egg itself, and therefore that the rupture of the follicle may take
place at various phases of maturation ; but he says that at the earliest the
egg leaves the ovary in the stage corresponding with the beginning of
the first spindle, and at the latest in that of the second spindle; but this

1 Cf. Kirkham, 19076, p. 79.

50 THE MATURATION OF THE EGG OF THE MOUSE.

statement is based on his assumption that oviducal eggs without polar
cells contain the first spindle, a view which arises from his being unable
to distinguish between the two spindles in the monaster stage. This
statement of Gerlach's has been disproved by Sobotta.

Lams et Doorme (1907, p. 284) maintain that ovulation takes place
only during the stage of the second spindle; but, as Sobotta (1908,
p. 259) points out, they contradict themselves by describing as a first
maturation spindle one found in an ovum occupying the oviduct. Ac-
cording to Kirkham, the first polar cell is always formed in the ovary;
but, as we have seen, this statement is supported, in part at least, by
false evidence. In spite of some diversity of opinion regarding the pre-
cise state of the egg at ovulation, all agree that ovulation occurs during
the stage of the second spindle. We, too, find this to be generally but
not invariably true. It is probably owing to the unusually large number
of eggs in the earlier stages of maturation studied by us that we have
found in the periovarial space eggs in the stage of the first spindle, and
also in the oviduct others that have already formed the first polar cell
but have not yet developed the second spindle. It might be maintained
that these eggs had been abnormally retarded in their development,
and it must be admitted that such cases are not numerous enough to
allow one to say that it is a common condition. On the other hand,
nothing else about these eggs pointed to their being in any way abnormal,
and no signs of degeneration were discoverable. These cases seem,
therefore, simply to prove that the general rule regarding the time of
ovulation in relation to maturation is not so inflexible as one wTould
infer from the observations hitherto published.

E. SIZE OF EGG.

Sobotta and Kirkham alone have published measurements of the
egg, Sobotta on fixed material and Kirkham on living material. Sobotta
(1908) states that ovarian eggs before the formation of the first polar
cell measure from 65 to 70 micra in diameter, and oviducal eggs 60 micra;
but he does not say what is the average in the former case, nor that the
latter measurement is an average, though such is presumably the case.
Gerlach thinks there is considerable individual variation, and Lams et
Doorme hold that oviducal eggs are smaller than ovarian ones. Our
conclusions (see table 2, p. 14, also p. 24) substantially confirm the above,
except that the averages we give are a little less than the dimensions
published by Sobotta. Kirkham (19076, p. 72) arrives at a different con-
clusion, namely, 80 micra as the diameter of ovarian eggs and 73 to 78
micra of oviducal eggs; but there may be some doubt concerning the
reliability of his measurements because his methods may have been
somewhat faulty, as we shall explain directly. Tafani, who was the first
to study living eggs, carefully states (1889, p. 6) that he collected them
from the oviduct and kept them at the proper temperature in the fluid

CRITICISMS AND CONCLUSIONS. 5 1

from the ovarian capsule or oviduct; but, unfortunately, he does not
give the dimensions, and his figures are too diagrammatic to serve as a
means of determining size. Kirkham has apparently overlooked the
above statement, for he says that Tafani makes no mention of the method
used to obtain living eggs. Kirkham (19076, p. 70) procures them by
killing a female soon after ovulation is supposed to have occurred, re-
moving the ovaries and Fallopian tubes to a slide, and gently teasing
them with fine needles until the eggs are seen to drop out ; he then trans-
fers them to the stage of the microscope for study. Kirkham does not
state in what fluid he studied the eggs. The medium, however, is impor-
tant, since it might, if not like the natural fluid in osmotic action, either
swell or shrink the egg. We have already shown that a prolonged stay
of eggs in the oviduct in the several cases results in an increase in their
size, the eggs used for comparison being also subjected to precisely the
same treatment as those from the oviduct. Since Kirkham's determina-
tion of the time of ovulation is in error by 10 hours or more, it is a little
doubtful whether all his eggs were in a normal condition.

F. MATURATION PROCESSES.
1. Germinative Vesicle.
It is agreed by all investigators that the germinative vesicle is at
first very near the center of the egg, and that it becomes more eccentric
as the time of its transformation into the first spindle approaches.
Tafani and Gerlach both state that its membrane becomes irregular
and disappears soon after the chromosomes have begun to form.

2. First Spindle.

Chromatin.

Tafani (1889, p. 21) believed that by the rupture of the germina-
tive vesicle the nucleolus escaped as an angular chromatophilous mass
and moved toward the surface of the egg, where it gave rise to the chro-
mosomes, while the remnants of the vesicle degenerated in the cyto-
plasm. We have observed that the cluster of chromosome fundaments
sometimes has the appearance of such an angular mass, and it is possible
that Tafani mistook this for the nucleolus. He figures it as in the act
of slipping out of the germinative vesicle. In Sobotta's opinion (1895,
p. 44) the chromosomes in eggs which produce but one polar cell are
formed from the chromatin of the whole nucleus, not merely from that
of the nucleolus as was claimed by Holl (1893), whose conclusions are,
in Sobotta's opinion, unreliable because of the poor preservation of his
material. Sobotta's statement (1895, p. 44) that the chromosomes are
very irregular in form before they become arranged in the equator of
the spindle and his illustration of the condition (Taf. 4, fig. 9, 9a) must
really relate to the second spindle, for they are both based on eggs from
either the periovarial chamber or the beginning of the oviduct ; but such
eggs must have already passed beyond the stage of the first spindle, as

52 THE MATURATION OF THE EGG OF THE MOUSE.

Sobotta himself admits in a more recent paper (1907). Although he
makes no mention of having seen the beginning of the (large) first spindle,
he states (1895, P- 52'> I9°7- P- 507), without qualification or conclusive
evidence, that it originates about 24 hours before ovulation. According
to Gerlach (1906, p. 9) the nucleolus disappears completely, and from
the chromatin spherules (which he believes owe their origin to the nucleo-
lus) the chromosomes are differentiated before the disappearance of the
nuclear membrane. Kirkham (19076, p. 73), describing the prophase
of the first maturation, says that in a few cases there were traces of the
nuclear membrane, though more often it had entirely disappeared. His
fig. 1 (plate I), though described as that of an ovarian egg before the
formation of the first maturation spindle, looks more like the cross-
section of a spindle in the monaster stage than an early stage in the meta-
morphosis of the germinative vesicle, and the two detached chromosomes
may possibly owe their peculiar position to the displacement which
sometimes is caused by the knife in sectioning.

It will be remembered (p. 25) that the wall of the nucleolus is thick
and deeply stained, and that the chromatin bodies of the germinative
vesicle are especially numerous around the nucleolus, which lies at one
side of the vesicle. Since, in the next stage, the chromosome fundaments
(see p. 26) are also at one side of the nucleus, it is probable that they
replace both the vesicular nucleolus and the chromatin bodies. This is
rendered the more probable by the fact that these fundaments are ar-
ranged at one side of a slightly denser part of the nucleoplasm. Such
conditions lead one to think it possible that the fundaments arise from
both the wall of the nucleolus and the chromatin bodies, while the achro-
matic spindle comes from other parts of the nucleus, or possibly originates
in the inner part of the nucleolus.

Precisely how the chromatin of the germinative vesicle is metamor-
phosed or differentiated into the fundaments of the chromosomes is
unknown ; but in three cases the arrangement of the curved fundaments
(as in fig. 36) suggests the possibility that they lie end to end and may
therefore be regarded as parts of a potential thread or spireme. This
possibility is perhaps strengthened by the fact that these fundaments
usually show a longitudinal division first and the transverse division later.
These observations suggest that the longitudinal division may corre-
spond to the longitudinal split in the spireme of the synapsis stage ob-
served in many invertebrates, and that each fundament consists of two
univalent chromosomes united end to end. The univalent chromosomes
would then be sometimes indicated by the cross-division, and would be
separated at the first mitosis, as described on page 30.

An inspection of the figures of the chromosomes of the first spindle
in the papers of Sobotta (1895, ^99, 1907), Gerlach (1906), Lams et
Doorme (1907), and Kirkham (19076) reveals the fact that there is no
essential disagreement in regard to the general forms of the chromosomes,

CRITICISMS AND CONCLUSIONS.

although Gerlach (1906, p. 13) believes that the typical forms appear in
the prophase only and that, apparently as a result of shrinkage, the
chromosomes of the equatorial plate are short, rounded rods, like those
of the second spindle. This supposed change of form is explained when
it is noted that in Gerlach 's figures the chromosomes of the first spindle
of ovarian eggs (Gerlach 1906, Taf. 1, fig. 2, 3) have the typical forms,
while the oviducal egg (fig. 4) with supposed first spindle has the rod-
like chromosomes; for, as pointed out before, what he calls first spindles
in oviducal eggs are really second spindles. Therefore, Gerlach's ma-
terial, after all, presents no real exception.

Gerlach (1906, p. 25) regards the chromosomes of the first spindle
as tetrads, those of the second as dyads. The conclusion that the chro-
mosomes of the first spindle are tetrads is based entirely on indirect
evidence and on reasoning from analogy with conditions demonstrated
in many invertebrates. Since in the first polar cell he finds that the
chromosomes sometimes seem to be present as dyads, he reasons that
those of the first maturation spindle must have been tetrads.

None of these observers has recognized and figured the quadripar-
tite structure of the chromosome of the first maturation spindle. Both
Tafani and Gerlach (1906, pp. 13-14), it is true, state that the chromo-
somes are composed of Pfitzner's granules embedded in a less deeply
stainable substance ; but that has no bearing on the question of quadri-
partite structure. That the first division is transverse is believed by all
authors except Tafani (1889, p. 22), who thinks it longitudinal, though
he has not directly observed it in the mouse. But, since he confused
the two spindles with each other, this statement applies to the second
spindle only. Sobotta (1899, 1907) alone gives illustrations of migrating
daughter chromosomes; but in none of his figures does he show their
longitudinal division. There is no doubt, as both Sobotta (1907, p. 511)
and Kirkham (19076, p. 73) state, that some chromosomes divide earlier
than others.

When one examines carefully the accounts of the first maturation
spindle given by Sobotta (1895, I907)> ft *s evident that in his first paper
he speaks of a relatively early stage (fig. 4a) of the spindle as showing
the equatorial plate, a stage which he later designates correctly as the
prophase. Subsequent writers — Gerlach, Kirkham — have figured simi-
lar stages, and Kirkham (19076, p. 73, fig. 2) has applied the expression
equatorial plate even to a stage in which the chromosomes are distrib-
uted over half the length of the spindle. Gerlach (1906, p. 13), however,
clearly states it as his opinion, and in this we believe he is right, that
such spindles are still in process of formation; but, in our opinion, he
fell into an error in ascribing to a later stage of the first spindle a condi-
tion which is to be found only in the second maturation spindle ; for he
says that when the equatorial plate is fully formed it presents in the side
view of the spindle a fairly uniform appearance, its chromosomes having

54 THE MATURATION OF THE EGG OF THE MOUSE.

the form of short rounded rods such as Sobotta shows in his (1895)
fig. 10a. But Sobotta, as we think, and as he would probably now admit,
made a mistake in supposing that his figures 10 and 10a represented the
first maturation spindle. The egg in question was taken from the ovi-
duct, and therefore exhibits the second maturation spindle. It may be
noted, in passing, that by some strange slip of the pen Sobotta (1895,
p. 91) describes his fig. 10c as representing the beginning of metakinesis
instead of an advanced anaphase. In his more recent paper he (Sobotta,
1907, pp. 508-511, fig. 2, fig. 3) has figured two spindles which may
more properly be said to exhibit an equatorial plate, though even here
the chromosomes do not assume that rigid, plate-like arrangement which
characterizes the equatorial plate in many other animals and also that
of the second maturation spindle in the mouse. This equatorial-plate,
or monaster, stage of the first spindle is distinguished (Sobotta, 1895,
pp. 508-511) from the prophase by the possession of smoother and
straighter spindle fibers and by the predominance of chromosomes
having a large one-sided protuberance. There is no disagreement among
authors concerning the orientation of the chromosomes on the spindle
nor concerning the fact that they vary in size. But as to the number
of chromosomes, there is a wide difference of opinion. Tafani and the
present writers count 20. Sobotta — whose view has been accepted by
all subsequent investigators, apparently under the influence of the large
amount of his material— maintained in 1895 that there were 12 chromo-
somes; but recently, stimulated by Dr. J. A. Murray to a reexamination
of his material, he has changed his opinion, and in two papers (1907, p.
512; 1908, pp. 248, 259) has stated that the number is certainly 16.
Holl (1893, P- 284) argued that since at an earlier stage there were 24
chromatic balls, there should be as many loop-like chromosomes, and was
able to count 20; but not much weight can be given to his conclusions.
He admits that it was impossible to count the chromosomes accurately.

The short account by Melissinos (1907, p. 584) is remarkably un-
critical. After stating that Tafani gave the number as 20, Holl as 18,1
Sobotta as 12, and others as 24, he remarks that Sobotta's counting seems
to him the more accurate, and then proceeds to state that he can make
out only 8. But his figures are too diagrammatic to inspire much con-
fidence on the part of the reader.

As already shown (p. 45), the number of eggs in which Sobotta could
possibly have counted chromosomes is really small. In 1895 (P- 4^) he
maintained on the strength of many successive countings of the same
material that the slender (second) spindle in all probability possessed
12 chromosomes, surely not over 14 or 15. Moreover, in the case of the
thicker first spindle (p. 51) there were three eggs in which he counted

1 It is not clear how Melissinos conies to make Holl responsible for the view
that the mouse egg shows t8 chromosomes, unless, perchance, his eye fell on the
page (280) where Holl reports that Ruckert found "about 18 chromatin rods" in
Selachian eggs.

CRITICISMS AND CONCLUSIONS. 55

"with absolute certainty" 12 chromosomes, and in many other instances
approximately 12. Now, however, apparently without any additional
material, he (1907, p. 512; 1908, p. 248) counts 16! Gerlach (1906, p. 23)
expresses himself as emphatically agreeing with Sobotta in his early
statement that the number is 12, he (Gerlach) having repeatedly counted
12 in both the first and the second spindle. Lams et Doorme count
the same number, 12, in two polar cells; but we have shown (p. 42) that
the number in the polar cell has no significance. Kirkham (1907b, pp.
74-78) likewise affirms that there are 12 chromosomes, and in those cases
where there are obviously more than 12 bodies he explains the higher
number as being due to the precocious division of some of the chromo-
somes. Nevertheless, in Kirkham's own preparations, which were so
generously loaned to us, out of four normal ovarian eggs in the stage of
the first spindle there were three cases in which we could count 20 with
certainty, and in the remaining one 17.

ACHROMATIN.

Gerlach (1906) and Sobotta (1908, p. 508) are the only writers on
the maturation of the egg in mice who give any opinion as to the precise
origin of the fibers of the first spindle. These they think arise from the
linin network of the germinative vesicle. But this seems improbable in
view of the fact that there is a stage before their appearance in which
only shreds of the linin network are left, while most of the vesicle is filled
with a clear fluid. It is possible that the linin plays some part in the
origin of the spindle; but, as has already been suggested, other parts of
the nucleus, including the nucleolus, are the more probable sources.

Tafani has pointed out that in its early stages the first spindle in
ovarian eggs is short and fat, a condition we also have found. Sobotta
(1895, 1899, I9°7) figures in a diagrammatic way the spindle with sharp
poles, the fibers converging to a point. Lams et Doorme (1907, p. 274)
say the fibers converge more or less to a point. Kirkham figures the
shape of the first spindle as elliptical.

According to Sobotta (1907) the largest spindle is 30 to 32 micra
long and 20 micra broad. The largest spindles we have found have the
following dimensions: 29.5 micra in length by 11 in breadth, and 22.6
in length by 14 in breadth. From Sobotta's paper of 1899 it must be
inferred that the size varies. The statement of Lams et Doorme (1907,
p. 275) and our own observations accord with this inference. Gerlach's
statement (p. 10) that the size depends in the main on the size of the
germinative vesicle can not be accepted as demonstrated, for the spindle
is not a result of the metamorphosis of a network confined in a rigid
vesicle; besides, the membrane of the vesicle has nearly disappeared
when the spindle is first differentiated.

Sobotta described the spindle fibers in 1895 (p. 51) as fine, wavy,
and branched; in 1907 (p. 508) as wavy with slight thickenings. His
latter description applies to the early stages of the first spindle, for later

56 THE MATURATION OF THE EGG OF THE MOUSE.

the fibers become thickened at the polar ends, as he and Lams et Doorme
figure them. Gerlach does not agree with Sobotta that there is a central
spindle. While we have no evidence of the existence of a central spindle
like that discovered by Hermann, we agree with Sobotta that there are
some fibers which run from pole to pole without being attached to chro-
mosomes. These probably persist as a part of the interzonal filaments.

Centrosomes, Circumpolar Bodies, and Clear Region.

No one (with the possible exception of Gerlach, fig. 2) has figured
the corpuscles near the poles of the spindles which we have called cir-
cumpolar bodies. Tafani (1889, p. 22), Sobotta (1907, p. 521, for the
second spindle only), and Gerlach (1906, p. 9), nevertheless, mention
granules at the poles, which, according to the two latter authors, form a
sort of mantle around the poles of the spindle and thus obscure its fibrous
structure. Gerlach describes them as occurring with both spindles and
adds that they sometimes have the form of tortuous threads, which
suggests to him that they may be mitochondria.

The first impression one forms of these bodies is that they are arti-
facts due to improper fixation; but when one reflects that they occur
in eggs fixed by different methods and that they are characteristic of
certain stages (see p. 33), this interpretation seems unwarranted. These
bodies were also seen in Kirkham's preparations, although he does not
himself mention them.

A study of the occurrence of these bodies brings out the fact that
they are characteristic of certain periods of morphological activity.
For example, they can be found for a short time before and during meta-
kinesis of the first spindle and during the early existence of the second
spindle when division is likely to occur as a result of semination. Con-
versely, they are absent during periods of morphological quiescence,
such as the telophase of both spindles, and when the second spindle
persists in the absence of semination. It will be remembered that these
periods of activity are very short (p. 16), while the quiescent periods
are comparatively long; therefore these bodies exist during only brief
periods. The question naturally arises, Are they the result or the cause
of the morphological changes? Unless it can be shown that they are
handed on from cell to cell, it seems reasonable to suppose them products
rather than causes of spindle activity. On the other hand, the absence
of typical centrosomes leads one to ask whether they may not in some
way fulfill the function of centrosomes, especially since they are situated
very close to the poles of the spindle. Such inquiries can not be answered
at present; these bodies, the existence of which is beyond dispute, are
worthy of more extensive study, and their possible relation to mito-
chondria should certainly be investigated further.

Tafani, Sobotta, and Gerlach deny the regular existence of centro-
somes. Gerlach (1906, p. 26) saw in one case two centrioles at the pole
of a spindle, and Sobotta (1907, p. 524, fig. 8) figures a disk-shaped

CRITICISMS AND CONCLUSIONS. 57

body at one pole of a spindle, where a centrosome might be expected;
but he declines to regard it as such, because it is an isolated case. Lams
et Doorme (1907, p. 274) and Kirkham (19076, p. 74) alone assert the
occasional presence of these structures, the former saying that there
are usually none with the first spindle. Lams et Doorme illustrate two
first spindles in side view, one in an egg from the ovary (fig. 2) and one
from the oviduct (fig. 5), the latter being the case to which Sobotta
calls attention as the exception to the rule that the first spindle is con-
fined to ovarian eggs. In the first case (fig. 2) they show no centrosomes,
but in the case of the egg from the oviduct (fig. 5) a curved rod occupies
one pole of the spindle. The latter, however, is probably a second spindle,
since the egg is in the oviduct and since all the second spindles figured
by them have somewhat similar centrosomes; furthermore, the chromo-
somes of this spindle resemble the chromosomes of the second spindle
rather than those of their fig. 2. As for the centrosomes drawn by Kirk-
ham, their presence is probably referable to the condition of the eggs,
many of which, as judged from an examination of his slides, were not
normal. It will be noted that some of his spindles do not show centro-
somes; they, we believe, are normal. There seems, then, to be no good
ground for the assertion that centrosomes exist in connection with the
first spindle.

Sobotta (1895, P- 44) states that the clear region around the chro-
mosomes of the spindle of eggs which produce only one polar cell has
almost precisely the extent of the vanished germinative vesicle. Since
this statement really relates to a spindle which does not originate from
the germinative vesicle directly (as Sobotta himself now admits), it
loses its significance. Lams et Doorme (1907, p. 274), who make a
similar assertion in connection with the first spindle, apparently have
not themselves seen the early stages (their fig. 3 being that of the second
spindle) , and consequently have no other ground than Sobotta for their
assertion. According to our descriptions (pp. 26, 27, 33) this clear region
has no direct relation to the germinative vesicle. Since it exists, as the
circumpolar bodies also exist, during the periods of morphological
activity of the spindle, it also is probably a manifestation of such activity.

Position and Orientation.

Sobotta (1895, I899, 1907) places much emphasis on the position
of the first spindle, which is situated deep in the egg. Our specimens
substantially corroborate his statement. Regarding the angle which the
axis of the spindle makes with the surface of the egg, there is some dis-
agreement among authors, arising, as it seems to us, from the paucity
of proper stages in the material which most of the investigators have
studied. It has been shown (p. 33) that the spindle may be parallel or
oblique to the surface, but that it is only rarely perpendicular at any
stage. Tafani (1889, p. 22) says that the spindle is from the first oblique,
not perpendicular, and figures it in an oblique position during the abstric-

58 THE MATURATION OF THE EGG OF THE MOUSE.

tion of the polar cell. Although the statement may be based upon the
sscond spindle, which Tafani mistook for the first, it nevertheless is
true of the first spindle. Sobotta (1895, p. 48) makes the unqualified
statement that the slender spindle (which he now calls the second)
turns from the paratangential position to the oblique and finally to the
radial just before the polar cell is cut off. He saw three cases of meta-
phase spindles, all oblique, but all of those in the telophase were radial.
Therefore, although he had not actually seen the process of abstriction,
he thought the first spindle was radial at the time the polar cell was
cut off. In a later paper (1899, p. 190) he describes the same process
for the first spindle and gives a figure (fig. 4) of the spindle during the
dyaster stage in what appears to be a radial position with one pole in
the polar-cell protrusion. The figure has a somewhat rigid diagrammatic
appearance and is not accompanied by any explanation to prove that the
spindle is radial with respect to the center of the egg as well as the center
of the section in which it lies. The relative shortness of the spindle
suggests the possibility that its axis is oblique to the plane of the section
and that consequently it may not be strictly radial in position. He
mentions having three other spindles in the stage of his fig. 4, but does
not state what their position is. In one of his recent papers Sobotta
(1907, p. 517) figures a dyaster stage of the first spindle (fig. 4) and
states that it is in an oblique position, having begun the rotation from
the tangential to the radial position. In a foot-note, however, he admits
that it really is never met with in a strictly radial position! He (1907,
p. 517) finds it difficult to decide whether the first spindle always rotates,
yet he argues that it must remain tangential in most cases (one polar
cell) because it is transformed in the monaster condition directly into
the monaster of the second spindle, which is likewise tangential. He
is not sure whether even in one-fifth of the cases (those in which it divides)
it may not be oblique when the polar cell is formed, but thinks it may be
assumed that as a rule it rotates, because the second spindle always
rotates, and because it (the first) takes up a position so near the surface
of the egg that no polar cell could be produced without its rotation.
Sobotta does not give any proof, except that contained in his first paper
(1895), that the second spindle is radial at the moment the polar cell
is abstricted. Moreover, he figures (1907, fig. 9) a dyaster of the second
spindle in a paratangential position and says (p. 525) that its not being
radial is purely accidental! Thus, except for his 1899 paper, which he
does not mention in this connection, there is no evidence that either
polar cell is cut off while the spindle is in a strictly radial position. Ger-
lach (1906, p. 10), while he does not take exception to the general conclu-
sion of Sobotta that there is a rotation of the spindle from a tangential
toward a radial direction, thinks that the strictly radial position is not
necessary to the formation of the polar cell. Neither Gerlach, Lams et
Doorme (p. 275), nor Kirkham (19076, p. 75) mention having seen any

CRITICISMS AND CONCLUSIONS. 59

stages of the metaphase, and the latter two, having seen spindles in
oblique positions, apparently assume that Sobotta is right in his opinion
that the spindle becomes radial and that the oblique position is simply
an intermediate one.

Division of First Spindle and Abstriction of First Polar Cell.

Sobotta (1899) is the only observer who has figured stages in the
migration of the daughter chromosomes towards the poles of the spindle.
Because of the scarcity of such stages in his material he concludes that
the first spindle divides in only one-fifth of the eggs. In the other four-
fifths, therefore, the spindle does not divide and the first polar cell is
not cut off. This may possibly be due to a failure of the spindle to rotate
(Sobotta, 1907, p. 5 1 8, footnote). This he thinks agrees with his obser-
vation that 80 per cent of the fertilized eggs have only one polar cell, this
one being in his opinion the equivalent of the second polar cell of those
eggs which form two such cells.

It has been shown (p. 16) that this stage is of very short duration.
Hence we draw the conclusion that the infrequent occurrence of this
stage is due, not to the failure of the spindle in some cases to divide, but
to the fact that the chances of meeting with it are few.

Gerlach (1906, fig. 5) figures a recently formed polar cell in an ovarian
egg, but he says nothing about the division of the supposed first spindle
in oviducal eggs. As Sobotta points out, supposed first spindles in the
oviduct have had as much time in which to divide as have the first spin-
dles of adjacent eggs which have produced the first polar cells. These
considerations go to show that Gerlach misinterpreted the spindles in
oviducal eggs.

In the opinion of Sobotta the "Zwischenkorperchen," sometimes
in two rows, are finally inclosed in the polar cell when it is cut off. He
describes and illustrates this condition in his papers of 1895 and 1907.
Although his observations were really made on the second spindle, they
hold also for the first. It is difficult to account for this conclusion except
on the ground of variable conditions or poorly preserved material, for,
as Lams et Doorme (for the second spindle) and Gerlach show, and as
our material so clearly proves, the bodies in question do not lie inside
the membrane of either egg or polar cell. Gerlach, however, thinks they
are at first in two rows which then fuse.

In the process of abstriction, as described on pp. 34 and 40, there
appears to be an attraction between the "Zwischenkorperchen" and
the vitelline membrane. Naturally any attraction between the mem-
brane and these bodies would be exerted more readily with the spindle
in an oblique or tangential position and its effect would be first manifested
on the side of the spindle nearest the surface. It is perhaps possible,
then, that the "Zwischenkorperchen" have some part to play in the
abstriction of the polar cell.

60 THE MATURATION OF THE EGG OF THE MOUSE.

3. Second spindle.

Chromatin.

It was Tafani (1889, p. 23) who first announced that in the greater
number of cases in mice only a single polar cell is formed. It was
therefore his opinion that the chromosomes which remained in the egg
after the formation of the first polar cell gave rise either to the second
spindle (few cases) or to the female pronucleus (greater number of cases) .
This opinion would be the natural consequence of his probable confusion
of the second spindle with the first. Sobotta in his early paper (1895,
p. 44) also held that in those eggs which produced but one polar cell
(in nine-tenths of the cases, in his opinion) the spindle was formed
directly from the germinative vesicle, and (1895, p. 53) that in all other
eggs (one-tenth of the total number) the second spindle was produced
from the chromosomes which remained in the ovum after the first polar
cell was abstricted. Since Sobotta considered the spindle in the former
instance to be the equivalent of that in the latter, it follows that, accord-
ing to his view, the second spindle was formed in some cases directly
from the germinative vesicle. In a later paper (1907, p. 514) he says
that he has no observations to prove this view and that it is erroneous.
As stated in this paper (1907, p. 519), he now believes that (in a larger
proportion, about one-fifth of the cases) the second spindle originates
as previously described for one-tenth; but in 4 out of every 5 eggs the
monaster of the second spindle is derived directly from the monaster of
the first, i.e., without the formation of a polar cell. That is, the first
spindle in a large proportion of ova does not divide, but, in some way
which involves a degeneration of half of the chromosomes within the
cytoplasm of the egg (1907, p. 541; 1908, p. 250), is transformed into
the corresponding condition of the second spindle. This belief he thinks
accords with his observation that in preserved material the occurrence
of the division of the first spindle is very infrequent.

This is Sobotta's explanation of the occurrence of only one polar
cell in many oviducal eggs in the late stages (the ones he worked with
chiefly. See pp. 14, 45). It is not based on any observation of degen-
erating chromosomes or of the supposed stages of transformation. In
fact, Sobotta repeatedly says that he has seen no such stage, although he
believes that in a single instance (1907, fig. 8, a spindle with more than
16 chromosomes, which occurred in an oviducal egg) he may have had
an example. It should be noted that, if this transformation occurs in
four-fifths of all the eggs, the chances of meeting with it must be four
times as many as the chances of encountering the division of the first
spindle. In view of these considerations one may be warranted in ques-
tioning the existence of such a condition.

Gerlach (1906, fig. 6) illustrates an early stage in the origin of the
second spindle , with which the description of the same stage in the pres-
ent paper agrees.

CRITICISMS AND CONCLUSIONS. 6l

The chromosomes of the second spindle are not described by
Tafani, except as the description which he gives of those of the supposed
first spindle really applies to those of the second. Sobotta (1907, p. 521)
holds that they are short rounded rods, similar in form to the daughter
chromosomes of the first spindle, though generally somewhat smaller, or
at least slimmer. Gerlach (1906, p. 14) is unable to distinguish between
the chromosomes of the first and second spindles, except that the latter
are the smaller; he figures the same shapes as Sobotta, and also a
spindle (fig. 16) having elongated granular chromosomes. We have
found in many spindles in which the chromosomes are closely packed
that the appearance — especially of those chromosomes which are seen
in end view, without careful, critical study and comparison with more
favorable examples — seems to be about like that figured by Sobotta
and Gerlach. Lams et Doorme (1907, p. 283) think that the presence
of the first polar cell is the only reliable criterion for identifying the second
spindle. Kirkham (19076, p. 78), Sobotta (1895, p. 48), and Gerlach
(1906, p. 19) state that the daughter chromosomes elongate, but they
describe no other structure. We have shown this lengthening to be
characteristic of old spindles.

So far we have made no definite statement concerning the homol-
ogies of the chromosomes of the second spindle with those of the first.
Whether the mother chromosomes of the second spindle are identical
with the daughter chromosomes of the first it is impossible to say with
certainty, for the reason that there is no way of determining directly
whether or not the chromosomes which become fused into a single mass
in the egg after the first polar cell is cut off keep their individuality and
reappear when the mass breaks up preparatory to the formation of the
second spindle. The striking similarity between the daughter chromo-
somes of the first spindle and the mother chromosomes of the second in
certain cases, and also analogy with those invertebrates in which the
daughter chromosomes of the first spindle are known to pass directly
to the second spindle without undergoing an intervening nuclear or
resting stage, make it seem highly probable that in the mouse the daugh-
ter chromosomes of the first spindle are identical with the mother chro-
mosomes of the second. If this is true, then the division between the
parts of the chromosome of the second spindle is the same as the longi-
tudinal division in the daughter chromosome of the first spindle and is
therefore apparent in the fundaments. On this ground it is proper to
call the chromosomes of the first spindle "tetrads," because they pos-
sess the two divisions which mark the planes of separation of the daughter
chromosomes of two quickly ensuing mitoses, and to designate those of
the second spindle "dyads." The division of the dyad, then, is a longi-
tudinal splitting, and the reduction is a so-called prereduction.

Tafani (1889) makes the statement that the chromosomes of the
first spindle divide longitudinally; but, as we have seen, this statement
5

62 THE MATURATION OF THE EGG OF THE MOUSE.

probably relates to those of the second spindle. Sobotta (1895, p. 46;
1907, p. 522) and Gerlach (1906, p. 14) state that the division is trans-
verse, but for theoretical reasons they believe that the division of the
chromosome of either the first or second spindle must be longitudinal.
Sobotta (1908, fig. 7) alone figures a dividing second spindle. His
"biscuit" shaped chromosomes remind one very much of some of the
dyads we have described (p. 37) as constituted of 4 parts, inasmuch as
the "biscuit" forms are in some instances in groups of 4. What he calls
a whole chromosome looks more like half of a dyad.

The same criticisms which have been made regarding the number
of chromosomes of the first spindle apply also to those of the second. It
was in polar views (the most favorable for counting) that Tafani found 20.

ACHROMATIN.

In his paper of 1895 (p. 45) Sobotta stated that the spindle fibers
of the single spindle (which occurred in nine-tenths of the eggs) were
derived in part from the achromatic portion of the germinative vesicle.
As already pointed out, he no longer holds this view.

The second spindle as drawn by Sobotta (1895, 1907) is barrel-shaped,
the ends being somewhat truncate, the fibers only slightly curved, and the
poles open. As illustrated by Gerlach, Kirkham, and the present writers,
this spindle is elliptical, with fibers incurving at the poles.

The flattening of some of the second spindles described on page 38
is apparently a result of their lying close to the surface of the egg. There
is a possibility that the flattening is caused by shrinkage due to fixing
and dehydrating. Shrinkage to produce this result would have to be
greater in a radial than in other directions, and could be explained only
on the supposition that the substance in which the spindle lies, being
probably more fluid than the surrounding cytoplasm, is extracted more
rapidly on the side nearest to the surface of the egg. However, were the
flattening due to shrinkage the chromosomes should be crowded in a
radial direction ; but that this crowding does not exist is clear from plate
4, fig. 20, in which the spaces between the chromosomes are as uniform
as in fig. 21.

All investigators agree that the second spindle is smaller than the
first. Sobotta (1907, pp. 508, 520) insists that the second is but half the
size of the first, although he does not state whether he used averages for
his conclusion. It seems unlikely that he did, since he says that his fig. 3
is the broadest first spindle. It must be admitted that a first spindle may
be about twice the size of a second spindle, for we have found that the
largest two first spindles measure 29.5 Xn micra, and 22.6X14 micra,
respectively, and the smallest second spindles 14X6.5 micra and 18X5.5
micra, respectively.

All who have published papers on the mouse, except Kirkham,
figure the polar ends of the fibers as thickened. In regard to the fibers
which are not attached to chromosomes, there is no conflict between the

CRITICISMS AND CONCLUSIONS. 63

statement of Sobotta, that there are fibers stretching from pole to pole,
and our own results. However, he gives the idea that such fibers form
a bundle on the outside of which the chromosomes rest and on which
they are drawn to the ends of the spindle, whereas the distribution of the
chromosomes in the plane of the equator in our preparations forces us
to conclude that such fibers, if present, must be interspersed among
the chromosomes. Sobotta (1895, p. 47) places the number at 12 (later
as probably 16). As it has not been possible to count them in our
preparations, we can not state what the number is.

Centrosomes, Circumpolar Bodies, and Clear Region.

The circumpolar bodies and the clear region have already been con-
sidered. The former dwindle away in old second spindles, leaving what
might be mistaken for centrosomes (p. 39). Such remnants may well be
what Sobotta (1907) and Gerlach (1906) occasionally saw and what Lams
et Doorme (1907, figs. 6 to 8) and Kirkham (1907) found more regularly.
Lams et Doorme say that in the second spindle the "centrosomes" vary
according to the method of fixing. But in our opinion these are not to
be regarded as centrosomes.

Position and Orientation.

Gerlach (1906, pp. 18 to 20) and Kirkham (19076, p. 78) have
observed that the second spindle or second polar cell may be at various
distances from the first polar cell. Sobotta (1907, p. 532) finds only one
such condition in 1,000 eggs and thinks the difference between Gerlach's
material and his may be due to the fact that he and Gerlach used eggs
of different ovulations. We are at a loss to account for the difference
in Sobotta's material; but the fact nevertheless remains that the polar
cells may be found at various distances from each other. Gerlach
(1906, pp. 18 to 20) accounts for this by supposing the spindle to migrate
through the cytoplasm, and he figures a path which he thinks was made
by such a moving spindle. The distance, he believes, is determined by
the epoch of semination, because with that event the second spindle,
wherever it may be, stops in its migration and forms the second polar
cell (or at least divides) . There is no final proof that this migration does
not occur, but, from the evidence adduced (p. 43) in connection with
the position of the first polar cell, it seems simpler and more reasonable
to suppose that the polar cell shifts its position under the zona. This
shifting might be aided by the power the polar cell has of changing its
shape, as was observed by Tafani. Such an explanation makes it unnec-
essary to assume changes in the cytoplasm and a migration of the spindle
that is so out of harmony with what is known in other animals, where
the conditions are so favorable as to leave no doubt as to the events.

The orientation of the second spindle is like that of the first and
needs no further discussion.

64 THE MATURATION OF THE EGG OF THE MOUSE.

Division of Second Spindle and Abstriction of Second Polar Cell.

The only illustrations showing the division of the second spindle
in the maturation of the mouse egg are those of Sobotta (1895, I9°7)-
The criticisms which we have made in connection with the division of
the first spindle and the formation of the first polar cell (p. 59) are appli-
cable to the corresponding processes in the second oocyte.

4. POLAR CELLS.

There is agreement among the investigators of the mouse egg that
not all fertilized eggs have both polar cells. According to Tafani and
Gerlach the first polar cell is always formed, but the second in a large
proportion (respectively four-fifths and three-fourths) of the eggs is sup-
pressed. Tafani does not state how the suppression is effected. Gerlach
thinks that in the event of late semination the second spindle divides
so quickly as to inhibit the formation of the polar cell and that the chro-
mosomes which would have been contained in the second polar cell
remain in the cytoplasm of the egg and degenerate. Although he avers
that he has seen such degenerating chromatin, it should be borne in
mind that it is possible he mistook for chromosomes cytoplasmic bodies
which sometimes stain deeply like chromatin. Sobotta, on the other
hand, believes that in most cases the first polar cell is never formed.
In 1895 he stated that even the first spindle did not come into existence.
Now (1907) he believes that the spindle is formed in all eggs, but that in
4 out of 5 eggs it is immediately metamorphosed into the second spindle,
half of the chromatin disintegrating in the egg. As he has not seen
either the metamorphosis or the degeneration of the chromatin he has
no direct evidence for his belief. Kirkham states, but on evidence that
in part at least is unsound, that all eggs produce the first polar cell.
His explanation (19076, p. 80) of the absence of one polar cell is appar-
ently suggested by a single case in the bat, in which, according to van
der Stricht, both polar cells lay outside of the zona pellucida. It is
supported by one observation (Kirkham, 19076, p. 81), according to
which the polar body of a living mouse egg (which he stained and dehy-
drated under the microscope) was forced through the zona pellucida by
the contraction of the latter under the influence of changing osmotic con-
ditions.

While the case in the bat is suggestive of a possible explanation for
the loss of the first polar cell in the mouse, it can scarcely be admitted
as evidence of the occurrence of such conditions in the mouse. As for his
observation on the living egg, Kirkham does not say with what strength
of solutions he stained and dehydrated the egg under the microscope.
Although he may have seen the polar cell forced through the zona under
direct action of reagents, the same thing need not necessarily occur under
natural conditions, since eggs in the oviduct, and still more those in the
ovary, are protected from the full vigor of osmotic action by the sur-

CRITICISMS AND CONCLUSIONS. 65

rounding fluid in the oviduct or follicle and by the tissues of the oviduct
or ovary. Kirkham, furthermore, states that this loss of the polar cells
occurs during ovulation ; but, since he has not seen any instances in which
the eggs are passing from the follicles, this conclusion must be based on
the presence of these bodies at one stage (viz, before ovulation) and their
absence at another (viz, after ovulation). But, unfortunately for this
explanation, they are not universally absent in the latter case.

None of the figures of mammalian eggs escaping from the follicle—
the only ones known to us being those given by Barry (1839), Sobotta
(1895), van der Stricht (1901), and the writers (figs. 38, 39, 40) — furnishes
any evidence whatever that the polar cell is being pressed through the
zona pellucida. Our preparations show, on the contrary, an increased
space between the zona pellucida and the vitellus. The change in osmotic
conditions in passing from the ovary to the periovarial space or to the
oviduct in a living mouse can scarcely be great enough to cause the
polar cell to be forced through the zona by shrinkage of the latter.
Furthermore, if the loss of the first polar cell is caused by the action of
reagents, why should not the second polar cell also be forced through
the zona? In the case of the bat van der Stricht had the evidence of
both polar cells lying outside the zona. There is not even this evidence
in the case of the mouse, for, as Sobotta (1908, p. 253) has observed, no
one has ever seen such a condition, though, if it occurs, the polar cells
should be easily recognizable among the surrounding follicle cells.

There is, then, no good evidence of the suppression of either polar
cell or of the loss of the first polar cell by extrusion through the zona
pellucida. Lams et Doorme (1907, pp. 276, 287) were the first to offer
the explanation that, while both polar cells are formed, the first under-
goes degeneration within the zona and disappears. Their figures show
this clearly, yet they suggest that what they call degenerating polar
cells may possibly be bodies (follicular cells) which have slipped under
the zona! Independently of Lams et Doorme, and before their paper
was published, we, also, had come to the conclusion that the first polar
cell degenerates, and can therefore support the view with unbiased
observations. We have already described the decrease in size of the first
polar cell and the evidence of the degeneration of its chromatin, using
polar cells of eggs which contain the second spindle in order to avoid
even the possibility of confusing the first polar cell with the second.
Tafani (1889, p. 24) mentions that the first polar cells vary in size and
also calls attention to cases where they are very small. Sobotta (1907, p.
544) alludes to these small forms by warning his readers not to mistake
for polar cells what he says may be follicle cells under the zona, or bodies
formed from the zona. He does not show why follicle cells should be
under the zona, or in what manner they could get into such a position,
or how the zona could give rise to bodies with nuclei. It must be remem-
bered that, since Sobotta's material contained a large proportion of the

66 THE MATURATION OF THE EGG OF THE MOUSE.

late stages (pronuclei and cleavage stages) , it presented few of the degen-
erate polar cells (see p. 41), those that persisted being of the larger size.
Again, eggs fixed in osmic-acid mixtures (which he used chiefly) have the
zona dark, which makes it difficult and often impossible to interpret or
even to see such small objects. Upon consideration, it is not surpris-
ing that the first polar cell should degenerate, for usually both polar cells
do so in time, forming no part of the embryo. It is quite possible that
the substance of the polar cell is absorbed by the egg.

The decrease in size of the degenerating polar cell explains the dis-
agreement of authors concerning the relative size of the first and second
polar cells. Sobotta (1907, p. 536) maintains that sometimes one, some-
times the other, is larger. Gerlach (1906, p. 13) says the first is larger;
Lams et Doorme (1907, p. 287) that the second is. It seems fairly cer-
tain that Lams et Doorme must have seen old first polar cells and young
second ones, for they have few of the earlier stages, even though they
show the first polar cell decreasing in size.

Gerlach (1906, p. 25) thought that in one first polar cell the chromo-
somes were dyads. Sobotta (1907, p. 537) says that both polar cells may
have either scattered chromatin or a nucleus, which is formed later than
the egg nucleus. In our opinion this statement must mean that he con-
fused the polar cells, for, of the 507 eggs with the second spindle that
we have studied, none have a first polar cell with a nucleus; whereas the
second, in seminated eggs, always forms a nucleus without its chromo-
somes becoming scattered and distinct. Kirkham (19076, fig. 14), also,
has probably mistaken the first polar cell for the second in the figure in
which he shows the monads much separated.

The difference in chromatin contents of the two polar cells accords
with the well-known fact that the first polar cell corresponds to the first
oocyte, while the second is a homologue of the second oocyte; for, on the
one hand, the chromatin of the first polar cell does not form a resting
nucleus, but may divide (as it occasionally does), and, on the other hand,
the chromosomes contained in the second polar cell immediately become
metamorphosed into a nucleus corresponding to the egg nucleus. The
first, being a cell which degenerates, divides not regularly and normally,
but with what seems to be imperfect mitosis or even amitosis.

5. Reduction.

It is fair to assume from the preceding account that the longitudinal
division in the tetrads corresponds to the longitudinal split in the spireme
of a synapsis stage, and that the transverse division marks the place of
union, end to end, of two somatic chromosomes. Since the tetrad gives
rise to two dyads by parting along the transverse plane of division, and
since the dyads form their daughter chromosomes by means of the longi-
tudinal division, the maturation of the mouse egg belongs to the class of
prereduction divisions.

SUMMARY OF PRINCIPAL RESULTS. 67

IX. SUMMARY OF THE PRINCIPAL RESULTS IN THE STUDY OF
THE MATURATION OF THE EGG OF THE MOUSE.

1. Parturition occurs at any time during the 24 hours of a day, but
more frequently in the early morning.

2. The stages of the formation of the first spindle, the division of the
first spindle, the formation of the second spindle, and the division of the
second spindle are relatively, and probably absolutely, very short.

3. The whole maturation process requires not less than 4 nor more
than 15 hours.

4. Maturation usually occurs at some time during the period ex-
tending from 13! to 28^ hours after parturition.

5. Ovulation may occur at any time during a period beginning at
14^ and ending at 28^ hours after parturition.

6. Ovulation may occasionally take place in the stage of the first
spindle, sometimes during that of the telophase of the first spindle and
the formation of the second polar cell, but usually not till the egg con-
tains the second spindle.

7. Insemination is most successful when it occurs between the 18th
and 30th hours after parturition.

8. The spermatozoa reach the egg in from 4 to 7 hours, or more,
after insemination.

9. The pronuclei are formed probably within a few minutes after
the penetration of the spermatozoon.

10. The diameter of the egg decreases from the stage of the germina-
tive vesicle until it reaches the oviduct, when it increases slightly.

11. The chromosomes of the first spindle are formed from the chro-
matin of the germinative vesicle, and possibly also from the wall of the
nucleolus.

12. They are formed before the nuclear membrane disappears.

1 3 . They show indications of both transverse and longitudinal divi-
sions, and are therefore "tetrads."

14. In the first maturation division the tetrads divide transversely.

15. All first spindles divide.

16. The spindle fibers are probably derived in part from the nu-
cleolus.

17. The chromosomes of the second spindle are " dyads" and divide
longitudinally, separating along a plane which is probably identical with
the longitudinal division-plane of the tetrads.

18. The chromosomes of each spindle number twenty.

19. Typical centrosomes are wanting in both spindle figures.

20. Bodies surrounding the poles of the spindles, here called cir-
cumpolar bodies, and the clear region surrounding the spindle are char-
acteristic of morphologically active stages of the spindle.

68 THE MATURATION OF THE EGG OF THE MOUSE.

21. Each spindle is oblique to the surface of the egg at the beginning
of the abstriction of its polar cell.

22. All eggs form two spindles and a first polar cell.

23. All seminated eggs form a second polar cell.

24. The first polar cell probably migrates in the peri vitelline space
inside the zona pellucida, and is aided in this movement by the process
of ovulation.

25. The first polar cell may or may not degenerate.

26. Maturation division in the mouse egg belongs to the type known
as prereduction division.

BIBLIOGRAPHY.

Allen, G. M.

1904. The Heredity of Coat Color in Mice. Proc. Amer. Acad. Arts and Sci.,

Vol. 40, No. 2, pp. 59-163.
Barry, M.

1839. Researches in Embryology. Second Series. Phil. Trans. Roy. Soc,
London, 1839, pp. 307-380, pi. 5-9.
Bellonci, G.

1885. Del fuso direzionale e della formazione di un globulo polare nell' ovulo
ovarico di alcuni mammiferi. Atti della R. Accad. dei Lincei, Roma,
Ser. 4, Rendiconti, Vol. 1, pp. 285, 286.
Boveri, T.

1892. Befruchtung. Ergeb. Anat. u. Entwick., Bd. 1, pp. 386-485.

COE, W. R., AND KlRKHAM, W. B.

1907. The Maturation of the Mouse Egg. Science, Vol. 25, p. 778, 779.
Gerlach, L.

1890. Beitrage zur Morphologie und Physiologie des Ovulationsvorganges der
Saugethiere. Sitzungsb. physik.-med. Societiit in Erlangen, Heft 22,

.. PP- 43~61-

1906. Uber die Bildung der Richtungskorper bei Mus museums. Festschr.

f. J. Rosenthal. Wiesbaden, 1906. vii+31 pp., 2 Taf.
Gregoire, V.

1905. Les resultats acquis sur les Cineses de maturation dans les deux Regnes.

(Premiere Memoire.) La Cellule, Tom. 22, pp. 221-376.
Hacker, V.

1899. Die Reifungserscheinungen. Ergeb. Anat. u. Entwick., Bd. 8, pp.

847—922.
Heape, W.

1897. The artificial Insemination of Mammals and subsequent possible Fer-

tilisation or Impregnation of their Ova. Proc. Roy. Soc, London, Vol.

61, No. 370, pp. 52-63.
Hertwig, R.

1903. Kapitel: Eireife und Befruchtung. O. Hertwig's Handbuch der ver-

gleichenden und experimentellen Entwickelungslehre der Wirbeltiere.

Jena, 1906. Bd. 1, Lief. 10, n [1903], pp. 477-568.
Holl, M.

1893. Ueber die Reifung der Eizelle bei den Saugethieren. Sitzungsb. Akad.

d. Wiss., math.-naturw. CI., Wien, Bd. 102, Abth. 3, pp. 249-309, 3 Taf.
Iwanoff, E. J.

1903. Ueber die kunstliche Befruchtung von Saugetieren und ihre Bedeutung
fur die Erzeugung von Bastarden. Biol. Centralbl., Bd. 23, No. 19,
pp. 640-646.

KlRKHAM, W. B.

1907a. The Maturation of the Mouse Egg. Biol. Bull., Vol. 12, No. 4, pp.

259-265.
19076. Maturation of the Egg of the White Mouse. Trans. Conn. Acad. Arts
and Sci., Vol. 13, pp. 65-87, pi. 1-8.
Korschelt, E., und Heider, K.

1903. Lehrbuch der vergleichenden Entwicklungsgeschichte der wirbellosen
Thiere. Allgem. Theil, Lief. 2, pp. 539-750. Jena, 1903.
Lams, H., et Doorme, J.

1907. Nouvelles recherches sur la Maturation et la Fecondation de l'CEuf des

Mammiferes. Arch, de Biol., Tom. 23, pp. 259-365, pi. 9-1 1.
Lange, J.

1896. Die Bildung der Eier und Graaf'schen Follikel bei der Maus. Verh. d.
phys.-med. Gesellsch. zu Wurzburg, Bd. 30, Heft 2, pp. 55-76, 1 Taf.
Loukianow, S. M.

1898. Contribution a l'etude de la spermatog6nese chez la souris blanche.

Arch. Sci. Biolog., Inst, imper. M^decine expeY. St. Petersbourg, Tom.
6, No. 3, pp. 285-305, 3 pi.
Mallory, F. B.

1905. A Contribution to the Classification of Tumors. Jour. Med. Research,
Vol. 13, pp. 1 13-136, pi. 5-8.
Melissinos, K.

1907. Die Entwicklung des Eies der Mause von den ersten Furchungs-Pha-
nomenen bis zur Festsetzung der Allantois an der Ectoplacentarplatte.
Arch. f. mikr. Anat., Bd. 70, pp. 577-628, Taf. 32-34.

70 THE MATURATION OF THE EGG OF THE MOUSE.

RUCKERT, J.

1894. Die Chromatinreduktion bei der Reifung der Sexualzellen. Ergeb.

Anat. u. Entwick., Bd. 3, pp. 517-583.

SOBOTTA, J.

1895. Die Befruchtung und Furchung des Eies der Maus. Arch. f. mikr. Anat.,

Bd. 45, PP- 15-93. Taf. 2-6. .

1899. Ueber die Bedeutung der mitotischen Figuren in den Eierstockseiern
der Saugetiere. Festschr. d. phys.-med. Gesellsch. zu Wiirzburg, pp.
185—192, 1 Taf.

1907. Die Bildung der Richtungskorper bei der Maus. Anat. Hefte, Bd. 35,
.. pp. 493-552, Taf. 21, 22. _

1908. Uber die Richtungsteiltmgen des Saugetiereies, speziell tiber die rrage

der Zahl der Richtungskorper. Verhandl. d. phys.-med. Gesellsch.

zu Wiirzburg, Bd. 39, pp. 241-261.
Stricht, O. van der.

1901. La ponte ovarique et l'histogenese du corps jaune. Bull, de l'Acad.

R. de Med. de Belgique, seY. 4, Tom. 15, pp. 216-236, 1 pi., 1901.
Tafani, A.

1889. I primi momenti dello sviluppo dei mammiferi. Studi di morfologia

normale e patologica eseguiti sulle uova dei topi. Arch. Anat.

norm, e patolog., Vol. 5, Fasc. 1, pp. 1-59. (Publ. del R. 1st. di Studi

Sup. Prat, e di Perfez. in Firenze, Sez. di Med. e Chir.)
1889a. I primi momenti dello sviluppo dei mammiferi. Studi di morfologia

normale e patologica eseguiti sulle uova dei topi. Atti R. Accad.

Lincei, Roma, Ser. 4, Rendiconti, Vol. 5, semestre 1, pp. 1 19-125.
18896. La fecondation et la segmentation etudiees dans les ceufs des rats.

Arch. Ital. de Biol., Tom. n, pp. 112-117.

EXPLANATION OF PLATES.

All drawings were made with the aid of a camera lucida. The figures as repro-
duced are four-fifths the diameter of the original drawings. The magnification
appended to the description of each figure is that of the reduced reproduction, the
magnification of the original drawing being in parenthesis.

The magnification of 2500 diameters (reduced = 2000) was obtained with a
Zeiss 2mm. homog. immersion apochromatic objective and No. 12 compensating
ocular; that of 1200 (reduced = 960) , with 2mm. objective and No. 6 compensat-
ing ocular; that of 880 (reduced = 704), with 2mm. objective and No. 4 compen-
sating ocular; and that of 170 (reduced = 136), with Zeiss A objective and No. 4
Huyghenian eyepiece.

PLATE 1.

Origin of First Maturation Spindle.

Fig. 1 . Germinative vesicle shortly before the disappearance of its nucleolus and
the transformation of its contents into the fundaments of the chromo-
somes and the spindle fibers. Ovarian egg. X(25oo) 2000.

Fig. 2. Early stage in the formation of the chromosome fundaments. Ovarian
e&g- X(25oo) 2000.

Figs. 2a, 26. Fundaments of chromosomes in sections adjacent to that of fig. 2.

Figs. 3a, 36. Two consecutive sections showing a somewhat later stage than the
preceding. Ovarian egg. X (2500) 2000.

Figs. 4, 4a. Chromosomes (20 in number) more completely differentiated. Spindle
not yet formed. Nuclear membrane still intact. Ovarian egg.
X (2500)2000.

Fig. 5. Section of a young spindle showing faint fibrillations. There are 20 chro-
mosomes scattered over its surface. Nuclear membrane is dissolved
at some points. Ovarian egg. X (2500)2000.

Fig. 6. Composite drawing of a spindle cut into three parts. There are 20 chromo-
somes. Stage slightly more advanced than that illustrated in fig. 5.
Nuclear membrane completely vanished. Ovarian egg. X (2500)2000.

Figs. 7, ya. Two consecutive sections of a spindle, like that shown in fig. 6, seen in
end view. There are 20 chromosomes, 10 in each section. The cyto-
plasm shows faint radiations about the spindle. Ovarian egg.
X (2500)2000.

PLATE 2.

First Maturation Spindle.

Fig. 8. Ovarian egg. The chromosomes have become arranged in the plane of
the equator. X (880) 704.

Figs. 8a, 86. Enlarged views of the two sections into which the spindle in fig. 8 is
cut. There are 20 chromosomes. X (2500) 2000.

Fig. 9. Section of a spindle like that in fig. 8. X (2500) 2000.

Figs. 10a, 106. The two sections of a spindle of which the fibers at one pole converge
to a point. There are 20 chromosomes. Ovarian egg. X (2500)2000.

Fig. 11. Section of a spindle similar to the preceding. Ovarian egg. X (2500)2000.

Fig. 12. Ovarian egg. The polar ends of the spindle fibers are becoming thickened,
and the clear region about the spindle is visible. One of the 20 chro-
mosomes (some of which are in adjacent sections) has been displaced
into the cytoplasm. X (1200) 960.

Fig. 1 3 . Ovarian egg. The circumpolar bodies are formed at the poles of the spin-
dle, and the clear region is evident. X (1200) 960.

Fig. 13a. More highly magnified view of the spindle shown in fig. 13. X(25oo) 2000.

Fig. 136. View of that portion of the spindle seen in fig. 13a which falls in the fol-
lowing section. X (2 500) 2000.

PLATE 3.
Division of First Spindle and Abstriction of First Polar Cell (Figures

14 to 18, Inclusive).

Fig. 14. Ovarian egg containing an oblique spindle. Several of the chromosomes
have already divided. Circumpolar bodies numerous and conspic-
uous. X (1200)960.

Fig. 14a. One chromosome from the spindle in fig. 14.

Figs. 15a, 156. An oblique spindle in two consecutive sections, showing the mi-
gration of the daughter chromosomes. Ovarian egg. X(25oo) 2000.

Figs. i6a-i6d. Four consecutive sections of a spindle similar in stage of division to
that of fig. 17. See fig. H (p. 34). Ovarian egg. X(25oo) 2000.

72 THE MATURATION OF THE EGG OF THE MOUSE.

PLATE 4.
Second Maturation Spindle (Figures 190-236, Inclusive).

Figs. 1 7a, 170. The two sections show a spindle in a more advanced stage of divi-
sion than that in figs. 15a, 15&. The abstriction of the polar cell has
begun in the vicinity of the "Zwischenkorperchen." Ovarian egg.
X (2500)2000.

Fig. 18. Polar cell recently abstricted. Ovarian egg. X (2500)2000.

Figs. 190, igb. Two sections of an oviducal egg showing polar cell and egg nearly
severed from each other. Prophase of second spindle. X(2 5oo)
2000.

Figs. 20, 21. Polar views of chromosomes of second spindle. Fig. 20 from an ovi-
ducal egg. Fig. 2 1 from an egg in periovarial space. X (2500)2000.

Fig. 22. Side view of second spindle. Large first polar cell on nearly opposite side
of egg. Oviducal egg. X (1200)960.

Figs. 23a, 236. Spindle in paratangential position, cut obliquely into two sections.
There are 19 chromosomes. Circumpolar bodies not stained deeply.
First polar cell very small and near the spindle. Oviducal egg.
X (2500)2000.

PLATE 5.

Second Spindle and Formation of Second Polar Cell.

Figs. 24a, 246. A spindle similar to that of fig. 23, cut into two parts. There are
20 chromosomes. First polar cell absent. Oviducal egg. X(2 5oo)
2000.

Figs. 25-27. Old second -spindles from three eggs showing diminution of circum-
polar bodies. All from oviducal eggs without first polar cell. X (2500)
2000.

Figs. 28a, 286. Polar views of the two daughter plates of a dividing second spindle
in a stage corresponding to that in fig. 16, plate 3. The first polar cell
is very small. Oviducal egg. X (2500) 2000.

Figs. 29a, 296. Two sections of an oviducal egg. The oblique spindle is more ad-
vanced than the one in fig. 28. The stage of the abstriction of the
polar cell (see also fig. J, p. 40) corresponds to that of figs. 16-17. The
first polar cell is seen lying at the left of the second in fig. 296. The
egg contains the heads of two spermatozoa. X(25oo) 2000.

Fig. 30. Oviducal egg showing second polar cell newly abstricted, the first polar
cell, and the head of a spermatozoon. X (1200) 960.

Figs. 31a, 31&. Two sections of an egg (another section of which, less highly mag-
nified, is shown in fig. 40) exhibiting in fig. 31a the first polar cell
lying in the enlarged perivitelline space. X(i2oo) 960.

PLATE 6.

Figs. 32-37. First polar cells from oviducal eggs which contain the second spindle.
They form a series of steps which illustrate the degeneration of the
first polar cell. Figs. 32 and 33 are of polar cells which have divided
into two or more parts. X (2500) 2000.
Figs. 38—40. Three stages in the process of ovulation. In all three cases the egg con-
tains the second spindle and is accompanied by the first polar cell.
X(i7o) 136.
Note. — A minute body appearing in the clear space between zona pellu-
cida and vitellus in fig. 38 is due to a defect in the plate.

PLATE A.

Fig. A. Mouse cage. For description see p. 6.

Figs. B, C. Suspended mouse cages, with self-recording apparatus to indicate ap-
proximately the time of parturition of a gravid female. For descrip-
tion see pp. 7-10.

Fig. G. Chromosomes of first maturation spindle. See pp. 28-30.

(Plate A faces page 6.)

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965 The maturation of the
L7 egg of the mouse

BioMed.

PLEASE DO NOT REMOVE
CARDS OR SLIPS FROM THIS POCKET

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