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UNIV.OF
Toronto
LIBRARY

http:/www.archive.org/d et

MEMOIRS
OF
THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY

No. 5

THE DEVELOPMENT OF THE ALEING
RAT, MUS NORVEGICUS ALBINUS

Pee oROW sth, PRONUCLEAR STAGE VO GEE
STAGE OF MESODERM ANLAGE? END
OF THE FIRS lO THE EN
OF EE aNgIN IPED Ag

THIRTY-TWO FIGURES

lk ABNORMAL OVA; END OF THE Fiksie lS
Eh END OF LEE NINTH Daw

TEN FIGURES

G, CARL HUBER

FROM THE DEPARTMENT OF ANATOMY, UNIVERSITY OF MICHIGAN,
AND THE DEPARTMENT OF EMBRYOLOGY, THE WISTAR INSTITUTE
OF ANATOMY AND BIOLOGY

PHILADELPHIA, PA.

Reprinted from the JouRNAL oF MorpHotocy, Volume 26, No. 2
June,

4 bal des

47
oar? CV ae

Baa an 6a. lf Mn

7 open io

THE DEVELOPMENT OF THE ALBINO RAT,
MUS NORVEGICUS ALBINUS

I. FROM THE PRONUCLEAR STAGE TO THE STAGE OF MESO-
DERM ANLAGE, END OF THE FIRST TO THE END
OF THE NINTH DAY

CONTENTS

Mt OCLC EL OTe iron eee cate OS eis a a eas pctee coe oleh 2 ale vias 3 3
Material amndineth ods sea Saat tse seen oe oo te 2 oe eee 5
OWVilaniGn, MEN OMAINON, Ail WEINGANOKOM so. s5onceonecesnseocicscoosrcencoan &
Pronucleansstage tek, noc tee his Seine cau Beet ook aa ke 13
Sepment avionustacessncme ce anleot oe Rie cineca oldie to 5 eee 21

DECC IIES AO Chet ete A te SER Nees See ae etuiciclla dha |S a1 roe 21

AZ COU S HAM CR sey s oe aes SAS ne ke asda e eb od 3 DA ee Bee,

S=Cellistiaea enone oh snes, hte bis Waite Mer via 2). ot on 31

avo mlG—celilustaice cc) oie ane s Seoeen eh kent a0 BE
Summary of segmentation stages, rate, and volume changes................ 36
Completion of segmentation and blastodermic vesicle formation............ 42
Blastodermic vesicle, blastocyst, or germ vesicle..........................- 56
Late stages of blastodermic vesicle, beginning o: entypy of germ layers.... 63
Development and differentiation of the egg-cylinder....................... 73
Late stages in egg-cylinder differentiation, and the anlage of the mesoderm 92
Conclusions'::4 Vara ot ik one Gey er Siniee Relais Ses oe oy 4S 3 ee 108
MEGeTratumeclhued i ware iemM Re hehe nn Set ti od Ae ey ce Fc ee Cree le

INTRODUCTION

The early developmental stages of placental mammals, em-
bracing the stages of sex cell maturation and fertilization, of
segmentation, of blastodermic vesicle and germ layer forma-
tion, though subject of numerous contributions extending over
many years, have in no form been completely investigated. The
literature dealing with the phenomena of maturation and fer-

3

4 G. CARL HUBER

tilization as observed in placental mammals has in recent years
been enriched by a number of studies to the extent that for cer-
tain of the mammals—bat, rabbit, guinea-pig, mouse, and
rat—the data at hand are sufficiently complete to enable a
clear and comprehensive presentation, based on observed facts,
and permit of comparison with similar phenomena as observed
in other vertebrate and invertebrate forms. As concerns the
process of segmentation in placental mammals, there are still
lacking sufficiently comprehensive observations embracing a
number of forms to enable a clear and succinct presentation of
the rate of blastomere formation, the cytomorphosis of the
cells, and of the relative position of the several segmentation
stages in the genital tract. This is no doubt owing to the diffi-
culty of obtaining the necessary material timed so as to admit
of proper staging, and the impossibility of making extended ob-
servations on living material. Our knowledge of the phenomena
of blastoderm vesicle formation, though comprehended in its
general phases, is lacking in detail, except for a very limited
number of forms. The process of germ laver formation is of
such fundamental importance to a clear comprehension of later
developmental stages, both in phylogeny and in ontogeny, that
a brief account of observed facts in any one form may not be
regarded as wholly without value.

Opportunity presented itself, while stationed at The Wistar
Institute of Anatomy and Biology, to collect and fix an extended
series of embryological stages of the albino rat. This material
has proven sufficiently comprehensive to enable a presentation
of the several developmental stages of this mammal, beginning
with the pronuclear stage and extending to the stage of the an-
lage of the mesoderm. For this period, which extends to about
the tenth day after insemination, only very few of the essential
stages are lacking, though for certain of the stages confirmatory
preparations would have been desirable. The material at
hand, however, seemed sufficiently complete to present a con-
nected account of the stages it is hoped to cover. The embryology
of allied forms, especially of the mouse, has received much more
extended study than has that of the rat, though the develop-

~

DEVELOPMENT OF THE ALBINO RAT 0

ment of the rat has received especial consideration by Fraser,
Christiani, Selenka, Duval, Robinson, Widakowich, and as
concerns maturation and ovulation, by Sobotta and Burekhard,
Kirkham and Burr. The pertinent literature will be considered
in connection with the presentation of my own results.

MATERIAL AND METHODS

The material on which this investigation is based was obtained
from albino rats (Mus norvegicus albinus, Donaldson)! taken
from the extensive rat colony of The Wistar Institute of Anatomy
and Biology. The experience gained in the breeding, feeding,
and growth experiments, extending over many years, conducted
by Donaldson and his associates and resulting in numerous ex-
cellent publications, was at my disposal while collecting this
material. The material used was all carefully timed, so that
sequence of stages was obtained with some degree of certainty.
With care and experience, it is possible to regulate and observe
insemination, so that stages may be approximated quite accu-
rately. Kirkham and Burr state that ‘‘on several different
occasions we have observed actual pairing’ of the albino rat.
Widakowich states that he was unable to obtain accurate data
as to the age of the embryos except by observing coitus. Ac-
cording to this observer, a female rat permits many males to
copulate in the course of several hours, receiving males 30 times
or oftener, when suddenly she drives them away. Sobotta and
Burekhard, on the other hand, admitted males a few hours
after parturition, depending on the fact that many mammals
ovulate soon after parturition. Though attempts were made,
they were unable to observe pairing, and they state that the
‘Dieners’ charged with the care and feeding of the rat colony
were only seldom able to observe attempts at pairing. At
The Wistar Institute no difficulty is experienced in pairing albino

1Melissinos and Widakowich state having used as material the albino rat,
variety Mus rattus albinus. Donaldson has conclusively shown, that by reason
of physical characters—blood crystals, shape of the skull, etc.—the albino rat
kept as pet or laboratory animal cannot be Mus rattus albinus, but must be Mus
norvegicus albinus.

6 G. CARL HUBER

rats. Dr. J. M. Stotsenburg, to whose experience and care-
ful records I am greatly indebted for the trustworthiness of
the material collected, made use largely of females who had
born one litter. Pairing was seldom attempted a few hours
post partem, as was done by Sobotta and Burckhard, but usually
about 30 days after the birth of a litter, which may have been
nursed or otherwise disposed of. The great majority of females
used in pairing were at the time free from ‘domestic cares.’
The females employed were kept in separate cages for some
time before giving birth to young and until the time of mating.
About 30 days after the birth of a litter, a male was placed
in the cage with the female. If the female was in heat, copu-
lation usually took place soon after. The male was left with
the female for an hour to an hour and a half, during which time
several pairings would occur, and at the end of which time the
female would try to hide from the male, climb the side of the
cage and defy him with her teeth. The male albino rat is not
prostrated by the sexual act, the same male serving for several
successive copulations. In case the female was not in heat, this
soon became evident and the male removed, to be again placed
into her cage 24 or 48 hours later. The time when the, copu-
lation was first observed was noted on the card attached to the
cage and gave the time from which the age of the embryo or
respective stage was reckoned. The time given is, therefore,
that of ‘insemination,’ a term which Long and Mark have intro-
duced to indicate ‘‘the introduction of the male sexual elements
into the genital tracts of the female by the act of coitus or other-
wise.’ This time could be accurately noted, while ‘semina-
tion’ which “applies to the access of the spermatozoa to the
eggs in the oviducts, the coming into contact of the male and
female reproductive cells’? can not be accurately timed. The
success attained in pairing albino rats as above stated, obviated
the necessity of depending upon chance material or resorting
to ‘artificial insemination’ as described for the mouse by Long
and Mark. Iam at loss to understand why Widakowich should
regard the age determinations of Sobotta and Melissinos (mouse
embryos) more accurate than his own, reckoned from the time

DEVELOPMENT OF THE ALBINO RAT 7

of observed coitus. The slight though observable variation in
the rate of development in a series of ova of the same animal,
more marked when supposedly similar stages of several animals
are investigated, precludes the accurate timing of stages.

As fixing fluids, there were used Zenker’s fluid, sublimate-
aleohol, Flemming’s fluid, Bouin’s fluid, and Carnoy’s fluid.
After a few trials, all were discarded in favor of Carnoy’s fluid,
prepared by mixing 6 parts of absolute alcohol, 3 parts of chloro-
form, and 1 part of glacial acetic acid. This somewhat illogi-
cally compounded fluid penetrates rapidly and does not cause
shrinkage. Tissues are fixed in it for several hours, then washed
in several changes of absolute aleohol in which it has been my
custom to store the tissues. The following procedure was prac-
ticed in all stages up to about 12 days after insemination: The
animals were anaesthetized and the head severed from the body,
to admit of free bleeding. The rat was then fastened to a
board, and thorax and abdomen opened by a mid-sagittal in-
cision, the abdominal walls pinned back, and the intestine ele-
vated toward the thorax. With as little manipulation as pos-
sible, the ovaries were separated from their attachment, the
mesometrium cut, the uterine horns elevated and the vagina
severed. The whole genital tract was then placed on a clean
slide and arranged in approximately normal position. Slight
tension was maintained by tying a thread to the connective
tissue removed with each ovary and bringing the threads along
the reverse side of the slide and tying them to the vagina. If
the slide is clean, the mesometrium of each uterine horn may be
spread out evenly and caused to adhere to the slide. Ovaries,
oviducts, and uterine horns may thus be spread out in normal
position and each uterine horn fixed as a straight tube. When
thus arranged on the slide, the preparation was placed in a
relatively large quantity of Carnoy’s fluid, fixed, and then trans-
ferred through several absolute alcohols. For nearly all the
material used in this study, the method of fixation was as here
given. In the earlier stages of material collection, attempts
were made to obtain segmentation stages in warm normal salt
solution. Several were thus obtained and were used to control

8 G. CARL HUBER

the observations made on sections, as will be discussed later.
By cutting the oviduct at about its middle, freeing it from its
mesosalpinx and cutting the uterus about | ecm. below the in-
sertion of the oviduct, a pipette fitted with a rubber bulb and
filled with warm normal salt solution can be inserted into the
uterine cavity and moderate pressure made. It is usually possible
to wash into a watch erystal a certain number of the contained
segmenting ova. Before reading the article by Widakowich,
essentially the same method as employed by him, for isolating
implanted blastodermic vesicles was developed. This may be
quite readily done after fixation in Carnoy’s fluid and teasing
under a stereoscopic binocular. Vesicles sectioned in situ,
however, gave on the whole more satisfactory results, so that
teasing out implanted vesicles was not resorted to.

The fixed tissues were imbedded in paraffin, using xylol as
a clearing fluid. For stages including those falling within the
period ranging from the first to the fourth day after insemination,
the ovary and oviduct to its insertion in the uterus, were em-
bedded en masse. For stages falling within the period of fifth
to sixth day after insemination, the uterine horns were divided
into segments measuring about 1.5 em., and sectioned parallel
to the plane of the mesometrium. For later stages, after the
enlargements in the uterine horns are distinctly evident, these
were removed and cut severally in the three planes. The great
majority of the sections were cut at a thickness of 10 1; certain ones
at a thickness of 5u;afew at athickness of 7u. The sections were
fixed to the slide by the water-albumen method. The great ma-
jority of the series were stained in hemalum, counterstained in
Congored. This solution, which presents certain advantages as a
counterstain for embryologic tissues, is prepared as follows: 0.5
ems. of Congo red (Griibler) is placed in 100 cem. of distilled
water and the water brought to boiling. This should give a clear
solution. Before cooling, add 100 cem. of distilled water and
10 cem. of absolute alcohol. The Congo red solution thus pre-
pared may be kept many weeks. After staining the series in the
usual way in hemalum, they are differentiated in acid alcohol,
and passed through several washes of ‘tap water’ into distilled

DEVELOPMENT OF THE ALBINO RAT Q

water. They are then stained in the Congo red solution, which
may be diluted with distilled water about five times. With the
diluted solution, the counterstaining requires one to two hours.
The sections are then rinsed in distilled water, differentiated in
80 per cent alcohol, dehydrated, cleared, and mounted in damar.
Certain of the series were stained in Heidenhain’s iron-hema-
toxylin and counterstained in Congo red. The drawings accom-
panying this contribution were nearly all drawn on coarse ‘Ross
board,’ with the aid of the camera lucida at a magnification of
1000 diameters, using pencil and India ink. Such drawings
admit of liberal reduction, and give a detail not readily obtained
otherwise. Free use has been made of the Born method of re-
construction, especially for earlier stages. The majority of
the models thus obtained are here reproduced.

I desire to express my sincere thanks and appreciation of the
very material aid given me by Mr. Wayne J. Atwell, then Assist-
ant in the Department of Histology and Embryology of the
University of Michigan, in the making of the reconstructions
of the oviducts included in this account.

OVULATION, MATURATION, AND FERTILIZATION

When this study was projected, it was the purpose to begin
it with the stages of maturation and fertilization. During the
time of material collection, there appeared the contribution of
Sobotta and Burckhard: “Reifung und Befruchtung des Eies
der Weissen Rate,’’ covering these stages fairly completely.
Duplication of their work did not seem necessary, so that my
own studies begin with the pronuclear stage, to which stage the
above mentioned investigators had carried their observations.
Therefore, as concerns the process of ovulation, maturation, and
fertilization as observed in the albino rat, I am confined for
my data to the literature; from which a brief résumé is here
made.

The normal gestation period for non-lactating albino rats may
be roughly estimated as from 21 to 23 days. As has been shown
by King, the period of gestation of lactating albino rats varies

10 G. CARL HUBER

from a minimum of 24 days to a maximum of 34 days. The aver-
age number in a litter is six. In lactating females suckling
five or less young and carrying five or less young, the period of
gestation usually does not exceed 23 days and may thus be con-
sidered as normal. In lactating females suckling five or less
young, while they are carrying more than five young, the period
of gestation may be prolonged from one to six days. In lactating
females suckling more than five young, the period of gestation
is always prolonged, and may be prolonged to a maximum of 34
days. Daniel’s studies on the white mouse lead him to formu-
late the following law: ‘‘The period of gestation in lactating
mothers varies directly with the young suckled.’ Such exact
relation between the number of young suckled and the extent
of the prolongation of the gestation period was not observed by
King for the albino rat.

In the albino rat, ovulation occurs spontaneously and is not
dependent on copulation, which act, however, may precede or
follow ovulation. Kirkham and Burr state that ovulation
usually occurs about 24 hours after parturition and that the
developing ova can be traced in the ovary through the two
oestrus cycles preceding their discharge. Long, in his study
No. 3, by Mark and Long, finds that ovulation must occur in
the albino rat on an average not less than 18 hours after par-
turition. Sobotta and Burekhard state that ovulation always
occurs within 36 hours post partem, though at very variable
periods, often only a few hours after the completion of parturi-
tion; again, much later. A second ovulation period apparently
occurs some 30 days post partem, as would appear from the
successful pairings conducted by Dr. Stotsenburg. This agrees
with the observations of Melissinos, who found that pairings were
more numerous when attempted 29 days after parturition, than
when attempted 20 to 21 days after parturition, as practiced by
Sobotta. Semination probably takes place in the ampullar
portion of the oviduct. Relatively few spermatozoa enter the
oviducts and Sobotta and Burekhard estimate that the life of
the spermatozoa in the genital tracts of the albino rat is only
about 10 hours.

DEVELOPMENT OF THE ALBINO RAT 11

The phenomena of maturation and fertilization in the albino
rat have been carefully studied by Sobotta and Burekhard,
from whose account the following brief summary is taken: The
behavior of the ovum of the albino rat with respect to the forma-
tion of polar bodies is very similar to that of most other mammals
studied. The first polar body is given off within the ovarian
follicle, the second in the oviduct and only after semination.
The first maturation spindle, developed from the nucleus of the
oocyte of the first order, forms usually immediately after par-
turition. Kirkham and Burr state “it is usually formed less
than 24 hours after parturition.” It is short and broad, with
the chromatin scattered. The first maturation spindle lies near
the center of the ovum, then passes toward the surface assuming
a tangential position, and only with the beginning of metakinesis,
takes a radial position. The chromosomes of the first maturation
spindle, estimated as numbering 16, appear in the form of modified
rings, which are divided transversely across to form short rounded
rods with a longitudinal direction in the diaster stage. The
first polar body is formed in the ovarian follicle and appears
to be relatively large. It is evident only in the ovarian ovum,
and appears to be lost soon after its formation. Its fate is
doubtful. The first polar body is nearly always missing in tubal
ova. Kirkham and Burr state that ‘the rare occurrence of
the first polar body associated with the egg in the tube is to be
attributed to its rapid disintegration, which begins as soon as it
is formed, and may lead to complete disappearance before
ovulation occurs.”’ The second maturation division begins
immediately after the completion of the first, without an inter-
vening resting phase. The spindle formed is narrower and
longer than the first, with the chromatin massed. In its monas-
ter stage, it lies in a tangential position, with the chromatin
in diads, and with the lines of division at right angles to the
axis of the spindle. The appearance of the second maturation
spindle in the monaster stage marks the end of the maturation
phenomena in the ovary. The monaster stage of the second
oocyte division was not observed in the ovary by Sobotta and
Burekhard, but was seen by Kirkham and Burr. The first

[2 G. CARL HUBER

division Sobotta and Burekhard regard as a reduction division,
a heterotypic longitudinal division; the second as an equatorial
division, a homeotypic longitudinal division. Ovulation prob-
ably occurs during the monaster stage of the second maturation
division.

The tubal ova are surrounded by a relatively thin oolemma
to which are adherent a variable number of discus cells. They
are smaller than the ovarian ova; the latter measuring 60 u to
65 u, the tubal ova 55 » to 60u. The recently discharged tubal
ova are to be found in the distended ampullar portion of the
oviduct, where they are found clumped together surrounded
by discus cells. Semination takes place in this region. The
spermatozoa usually enter while the tubal ova are in the mon-
aster stage of the second maturation division, after which meta-
kinesis begins. The second maturation spindle assumes a radial
position in the metakinetic phase. The second polar body is
smaller than the first, and usually les compressed between the
oolemma and the ooplasm, and is evident during fertilization and
segmentation. The spermatozoan head penetrates the thin
oolemma and the ooplasma; the long middle piece and _ tail
following the head into the ooplasma, as has been shown by Coe,
and Kirkham and Burr. The long middle piece, soon after
penetrating the ooplasma, presents an increase in stainability,
and its spiral thread becomes evident. The spiral thread, as
Duesberg has shown, has its origin in the mitachondria of the
spermatid. It may be, therefore, that the male sexual cell
introduces mitachondria to the egg cell at the time of fertilization.
Some little time after the penetration of the sperm head, this
enlarges and becomes vacuolated, and diplosomes with polar
rays become evident. As the sperm head begins to metamor-
phose, tending to the formation of the male pronucleus, the chro-
mosome group of the dispireme of the second maturation spindle,
undergoes metamorphosis to form the female pronucleus. This
enlarges rapidly to form a vesicular nucleus which lies free in the
ooplasm, while the metamorphosing male pronucleus, usually
smaller, is accompanied by a deeply staining thread-like struc-
ture, derived from the middle piece. The centrosomes of the

DEVELOPMENT OF THE ALBINO RAT 13

first segmentation spindle are by inference derived from the
sperm centrosome. The data here given, as concerns the matu-
ration and fertilization phenomena pertaining to the albino
rat, unless otherwise credited, have been drawn from the account
of Sobotta and Burckhard, whose account is accompanied by
excellent figures.

Long has studied in living ova of mice and rats the phenomena
of maturation and fertilization. Tubal ova were placed in Ring-
er’s solution on an especially constructed slide and spermatozoa
introduced. It was possible to seminate the ova of rats with
rat spermatozoa and to observe the formation of the second
polar body. The formation of the second polar body, ‘usually
near the first polar cell, may begin within five minutes to two or
more hours after the spermatozoa are introduced. The con-
striction may be finished three-fourths of an hour later.’ ““The
first appearance is an elevation clearer than the rest of the cell.
The swelling becomes higher, and at one side of the elevation
there appears a depression which is the beginning of the constric-
tion which presently encircles the whole swelling and cuts it
off from the egg.’”’ Nothing couldbe said as to the changes which
the chromatin undergoes after the spermatozoa have penetrated
the egg. The eggs remained alive and apparently normal for
about twelve hours, after which they began to degenerate.

PRONUCLEAR STAGE

As has been stated, my own observations on the develop-
ment of the albino rat (Mus norvegicus albinus) begin with the
pronuclear stage. The material at hand for this stage is listed
in table 1, page 258.

Thus there are present in the series 34 ova showing a pro-
nuclear stage and 9 ova showing the second maturation spindle
in the monaster phase. The latter may be dismissed with the
brief statement that they represent unfertilized ova. In rat
No. 108, with 7 ova in the stage of the second maturation spindle,
killed 24 hours after the observed copulation, there was found
no trace of spermatozoa in the oviduct. Two reasons may be
offered for the non-appearance of fertilization in this case:

14 G. CARL HUBER

TABLE 1
| | STAGE OF DEVELOPMENT
RECORD HOURS AFTER BEGINNING NUMBER OF | ees tS 2aaF uy
NUMBER OF INSEMINATION | OVA | | Second matu-
| Pronuclear ration spindle
eae : A | ats
106 24 hours 8 | 8
107 24 hours 11 | 10 1
108 24 hours if 7
109 24 hours, 15 min. 9 8 1
110 24 hours, 15 min. 8 s
Total 43 34 9

Ovulation may have occurred so late that the spermatozoa
may have died before the ova reached the ampullar portion of
the oviduct. This explanation, it would seem, is invalidated
by the fact that the position of the ova in the oviduct, as shown
by graphie reconstruction, is essentially the same as in the other
four rats studied, and in which fertilized ova were found, so that
ovulation must have preceded the killing of the animal by some
hours. The other reason, more plausible, attributes non-fertili-
zation to a pathologie condition of the genital tract. In this rat,
one ovary was distinctly pathologic, with periovarian capsule
greatly distended with a sanguinous liquid, while the upper end
of the uterine horn with adjacent oviduct on the other side,
as seen in sections, presented evidence of inflammation and epithe-
lial desquamation, in part occluding the lumen. It seemed
evident, therefore, that the spermatozoa introduced in the genital
tract were unable to penetrate to the oviduct and consummate
fertilization. The other two unfertilized ova, found with ova
in the pronuclear stage, were in oviducts in which no spermato-
zoa were found. Both in the mouse and the rat, relatively few
spermatozoa reach the upper end of the oviduct; too few, it would
seem, to consummate fertilization of all the ova in certain cases.
Tn all of the ova which contained the second maturation spindle,
this was in the monaster phase and in tangential position. In
size, shape, and chromatin configuration, all presented the char-
acteristics described and figured by Sobotta and Burckhard and
Kirkham and Burr, therefore, need not be considered further.

DEVELOPMENT OF THE ALBINO RAT 15

The stage of pronuclei was observed in over 100 ova of the
white rat by Sobotta and Burckhard. According to these ob-
servers, the two pronuclei show in the earlier stages of their
development, large chromatin-like nucleoli, the number of
which varies. Some little time later, one or several such chroma-
toid nucleolar bodies with irregularly formed chromatin masses
arranged on the linin network are to be observed. At a still
later time, the chromatin becomes distributed over the linin
network, throughout the nuclear space, giving the appearance
of a fine chromatin network. One of the pronuclei is, as a rule,
somewhat smaller than the other. This is regarded as the male

Fig. 1 Tubal ova, albino rat. X 200. A, rat No. 110, 24 hours, 15 min.,
ovum in pronuclear stage, larger nucleus female pronucleus; B, and C, rat No.
59, 2 days, 2-cell stages, thin oolemma showing in C, only partially seen in B;
D, rat No. 62, 2 days, 22 hours, 3-cell stage, the nucleus of the unsegmented
blastomere in the monaster phase, only one of the other two cells showing in the
figure.

pronucleus, since near it the ‘sperm centrum’ was now and
then observed. The pronuclei lie in about the center of the
ovum. The pronuclear stages of my own material, observed in
34 ova, obtained 24 hours after the beginning of insemination—
thus at the end of the first day of development—all present essen-
tially the same stage of metamorphosis. As may be seen in A
of figure 1, the nuclei are distinctly membraned, and are of
relatively large size. The ovum here sketched measures in the
stained preparation 70 u by 62 u, and is, therefore, of slightly oval
form. Sobotta and Burckhard give 55 u to 60 yu as the size of
the tubal ova, and 60 » to 65 uw as the size of the ovarian ova in

16 G. CARL HUBER

the white rat. JXirkham and Burr give the diameter of the
living unsegmented egg of the rat as of 0.079 mm. As may be
seen from A and B, of figure 2, the tubal ova, even when free in
the oviduct, are not of necessity spherical in shape, but often
slightly compressed, as may be clearly seen in four models of
tubal ova in the pronuclear stage, reconstructed at a magnifica-
tion of 1000 diameters, in my possession. Depending on the
plane of section, the diameter of a tubal ovum may thus vary
to the extent of 5 u to 8 uw. The two nuclei in the preparation
shown in A of figure 1, measure, the larger one, regarded as the
female pronucleus, 23 » by 16 yu, the smaller 17 uw by 15 u. Essen-
tially all of the chromatin is distributed over the linin network
in fine granules, the larger nucleus presenting one large, faintly-

Fig. 2. Models, made after the Born method, of two tubal ova of the albino
rat in the pronuclear stage. > 200. A,rat No. 106, 24 hours; B, rat No. 110,
24 hours, 15 min. Reconstructions made at a magnification of 1000 diameters,

figure reduced in reproduction.

staining chromatoid nucleolus, The ooplasm is finely granular,
distributed so as to give the section a slightly mottled appear-
ance. When compared with figures given by Sobotta and
Burekhard (figs. 21 to 24, plates 9-10) showing pronuclear
stages of the ova of the rat, my own seem to fall in about the
middle of this series, thus some little time after their formation,
but not immediately preceding the stage of segmentation spindle
formation. In the albino rat, and perhaps in other mammals, the
pronuclear stage, in its various phases of nuclear metamorphosis,
must constitute a stage covering a relatively long period. If it
is assumed that semination occurs about 10 to 12 hours after the
beginning of insemination, such assumption being justified
by the observations of Sobotta and Burekhard, according to
whom the life of the spermatozoa in the genital tract of the
white rat is only about 10 hours, and if it is recalled that in

DEVELOPMENT OF THE ALBINO RAT 17

living rat ova Long found that the constriction of the second
polar body may be completed three-fourths of an hour after
its inception, then it must be evident that the pronuclear stage
extends through a period which exceeds 10 to 12 hours, since in
none of my pronuclear stages obtained 24 hours after insemina-
tion was evidence of first segmentation spindle observed.

In order to determine accurately the relative position of the
ova within the oviduct during the pronuclear stage and the
stages of segmentation, oviducts containing ova were recon-
structed after the Born wax plate method. In form, relations,
and general structure, the oviduct of the albino rat is essen-
tially the same as that of the mouse as described by Sobotta. The
oviduct of the rat measures from fimbriated end to termination
in the uterine horn from 2.5 em. to about 3.0 em. It presents
eight to ten fairly constant major folds, the middle group of
which is closely applied to the ovarian capsule. The upper or
distal folds pierce the capsule, ending in the fimbriated end
found within the capsule, while the lower or proximal folds,
proximal with reference to the uterine horn, effect connection
with the uterine horn. These relations are essentially the same
as those described by Sobotta for the oviduct of the mouse. This
observer recognizes four segments in the oviduct of the mouse,
characterized by epithelial lining, nature and extent of folding
of the mucosa, and thickness of the musculature. The first
segment, which falls to the infundibulum, presents a thin muscu-
lature and high mucosal folds with epithelial lining consisting
of relatively short cylindrical cells with distinct cuticular border
and long cilia. As characteristic of this portion of the tube
there are further described accessory nuclei compressed between
the epithelial cells. Only this portion of the oviduct is ciliated.
In the second segment, the lumen is large and the folds of the
mucosa prominent. They are covered by a non-ciliated epithe-
lium, without distinct cuticular border. The musculature is
relatively thin. In the third segment the musculature is well
developed with circularly and longitudinally disposed cells. The
lumen is narrow and the folds are nearly absent, while the epithe-
lium is of a simple columnar variety. The fourth segment, not
so well characterized, consists of the loops which make con-

18 G. CARL HUBER

nection with the uterine horns, with folds and epithelium much
as in the third segment, and a prominent musculature. In all
essentials, this description applies to the oviduct of the albino
rat, except that in the first segment the accessory nuclei de-
scribed by Sobotta as found between the epithelial cells were not
evident in the rat. In figure 3, is reproduced a model of a wax
reconstruction of the right oviduct of rat No. 106, killed 24
hours after the beginning of insemination, and containing eight
ova in the pronuclear stage. This oviduct measured from
fimbria to termination in the uterine horn 3.2 cm. It pre-
sents 10 major folds, which folds may be recognized with more

Fig. 3 Model of right oviduct of rat No. 106, 24 hours. X 10. Fimbriated
end and infundibulum removed in the drawing so as to expose underlying loops;
their relative position given in dotted outline. The position of the ova, which
are outlined in circles, is shown as if seen through a transparent wall. ‘The rela-
tive position of three of the eight ova found within this tube cannot be revealed
in this view of the model.

or less clearness in all the models made and here reproduced.
The slight difference in the relative position of these folds as seen
in the several figures may be accounted for by the varying de-
grees of tension to which the tissues were subjected prior to
fixation. In rat No. 106, the ovaries with oviduct and upper
end of the uterine horn, were excised and placed in the fixing
fluid without applying any tension. Of these 10 major folds,
the four distal ones, those beginning with the fimbriated end,
fall to segments one and two of Sobotta’s designation, having a
wide lumen and folded mucosa. In the figure, the position of
the ova is indicated by small black circles. By reason of the
relation of the folds, only five of the.eight ova can be brought

DEVELOPMENT OF THE ALBINO RAT 19

to view in the aspect of the model sketched. The position of
the first and the last of the series is correctly given. The ova
are situated in a loop of the oviduct which is about 8 mm. from
the fimbriated end. By the end of the first day after the begin-
ning of insemination, the ova have thus travelled about one-
fourth the length of the oviduct. In figure 4 is reproduced a
model of a detailed reconstruction of that portion of the oviduct

Fig. 4 Model of the segment of the right oviduct of rat No. 106, 24 hours,
containing the ova the general position of which is shown in Figure 3. 50.
The wall is in part removed, so as to expose the lumen. Note the character of
the folds of the mucosa. ‘The relative position of the eight contained ova, all
in the pronuclear stage, is clearly shown.

containing the ova, representing a loop of the tube with one
side cut away, this to show the extent and character of the
mucosal folds, the width of the lumen and the relative position
of the several ova. The figure presents these facts so clearly
that lengthy description is deemed unnecessary. The several
ova are distributed through a tube segment measuring about
2.5mm. inlength. They lie free in the lumen, apparently bathed
in a fluid from which there is only a small amount of precipita-
tion at the time of fixation. Their position in the oviduct at

20 G. CARL HUBER

this stage, free in the lumen, is well shown in figure 5, which is
from a longitudinal section of a loop from the left oviduct of
rat No. 109, showing three ova, with but few remaining discus
cells and a thread of coagulum linking the ova together, an
appearance quite characteristic at this stage. The figure was
drawn by aid of camera lucida from a single section. All of the
ova, of which there are seven, distributed through this loop, con-
tain two pronuclei; in none of the ova figured do the two pronuclei

Fig. 5 Camera lucida drawing of a portion of a section of the left oviduct of
rat No. 104, 24 hours, 15 min. »X 100. Three ova with a few discus cells, are
shown as lying free within the lumen. The ova are in the pronuclear stage, not
shown in this section, but readily ascertained by tracing through the series.
The loop of the oviduct here shown in section is cut longitudinally, thus the
folds of the mucosa are not prominent.

fall in the same section. My series contains seven oviducts with
pronuclear stages, with accompanying ovary, cut serially. Only
one of the oviducts, rat No. 106, was reconstructed in wax. In
the other six, graphic reconstructions were made. This permits
analysing the loops, determines their sequence, but does not
readily admit of measuring their length. In the six oviducts
graphically reconstructed, the position of the ova, the number
of which varies from one to seven in the several tubes, is essen-
tially as in the wax reconstruction figured. It would appear,

DEVELOPMENT OF THE ALBINO RAT 21

therefore, that in the albino rat, 24 hours after the beginning of
insemination, the ova are to be found in the pronuclear stage,
with the ova distributed in the end of the third to the beginning
of the fourth major loop of the oviduct, a portion of the oviduct
having a relatively wide lumen and lined by a much folded
mucosa and possessing a relatively thin muscular wall, having
thus migrated about one-fourth of the length of the oviduct.

SEGMENTATION STAGES

2-cell stage. The material on which my own observations of
this stage are based is listed in table 2.

TABLE 2

HOURS AFTER BEGINNING
EC NUMBER NU) a r OV: STAGE =eVELOPMEN
RECORD NUMB OF INSEMINATION NUMBER OF OVA TAGE OF DEVELOPMENT

60 1 day, 18 hours vi 2-cell stage
59 2 days 8 2-cell stage
it. 9 ae yr .
58 2 days, 17 hours 8 J fy 2 IEE SESS;
’ | 1, 3-cell stage
61 2 days, 18 hours 8 2-cell stage
9_-np via
62 2 days, 22 hours 11 Be ac oe:

1, 3-cell stage

Thus in all 40 ova after the completion of the first segmenta-
tion division and 2 ova in the 3-cell stage, in each of which the
undivided blastomere presents a nucleus in mitosis.

My own material lacks stages showing the formation of the
first segmentation spindle, the conjugation of the two pronuclel,
and the first segmentation division. I am forced to proceed
from the pronuclear stage to that showing the first two blasto-
meres. It was not possible to supplement my material after this
was sectioned and the stages determined, since it was only after
leaving The Wistar Institute that this gap in my series was recog-
nized. This is the more to be regretted since neither Melissinos,
Sobotta and Burekhard, nor Kirkham and Burr, all of whom
have considered maturation and fertilization as observed in the
albino rat, discuss these stages in their account. In the albino
rat, the fusion of the two pronuclei on the first segmentation

D2, G. CARL HUBER

spindle, and the first segmentation division would appear to
fall to a period ranging from the beginning to near the middle
of the second dayafter the beginning of insemination, probably
about 30 to 32 hours after insemination. In the mouse, in which
these stages have been very completely and carefully investi-
gated by Sobotta, the conjugation of the pronuclei and the
first segmentation spindle formation falls to the end of the first
day after copulation. These phenomena appear to be passed
through rather quickly in the mouse ovum, covering a period of
only about one and a half to two hours.

The 2-cell stage with resting nuclei extends through a relatively
long period. In the mouse it extends through nearly an entire
day, as shown by Sobotta, who found 2-cell stages present through
a period ranging from 25 hours to 48 hours after copulation.
Melissinos often observed the 2-cell stage with resting nuclei in
both mice and rats in material gathered 24 hours after copulation
and to 44 hours thereafter. It is to be regretted that this ob-
server does not differentiate more specifically between ova of
mice and rats in his description. As a rule it is impossible to
determine except by inference to which of the two varieties of
ova his account refers. It may be assumed that the statements
made apply equally well to the ova of either the mouse or the
rat.

In my own material, the 2-cell stage was observed during a
period extending from 1 day, 18 hours to 2 days, 22 hours after
the beginning of insemination, thus for a period extending over
more than 24 hours. In the albino rat, the first two blastomeres
are equivalent cells of essentially the same size and structure,
as may be seen from B and C, of figure 1, drawn respectively of
ova found in the right and left oviducts of rat No. 59, killed two
days after the beginning of insemination, and regarded as repre-
sentative ova. The two cells of each ovum are not spherical,
but of shghtly oval form, with relatively large, distinctly mem-
branated nuclei, with fine chromatin granules scattered on the
linin network and a number of relatively large chromatoid nucleoli.
The cytoplasm presents a granular appearance, the granules
being evenly distributed throughout the cell. In my own
material, I seldom find the two cells lying in the same plane,

DEVELOPMENT OF THE ALBINO RAT 23

but one cell, as a rule, rises slightly higher than the other. This
is more clearly seen in reconstructions than in sections. In
figure 6 are shown reconstructions of the 2-cell stages, figured in
B and C of figure 1. In B, of the figures, the plane of section is
at right angles to the vertical axis of the reconstruction as shown
in B of figure 6, while in C of figure 1, the plane of section is
parallel to the vertical axis of the reconstruction shown in A
of figure 6. The equivalence or non-equivalence of the first
two blastomeres of the segmenting mammalian ovum has been
the subject of discussion since the time of Van Beneden’s funda-
mental observations on the segmentation of the ovum of the rab-
bit. This discussion has been summarized a number of times
in recent years, and need not be entered into here. Suffice to
say that the consensus of opinion of the more recent contributors

A B

Fig. 6 Models, obtained by reconstruction after the Born method, of the
2-cell stages of the albino rat. Rat No. 59, 2 days. X 200.

is, that the first two blastomeres of the mammalian ovum are
equivalent in size and structure if the stage is observed soon after
its formation. As above stated, the 2-cell stage of the mam-
malian ovum extends through a relatively long period, probably
about 24 hours. The two cells do not as a rule divide synchron-
ously, the division of one preceding the other by some little time,
resulting in a 3-cell stage. The cell to divide first increases
slightly in size and presents a clearer protoplasm prior to its
division. Ina2-cell stage, viewed in this phase of eytomorphosis,
one of the cells appears slightly larger with clearer protoplasm
than does the other cell, explaining the difference in size and
structure observed by Van Beneden and by other observers who
concur in his views. I am convinced that a difference in the
size of the two cells may be accounted for by the plane of section
in which they are cut, even though the nuclei of both cells are
included in the section. In the figures of sections of the 2-cell

24 G. CARL HUBER

stage of the mouse, given by Sobotta and Melissinos, the nuclei
of the two cells he in about their center and essentially in the
same plane. In my own material of the 2-cell stage of the albino
rat it is not unusual to find the nuclei of the respective cells
nearer the opposite poles of the two cells than at their centers,
as shown in C, of figure 1. In B of this figure, where the two
nuclei appear as lying much nearer the center of the cells,
they are in reality placed much as in C, as is shown by the
reconstruction.

od

Fig. 7 Model of the right oviduct of rat No. 59, 2 days. X10. Not quite the
entire oviduct was available for reconstruction, the upper end of the uterine
horn thus not shown in the figure. The position of the four 2-cell stages, each
of which is outlined in a circle, found within the tube, is shown as if seen through
a transparent wall.

To determine the position of the segmented ovum in the
2-cell stage in the oviduct, reconstructions were made of two
oviducts. In figure 7 is shown a reconstruction of the right
oviduct of rat No. 59, killed two days after the beginning of
insemination. In preparing the material for embedding, this
oviduct was cut not quite at its insertion into the uterine horn.
The portion of the oviduct reconstructed measures 2.29 cm.
Nine major loops are shown. The four ova in the 2-cell stage
found in this tube are situated in the sixth to the seventh loop
at a distance of about 1.4 em. from the fimbriated end. This
portion of the oviduct falls to segment three of Sobotta’s designa-
tion. It is lined by non-ciliated epithelium resting on a mucosa
with inconspicuous secondary folds, but presenting four or five
characteristic major folds. This portion of the oviduct is closely

DEVELOPMENT OF THE ALBINO RAT 25

applied to the outside of the ovarian capsule, and conspicuous
in all of the figures of models of the oviducts here presented. The
detail of the distribution of the ova in the tube is given in figure
8, a reconstruction under a higher magnification of the segment
of the oviduct containing the ova. The lumen is exposed so
that the character of the mucosal folds may be seen. The ova
are spaced in a segment of the tube measuring 3 mm., and are

Fig. 8 Model of the segment of the right oviduct of rat No. 59, 2 days, con-
taining the four 2-cell stages as shown in figure 7. X 50. Note the absence of
prominent folds in the mucosa. The segment presented in the reconstruction
measures 3 mm. The four 2-cell stages contained in this tube are relatively
widely spaced.

in this case more widely separated than is usual for this stage.
In figure 9, there is reproduced a reconstruction of the left ovi-
duct of rat No. 62, killed 2 days, 22 hours after the beginning
of insemination. This tube was also cut a little before its in-
sertion into the uterine horn. The portion reconstructed meas-
ures 2.45 ecm. In it there are found five ova in the 2-cell stage,
situated about 2 em. from the fimbriated end, and in the last
loop of the third segment of the oviduct. The five ova are closely

26 G. CARL HUBER

grouped between two opposing folds of the mucosa. Their
general relations are shown in figure 10, a reconstruction under
higher magnification of the segment of the oviduct containing

Fig. 9 . Model of the left oviduct of rat No. 62, 2 days, 22 hours. X 10. Not
quite the entire oviduct was available for reconstruction, thus the relative posi-
tion of the upper end of the uterine horn is not shown in this figure. Fimbriated
end and infundibulum removed in the drawing, so as to expose the underlying
loops; their relative position is given in dotted outline. The position of five 2-
cell stages, found within this tube, is given as if seen through a transparent wall.

Fig. 10 Model of the segment of the left oviduct of rat No. 62, 2 days, 22 hours,
containing the five 2-cell stages, the general position of which is shown in figure
9. 50. Note the compact grouping of the ova.

the ova, cut so as to expose the lumen. At the magnification
used it was not possible to reproduce in the model the exact
shape of the several ova, their relative position is, however,
correctly given. In all, ten oviducts, containing 40 ova in the
2-cell stage, are included in my series. Of these, two, as above
given, were reconstructed by the Born method. The other
eight were reconstructed graphically, beginning with the uterine

DEVELOPMENT OF THE ALBINO RAT 27

end of the tubes. In six of these, the ova are quite closely
grouped as given in the reconstructions shown in figures 9 and
10. In the remaining two they were more widely spaced, about
as shown in figures 7 and 8. In the oviducts taken from rats
Nos. 58, 61, 62, killed respectively 2 days, 17 hours, 2 days, 18
hours, and 2 days, 22 hours, after insemination, the ova are
found in a portion of the tube which corresponds very closely
to that shown in the reconstruction presented in figure 9. In
rat No. 60, killed 1 day, 18 hours after insemination, the ova
are more widely spaced and are situated in a segment of the
oviduct approximately one loop nearer the fimbriated end than
that given in figure 7, a model of the oviduct of rat No. 59, killed
two days after insemination.

In one of the segmented ova of rat No. 60, the two blastomeres
resulting from the first segmentation division are distinctly sep-
arated by a space equal to about one-half of the diameter of each
of the cells. No oolemma is discernible. The two separated
cells appear normal in size, shape, and structure, as do also their
nuclei. They lie free in a slightly distended portion of the lumen,
and appear not to have been separated as a consequence of ma-
nipulation. The possibility of each developing separately is
suggested, and may be offered as a possible explanation of the
occurrence of very small embryos now and then found among
others showing normal development. King states that “On
dissecting pregnant females (rats) one frequently finds one or
more embryos that are much smaller than the rest. While in some
instances such small embryos appear normal and are presumably
either runts or embryos that have resulted from superfecunda-
tion, in the majority of cases they are pathological, probably
because of faulty implantation of the ovum.” My own material
contains pathologic ova and embryos in different stages of
development. This portion of the material will be considered
in Part II, where the possibility of the occurrence of half em-
bryos will be discussed.

As may have been seen, the 2-cell stage of the albino rat covers
a period of somewhat more than 24 hours, extending from about
the middle of the second day until toward the end of the third

28 G. CARL HUBER

day after the beginning of insemination. During this period
the segmented ova migrate in the oviduct for a distance equaling
nearly half its length. The trustworthiness of the material, it
would seem to me, is shown by the fact that in the shorter time
stages the segmented ova are situated nearer the fimbriated
end, while in the longer time stages they approach the region
of the insertion of the oviduct into the uterine horn. This is
clearly shown in the reconstructions shown in figures 7 and 8.

A 3-cell stage was observed only twice: in one of eight ova
contained in the oviducts of rat No. 58 (2 days, 17 hours) and in
one of eleven ova found in the oviducts of rat No. 62 (2 days,

Fig. 11 Two views of each of three models of 4-cell stages of the albino rat.
Rat No. 50,3 days, lhour. X 200. A, B, and C, gives aside view, A’, B’, and C’
a vertical view, of each of the three models.

22 hours). All the other ova found in these two animals were in
the 2-cell stage. In the two 3-cell stages noted, the undivided
blastomeres of each ovum presented a nucleus in mitosis; in one,
in the monaster phase, in one, in the diaster phase. The divi-
sion of the first two blastomeres, resulting in the 4-cell stage, it
would appear, occurs in the albino rat toward the end of the
third day. The material gathered at the beginning of the
fourth day after insemination presents throughout a 4-cell
stage. In D of figure 1 is shown reproduced one of the sections
of a series of six sections including one of the ova in the 3-cell
stage. Only one of the two cells resulting from the division of
one of the first two blastomeres is included in the section; the
cell in mitosis represents the undivided blastomere.

DEVELOPMENT OF THE ALBINO RAT 29

4-cell stage. The material includes the oviducts of two rats,
Nos. 50 and 63, killed 3 days and 1 hour after the beginning
of insemination, with twelve ova in the 4-cell stage. In figure
11, there are shown two views of each of the models obtained
by reconstruction after the Born method, at a magnification of
1000, of the three 4-cell stages found in the oviducts of rat
No. 50. The drawing of the reconstructions do not present the
conventional figures of the 4-cell stage of the mammalian egg.
In none of the twelve ova of this stage was the plane of section
such as to include all of the four cells in one section. Nearly all

Fig. 12 Cross-section of right oviduct of rat No. 50, 3 days, 1 hour. ™ 100.
This section contains two cells of a 4-cell stage of the albino rat, slightly com-
pressed between the folds of the tubal mucosa.

lie in a portion of the tube which presents a relatively narrow
lumen, and appear as if slightly compressed between the folds of
the mucosa. I am not disposed to regard this as a resultant of
fixation, due to contraction at the time of fixation. In figure
12 is reproduced a cross section of the right oviduct of rat No.
50, passing through a 4-cell stage. It is evident that in shape
the two cells included in the section, conform in the main to
the form of the lumen, the mucosa appearing as slightly retracted
to one side of the egg mass. This conformity in shape of cell
mass to the form of the lumen I find quite general in my material
showing segmentation stages of the albino rat, to some extent

30 G. CARL HUBER

even in the 2-cell stage, more clearly shown in the 4-cell and later
segmentation stages, as will appear from further reconstructions
presented. It would seem to me reasonable to assume that these
cell masses are of such plasticity that they are molded by the
tubal mucosa rather than they would compress the mucosa and
maintain an inherent form. A number of segmented ova in
presumably the 6- and 8-cell stages were removed from oviducts
by injection and studied in warm normal salt solution, in a liy-
ing state. In the warm normal salt solution the morula masses

Fig. 13 Model of right oviduct of rat No. 50, 3 days, 1 hour. »X 10. A short
segment of the upper end of the uterine horn, lower part of the figure, is included.
The fimbriated end and a part of the infundibulum removed in the drawing so
as to expose the underlying loops; their relative position is indicated in dotted
outline. The position of the four ova in the 4-cell stage, at the beginning of the
last loop of the oviduct, is shown as if seen through a transparent wall.

presented a nearly spherical form, conforming to the conventional
illustrations of the same. In none of the sections of fixed material
of my series was this the case. The form of the cell mass, assumed
by the segmenting mammalian ovum in early stages of segmenta-
tion, therefore, seems to me a question more for academic dis-
cussion than one of fundamental importance. The right ovi-
duct of rat No. 50 (3 days, 1 hour) was reconstructed after the
Born method. This model is reproduced in figure 13, and
includes the uppermost end of the uterine horn. The oviduct

DEVELOPMENT OF THE ALBINO RAT 31

measures 2.8 cm. and contains four ova in the 4-cell stage, situ-
ated at the beginning of the last loop leading to the uterine
horn, 2.25 em. from the fimbriated end, thus in the fourth seg-
ment of the oviduct as of Sobotta’s designation. In figure 14
is reproduced a detailed reconstruction of the segment of the
oviduct containing the ova, with the convex portion of the wall
of this loop, as shown in figure 13, removed. The section re-
produced in figure 12, passes through the lower of the three
upper ova, shown in reconstruction in figure 14. In the figure of
the reconstruction as also in that of the section, is shown the
groove in which these three ova lie. The other oviducts con-

Fig. 14 Model of the segment of the right oviduct, rat No. 50, 3 days, 1 hour,
containing the four ova in the 4-cell stage, the general position of which is shown
in figure 13. The convex portion of the wall of the loop containing the ova is
removed, so as to expose the lumen.

taining 4-cell stages were reconstructed graphically, beginning
with the uterine end. The position of the ova in each is essen-
tially as given in the model reproduced in figure 13.

8-cell stage. In rat No. 57, killed 3 days, 17 hours after the
beginning of insemination, there are found in the left oviduct,
six segmented ova in the 8-cell stage and one segmented ovum
in the 11-cell stage. The right ovary and oviduct was injured
in the process of embedding and could not be used for sectioning.
The ova are spaced in the loop of the oviduct which terminates
in the uterine horn. Six of the segmented ova were recon-
structed, the seventh was not detected at the time the recon-
structions were made. The six models obtained are reproduced

By G. CARL HUBER

in figure 15, two views of each model being shown. Five of
the models, A to E, show 8-cell stages. In F, there is figured
an 11-cell stage, three of the cells having completed the next
following division. As may be seen from the figures, the form
of these morula masses is not spherical but in the main slightly
oval, with further irregularities better shown in the models than
in the illustrations, due to the fact that the egg masses con-
form to the shape of the lumen of the oviduct in the region in
which they are found. The mucosa lining the segment of this —

Fig. 15 Models, obtained by reconstruction after the Born method, of 8-
cell and 11-cell stages of the albino rat. Rat No. 57, 3 days, 17 hours. X 200.
Two views of each model is presented. A-A’, to E, E’ are of models of 8-cell
stages; F and F’ of a model of a 11-cell stage.

oviduct containing the ova presents four quite regular longitudi-
nal folds. In figure 16, there is presented a model of a detailed
reconstruction of the segment of the oviduct containing the ova,
their relative position in the tube and their relation to the major
folds is clearly shown. One of these folds it was necessary to
in part remove so as to bring to view in the drawing certain of
the ova. In figure 17, there is reproduced a portion of one of
the sections of the series from which the medel shown in figure
16 was made. The fold of the mucosa occupying the center

DEVELOPMENT OF THE ALBINO RAT 33

Fig. 16 Model of the segment of the oviduct, rat No. 57, 3 days, 17 hours,
containing the ova shown in fig. 15. > 50. A portion of the wall of the oviduct
and a part of the major folds of the mucosa are removed in the drawing so as to
expose the contained ova. The relative position of the seven ova found in the
tube is shown, as also the extent and character of the folds of the mucosa. The
exact form of each of the several ova could not be reproduced in the model at
the magnification used; their position is given correctly.

Fig. 17 Camera lucida drawing of a portion of a section of the left oviduct
of rat No. 57, 3 days, 17 hours. X 100. This section is of the series of sections
from which the models shown in figures 15 and 16 were made. Sections of four
8-cell stages, as seen in a single section, are included. The close proximity of
three of these ova, their relation to the tubal wall and mucosal folds is to be
noted.

34 G. CARL HUBER

of the drawing, and greatly occluding the lumen, is the fold re-
moved in the model. In this very fortunate secticn four of the
morula masses are included; all are of the 8-cell stage and repre-
sent in section the four ova which are placed closely together as
seen in the model figured in figure 16. In figure 19, A, there is
reproduced at higher magnification another of the sections of
the series, including the right one of the three ova in close apposi-

Fig. 18 Model of the left oviduct of rat No. 51, 4 days. x 10. A short
segment of the upper end of the uterine horn was included in the reconstruction,
lower end of the figure. The position of three of the morula masses, 12-cell to
16-cell stages, in the terminal part of the oviduct is to be noted, a further one is
located in the upper part of the uterine horn. These are shown as if seen through
a transparent wall. <A fifth morula, situated in the uterine horn about 1.5 em.
from the entrance of the oviduct, is not included in the figure.

tion as seen in figure 17, showing six of the eight cells, each cut
in the plane of its nucleus. In both of these figures (figs. 17 and
19) the morula masses, as seen in the sections drawn, present a
quite regular oval outline. In succeeding sections, in which
the mucosal fold and the wall of the oviduct approximate, the
cross diameter of each of the four morula masses becomes greatly
reduced, they appearing in the final sections of the series in which
they are included as narrow, non-nucleated bands of protoplasm.

DEVELOPMENT OF THE ALBINO RAT 35

This series, it seems to me, corroborates the statement previously
made, that the detail of form of the living segmenting ova of
certain mammals, while in transit through the oviduct, is in a
great measure dependent on the configuration presented by the
lumen of the oviduct in the particular region in which they are
found.

12-cell to 16-cell stages. Rat No. 51, killed 4 days after the
beginning of insemination, presents the end of the segmentation
stages in the oviduct. In the genital tract of this animal there
were found eight morula masses, five on the left side and three on

c D

Fig. 19 Sections of morula stages of the albino rat. > 200. A, 8-cell stage,
rat No. 57, 3 days, 17 hours; six of the eight cells, each cut in the plane of its
nucleus, are included in the section figured. B, C, and D, 12-cell to 16-cell stages,
from right oviduct, rat No. 51, 4 days.

the right side. Itis somewhat difficult to determine definitely
the number of cells constituting each of the morula. The number
appears to vary between 12 and 18, though nearly all of the moru-
la masses show certain nuclei in mitosis. The left oviduct with
a short adjoining segment of the uterine horn was reconstructed.
Slight tension was applied to the tissue prior to fixation, which
accounts for the elongation of the proximal loop of the oviduct.
The model is reproduced in figure 18. As is evident on study
of this figure, three of the morula masses are situated in a por-
tion of the oviduct just prior to its insertion in the uterine tube.
These are closely grouped between folds of the mucosa. <A
fourth morula is found in the uppermost part of the uterine

36 G. CARL HUBER

cavity, just distal to the opening of the oviduct, lying free in a
slightly distended portion of the lumen. This morula is of
irregular discoidal form, presenting an appearance which. sug-
gests that it was fixed soon after it escaped from the oviduct. A
fifth morula, of regular oval form, comprising very probably 18
cells, all of which present resting nuclei, is lodged in a shallow
pit of the uterine mucosa a little over 1 em. from the tubal open-
ing. This portion of the uterine horn was not included in the
reconstruction, the position of this morula is not, therefore, in-
dicated in the figure. It is evident that this tube was fixed while
the several morula masses were in transit from the oviduct to
the uterine horn, which occurs, to judge from the material at
my disposal, at the end of the fourth day after the beginning
of insemination. The morula masses of the right tube are sit-
uated in the oviduct just before its point of insertion into the
uterine horn, in about the same relative position as are the
three upper morula masses of the left side, as shown in the re-
construction. They are of discoidal form, in close relation and
appear to comprise, the one 12, the other two 14 to 16 cells. In
B, C and D of figure 19 are reproduced sections of each of these
three morula stages. The figures, however, are delusive in that
the section for each passes through the greatest diameters of the
respective morula.

The material at hand permits the conclusion that in the
albino rat the segmenting ova pass from the oviduct to the
uterine horn at the end of the fourth day after the beginning
of insemination, probably in the 12-cell to 16-cell stages. With
the beginning of the fifth day, as will appear from further dis-
cussion, all of the ova are to be found in the uterine horn.

SUMMARY OF SEGMENTATION STAGES, RATE, AND VOLUME
CHANGES
The following summary of the data (table 3) gained by a study
of the models of oviduets containing ova in stages from the pro-
nuclear to 12-cell to 16-cell stages in which latter stage transit
to the uterine horn occurs, is presented to indicate rate of
transit within the oviduct. The regularity of the rate of transit

DEVELOPMENT OF THE ALBINO RAT BYE
as revealed in the summary may perhaps speak for the trust-
worthiness of the age data as concerns my material.

It will be observed that the ova approach the uterine end of
the oviduct while in the 2-cell stage; transit through the last
portion of the oviduct, where the greater part of the segmentation
occurs, being relatively slow. It is hoped that these data, for
the accuracy of which I am dependent on reconstructions, may
be of service to others who may desire to collect segmentation

stages of the albino rat.

TABLE 3
DISTANCE RELATIVE
RECORD SIDE RECON- AGE NUMBER STAGE LENGTH OF OF OVA LENGTH OF
NUMBER STRUCTED OF OVA OVIDUCT FROM FIM- TUBE
BRIA TRAVERSED
cm. ecm
106 R 1 day 8 pronuclear 3.2 0.8 0.25
59 18 2 days 4 2-cell 2 .29* 1.4 0.61
62 L 2 days, 5 2-cell 2 .45* 2.0 0.82
22 hrs.
50 R 3 days, 4 4-cell 2.8 Pole 0.90
1 hr.
51 L 4 days 5 12- to 16- 2.86 2.86 1.00
cell

*Not the entire length of oviduct was available for reconstruction.

In order to obtain the volume changes of the ova during transit
through the oviduct, beginning with the pronuclear to 8-cell
to 11-cell stages, the following procedure was adopted. As has
been shown by my figures, reconstructions were made at a mag-
nification of 1000 diameters of ova presenting the stage in ques-
tion. The sections of my series measure 10 uw in thickness.
In order, therefore, to obtain the correct third dimension, it
was necessary to use wax plates 10 mm. thick, in actual prac-
tice, five superimposed 2 mm. plates. For the majority of the
sections of my series this procedure was relatively simple. How-
ever, there was usually a question as to the thickness to be
ascribed to the first and last section of any given series, since it
was evident, both from the appearance of the section, as seen
under the magnification used, and the appearance of the model,
that the end sections did not measure 10 u» in thickness, and it

38 G. CARL HUBER

was necessary to reduce proportionately the thickness of the
wax plate representing them. Asa rule, these were made about
one-half the thickness of the other plates. The irregularities
revealed by the rough model after superimposing the respective
plates, not so marked as might be supposed considering the
thickness of the plates used, were adjusted, not by trimming
the model and cutting away wax, but by smoothing with warm
irons. The possibility of error is admitted, but since all of the
models were made in the same way, errors if committed were
probably essentially the same for all of the models. The volumes
of the models were obtained by weighing the water displaced
by each, and after making the necessary temperature corrections,
reducing weight of water displaced to volume. The average
of several determinations is given in table 4.

TABLE 4
ACTUAL VOL. AVERAGE VOL.
RECORD NUMBER AGE STAGE OF EGG MASS PER STAGE GIVEN
IN C. MM. IN €. MM.
106 1 day pronuclear 0 00015058
106 1 day pronuclear 0 00014317
106 1 day pronuclear 0 00015775
106 1 day pronuclear 0 .00017127 0 000155693
59 2 days 2-cell stage 0 .00016240
59 2 days 2-cell stage 0.00018273 0 000172565
50 3 days, lhr.| 4-cell stage 0..00018338
50 3 days, in. 4-cell stage 0 .00015520 0 000162443
57 3 days, 17 hrs.| 8-cell stage 0 .00018893
57 3 days, 17 hrs.| 8-cell stage 0 .00016040
57 3 days, 17 hrs.| 8-cell stage 0 .00018653
57 3 days, 17 hrs.| 8-cell stage 0 00018193
57 3 days, 17 hrs.| 8-cell stage 0 .00019979 0 000183516
57 3 days, 17 hrs.| 11-cell stage 0 .00021025 0 .00021025

The uniformity of the figures giving the actual volume of the
egg mass, as determined by the weight of the water displaced
by the models of the respective ova reconstructed, leads me to
feel that the errors committed in reconstruction were not serious.
The last column of the table, giving averages, is of interest
since it shows a very slight increase in the volume of the egg
mass during segmentation and transit through the oviduct.
Following the pronuclear stage, which, as has been seen, ex-

DEVELOPMENT OF THE ALBINO RAT 39

tends through a relatively long period and into the beginning of
the second day, by which time the ova have migrated about one-
fourth of the length of the oviduct, there occur only three suc-
cessive mitotic divisions, including the first segmentation divi-
sion, namely mitoses resulting in 2-cell, 4-cell and S8-cell stages
while the ova are in transit in the oviduct. In making this
statement it is assumed that in the successive segmentations, the
several cells divide synchronously, which is not in conformity
with the fact. These three mitotic divisions are spaced at in-
tervals of about 18 hours. In the next following division, the
fourth, the ovum passes from the oviduct to the uterine horn.
Since the normal gestation period of the non-lactating albino
rat is only 21 to 23 days, this slow rate of increase in volume and
multiplication of cells during the first four days of development
is of especial interest and is very probably to be accounted for
by the inadequacy of the food supply of the ovum during its
transit through the oviduct.

The presence or absence of the oolemma has not been considered
in discussing the segmentation stages of the albino rat. In my
own material, the oolemma was clearly observed in certain of
the 2-cell stages, but not in the 4-cell nor 8-cell stages. Wida-
kowich reports that he has observed in the albino rat, loss of
the oolemma even in the 2-cell stage. Since all of the material
covering these stages was fixed in Carnoy’s fluid, a fluid with a
relatively large glacial acetic acid content, it may be questioned
as to whether the fixative used may not be in part responsible
for the early disappearance of the oolemma, though neither
Hubrecht nor Sobotta considers the presence or absence of an
acid in the conserving fluid of special moment in the fixation of
the oolemma. Sobotta finds that the oolemma disappears in
the ova of mice during the 8-cell stage. The early disappear-
ance of the oolemma in the albino rat may be offered as an ex-
planation of the fact that the egg mass during segmentation and
transit through the oviduct does not, as a rule, present a spherical
form but appears compressed and molded to fit the form of the
lumen. A similar explanation is offered by Sobotta to account
for the irregularity of form assumed by the ovum of the mouse
after loss of the oolemma. In the forms in which the oolemma

AO G. CARL HUBER

persists through the later stages of segmentation, as for instance
in the rabbit, the morula mass presents a spherical form. The
transit of the ova through the oviducts is effected, very probably,
through peristaltic action of the muscular coat, since only a
relatively short portion is lined by ciliated epithelium. Whether
or not there exists a rhythmic periodicity in the peristaltic action,
it is impossible to state. The fairly regular rate of transit argues
for the presence of some regulatory mechanism. The compact
grouping often presented by a series of ova in transit through the
oviduct, especially after reaching the portion with narrower
lumen, suggests peristaltic action.

The literature dealing with the segmentation stages of the
albino rat is very meagre. Grosser figures what is presumably
an 8-cell stage. His figure 27 is referred to only incidentally
in the text, but in the accompanying legend it is stated that the
figure shows ‘‘three ova of the white rat in process of segmenta-
tion, with zona pellucida, in transit through oviduct, three and
one-half days after insemination.” If I am right in interpret-
ing these ova as in the 8-cell stage, this corresponds very closely
to my own observation on rat No. 57, 3 days, 17 hours (figs.
15-17). It is impossible to draw definite conclusions as to
the segmentation of the ova of rats from the account of Melis-
sinos. This observer while he states that his material includes
the ova of mice and rats, and while considering segmentation
mentions the ova of both forms, discusses them without dif-
ferentiating between the two. His figures all refer to ova of
the mouse. Selenka, Robinson, and Widakowich, who have
contributed to our knowledge of the embryology of the albino
rat, do not include the segmentation stages, to be found in the
oviduct, in their account.

The rate of segmentation and the time of transit through the
oviduct, as given in the literature for certain other mammals
is as follows: Sobotta has shown for the mouse that the 2-cell
stage 1s reached about 24 hours after copulation, the ovum
remaining in this stage to about the 48th hour. The 4-cell
stage was observed at about 50 hours, the 8-cell stage at 60 hours,
and the 16-cell stage at 72 hours ‘post coitum.’ The ova of the
mouse pass into the uterine horn about 80 hours post coitum,

DEVELOPMENT OF THE ALBINO RAT 41

thus the beginning of the fourth day, in a stage in which 16 cells
up to 32 cells may be enumerated; the oolemma having been
lost in the 8-cell stage. The data furnished by Melissinos as
concerns the mouse, are as follows: The 2-cell stage is obtained
at the end of 24 hours after copulation, the 6-cell stage during
the first 12 hours of the second day, and the 28-cell stage during
the second 12 hours of the second day. The ovum is said to
pass into the uterine horn at the end of the third day after cop-
ulation, retaining its oolemma. The account of Sobotta seems
the more reliable. Hensen describes a 2-cell stage in the guinea-
pig 22 to 24 hours after copulation, and Bischoff records that the
ovum of the guinea-pig passes into the uterine horn while in the
8-cell to 16-cell stage, toward the end of the third day. Heape,
who has described very fully the segmentation stages of the
mole (Talpa europea) gives no data as to the rate of segmenta-
tion. In the explanation of the figures presented it may be noted
that the ova figured, showing 2-cell to 15-cell stages, were taken
from the oviduct. His figure 20, showing an ovum ‘fully seg-
mented’ was obtained from the anterior end of the uterus. Asshe-
ton gives for the rabbit the following data: The 2-cell stage is
obtained about 24 hours and the 4-cell stage about 26 hours
after coitus. The third series of divisions begins about 28 hours
after coitus, so that by the end of the second day a typical morula
of 16 cells to 20 cells is to be found. Between 73 hours and 96
hours the beginning of the blastodermic vesicle formation is to
be noted. Ova obtained 80 hours after coitus, still surrounded
by the oolemma, were removed from the uterine horn. Data
as to the relative position of the ova in the oviduct in the several
stages of development discussed, are given. As concerns the
sheep, Assheton states that the ova pass into the uterine horn
early on the third day after mating. The pronuclear stage is
to be observed the second day, and the first segmentation at the
end of the second day. By the fourth day, with the ova in the
8-cell stage, they are found in the upper end of the uterine horn.
The blastodermiec vesicle formation begins with the 16-cell stage.
Again, according to Assheton, the ova of the pig pass to the uterus
about the third day:after fertilization, if I read him rightly,
reaching the uterus in the 4-cell stage, although ova in the 2-cell

42 G. CARL HUBER

and 3-cell stages were obtained from the upper end of the uterine
horn. The presence of 2-cell stages in the uterine horn has also
been noted by Keibel, in Erinaceus europaeus, by Van Beneden
in the bat, and by Hubrecht in the insectivor Tupaya javanica.
Finally, it may be noted that according to the observations of
Bischoff, the segmenting ovum of the dog occupies 8 to 10 days
after insemination in transit through the oviduct.

COMPLETION OF SEGMENTATION AND BLASTODERMIC
VESICLE FORMATION

The material covering the end stages of segmentation and the
early stages of blastodermic vesicle formation is listed in table 5.

TABLE 5
D NUM-
RECOR AGE BER OF STAGE
NUMBER OVA
| = + : : .
64 4days,14hrs. 5 ~ Early stage of blastodermic vesicle formation
52 4 days, 15 hrs. 8 Morula, beginning of segmentation cavity, early

stage of blastodermic vesicle
55 4 days, 16 hrs. 1 Early stage of blastodermie vesicle
68 | 4days, 16hrs. 4 Early stage of blastodermie vesicle
7 | Early stage of blastodermic vesicle
5 Early stage of blastodermic vesicle

53° | 5 days
56 5 days

Thus there are at hand 30 ova, showing late morula stages,
the beginning of segmentation cavity formation and _ early
stages of the blastodermic vesicle, falling in the latter half of
the fifth day after the beginning of insemination. Jn all of
the uteri from which this material was taken, the ova are spaced
in the uterine horns about as in later stages of development; they
lie free in the uterine lumen, are in the main ovoid in form, their
long axis presenting no definite relation to the long axis of the
uterine horn. In preparing this material for sectioning, it was
the custom to cut an entire uterine horn into segments measur-
ing about 1.0 em. to 1.5 em. in length. These segments were
then embedded so as to admit cutting longitudinally and in a
plane parallel to the plane of the mesometrium. Cut in this
way, the majority of the ova were cut longitudinally or nearly
so, others in an oblique plane, others again, crosswise. Since it

DEVELOPMENT OF THE ALBINO RAT 43

is impossible to orient the ova prior to sectioning, the securing
of desirable sections is a matter of chance. The difficulty is
further enhanced by reason of the fact that owing to shrinkage
as a result of the action of the fixing fluid, the ova in the vesicle
stage are apt to be more or less folded, so that even though the
plane of section may be that desired, the resultant sections lose
in value by reason of this folding.

It has been shown that in the albino rat, the ova pass from the
oviduct to the uterine horn toward the end of the fourth day.
During the first half of the fifth day, the migration of the ova
from the oviduct to the uterine horn appears to be completed,
so that by the second half of the fifth day the ova are spaced
in the uterine horn about as after fixation to the uterine
mucosa. As to the factor or factors which play a réle in
the descent of the ova through the uterine horn and _ their
fairly regular spacing, my own material gives no data; these
changes occurring, apparently, during the first half of the
fifth day, covering which my material is lacking. Widakowich,
who has given especial study to these questions, presents the
following considerations: In the downward migration of the ove
in the uterine horn, it cannot be assumed that the ova are capable
of active movement nor can their motion be ascribed to the action
of gravity. While peristaltic action may play a part, it is diff-
cult to see how peristalsis could be so regulated as to space the
ova fairly regularly within the uterine cavity. The presence of
a ciliated epithelium in the human uterine cavity during the
intermenstrual period suggested the presence of a ciliated epithe-
lium in the uterine horn of the rat. After many preparations
had been searched in vain for its presence, Widakowich found
short cilia, not more than 2 » long in the epithelium lining the
uterine cavity of a rat killed four days after copulation, and
containing ova in the blastodermic vesicle stage. It would ap-
pear, therefore, that the uterine epithelium of the rat presents
a ciliary border for only a relatively short time, and that the
transportation of the ova within the uterus is effected by the cilia.
Mandl also found, his material however not including the rat,
that cilia are present in many animals on the epithelium lining
the uterus only at certain periods, and perhaps only relatively

44 G. CARL HUBER

short periods. While the presence of cilia may explain the
migration of the ova in the uterine tube, Widakowich ean offer
no conclusions as concerns the regulatory mechanism by means
of which the ova are spaced at fairly regular intervals in the
lumen of the uterus. In none of my sections of the uteri of
albino rats, obtained during the fifth day after insemination,
have I been able to note the presence of cilia on the uterine
epithelium, even when sections were studied under the oil im-
mersion. After reading the account of Widakowich, their
presence was looked for in all pertinent stages, but without
success. Especially in rat No. 50, in which the ova were pass-
ing from the oviduct to the uterine horn was careful search made,
but nothing like a distinct ciliary border, composed even of
short cilia, was ascertained. In the left genital tract of this
rat, as has been stated, three ova were found in the terminal
part of the uterine end of the oviduct, one in the uterine lumen
just distal to the mouth of the oviduct. and one a little over
a centimeter from this opening. The latter was lodged in a
shallow depression of the uterine mucosa, as is characteristic
for stages lying free in the lumen. The question as to whether
this ovum was permanently lodged is difficult to answer. If
this is assumed, it is further necessary to assume that the other
ova would need to pass it to reach the more distal parts of the
uterine lumen.

The literature contains no definite statements as concerns
the reactions of the epithelium and mucosa of the uterus to the
ova soon after their appearance in the uterine cavity. _Widako-
wich summarizes the views by stating that “It is generally
stated, that so long as the ova lie free, the uterus shows no
changes.’’ He himself notes that at this time the mucosa pre-
sents evidence of marked new formation of capillaries. Burck-
hard, who had at his disposal a large number of stages showing
implantation of the ovum of the mouse, discusses at length the
appearance presented by the uterus soon after the ova enter
the same and the lodgment of the ova therein. This observer
notes that in the non-gravid uterus of the mouse, the lumen
lies more or less eccentric, and towards the mesometrial border.

DEVELOPMENT OF THE ALBINO RAT 45

The lumen is not smooth, but presents numerous radially ar-
ranged folds, certain of which are relatively deep. Essentially
the same characteristics pertain to the mucosa of the uterus,
soon after the beginning of gravidity. As the ova pass from
the oviduct to the lumen of the uterus they become lodged in
certain of the mucosal folds, and generally in certain of the
deeper ones to be found along the anti-mesometrial border.
I find Burekhard’s account of the form of the lumen of the
uterine horn, of the structure of the mucosa in early stages of
gravidity, and the lodgment of the ova, pertaining to the mouse,
applies equally well to the albino rat. No reason can as yet be
given as to why the ova are lodged in the mucosal folds in which
they are found, and not.in others. So far as may be ascertained
from the sections, the particular mucosal folds in which the ova
are found, do not differ in form and structure from neighbor-
ing folds. It is possible that by reconstruction of the epithelial
lining of the entire uterine horn in pertinent stages, certain
characteristics of form and position might be revealed as pos-
sessed by certain mucosal folds which make them especially
favorable for the lodgment of the descending ova. Such re-
constructions, however, have not been made. Burckhard states
that in the mouse, about the middle of the fifth day, after the
ova have been in the uterine cavity for a number of hours, there
may be observed the first changes in the uterine wall. The
changes consist primarily in a flattening of the uterine epithelium.
In the immediate region where implantation is to occur, the
lining epithelial cells present instead of a cylindric form, a cubic
form. The area is sharply demarked from the surrounding
epithelium, the transition of cubic to cylindric epithelium being
marked by a sharp-lipped epithelial ledge. In my own material
of the rat covering these stages, the uterine mucosa likewise
presents shallow pits, in the immediate regions where the ov:
are lodged, lined by slightly flattened, cubie epithelium, very
much as described by Burckhard for the mouse. Widakowich
presents an excellent figure (fig. 2 of his contribution, rat
four days after fertilization—‘Befruchtung’) showing clearly
the relations of the ova to the uterine wall. In this rat, the

46 G. CARL HUBER

uterine epithelium presented a ciliary border, present even in
the shallow pit lodging the ovum sketched. He argues from this
that the shallow depression and the flattening of the epithelium
are not a result of pressure exerted by the vesicle, as thought by
Sobotta and Melissinos, but must be due to an active change in
the epithelium itself. The mucosa underlying the shallow pits
presents at this stage no change of structure. J am thus in ac-
cord with Widakowich when he states that he was not able to
observe in the mucosa of the rat in the early stages of gravidity,
the giant cells described by Disse as found in the uterine mucosa
of Arvicola arvilis, in similar stages.

The form presented by the ova of the albino rat, in the late
morula stages and the early stages of blastodermic vesicle, is
ovoid, as may be seen from the figures to be presented. Wida-
kowich is inclined to believe that the form of the blastodermic
vesicle of the rat is in a measure dependent on the general form
of the space in which it is lodged. He figures two vesicles (figs.
1—2) one of which is nearly spherical, the other of distinctly oval
form. Duval (figs. 73-83) presents vesicles having ovoid, trian-
gular, and spherical forms. Christiani’s figures covering these
stages, are too schematic to be of any value in drawing con-
clusions. I fear Robinson’s account is based on imperfectly
fixed material. He states that ‘‘toward the end of the fifth day,
or the commencement of the sixth day, the longitudinal axis
of the blastodermic vesicle is 125 uw long. During the sixth
day, that axis is diminished, first to 95 u, and then to 64 u, after
which it again increases, and at the commencement of the seventh
day, it is 121 y.’’ Neither Fraser nor Selenka describes nor fig-
ures the stages here considered. In the mouse, according to
the accounts of Melissinos, Burckhard, and Sobotta, the form of
the blastodermic vesicle in early stages is spherical.

The more specific consideration of my own material I shall
introduce with a discussion of three stages taken from the uterus
of rat No. 52, killed 4 days, 15 hours after the beginning of
insemination. In A, of figure 20, there is reproduced the mid-
dle one of seven sections of a late morula stage. This morula
is of ovoid form, measuring 85 u in its long diameter, 54 » in its

DEVELOPMENT OF THE ALBINO RAT 47

broad diameter—that is, in plane of sections, and since it passes
through seven sections of 10 thickness, measures approximately
70 w in its third dimension. It consists of 24 cells, as estimated
by counting the nuclei contained in its several sections. The
cells vary in size as well as in shape. The nuclei are for the main
of spherical form, presenting one or several large nucleoli and
fine chromatin granules. This morula is found within a fold
of the mucosa, each side of which presents a slight depression,
lined by slightly flattened epithelium. This morula mass lies

Fig. 20 Sections of morula mass and early stages of blastodermie vesicle of
the albino rat. X 200. A, B, C, rat No. &2, 4 days and 15 hours. D and E, rat
No. 68. 4 days and 16 hours. A, late stage of morula; B, shows the very be-
ginning of the formation of the segmentation cavity; C, D, E, early stages of
blastodermic vesicle, in E, a distinct covering layer in the thicker portion or
floor of the vesicle is evident.

free in the lumen of the mucosal fold, and not in contact with the
uterine epithelium. The outline and extent of the shallow
depressions found in the opposing walls of the mucosal folds
conform to shape and size of the morula mass contained, which
appear as if slightly retracted as a result of fixation.

In B, figure 20, is figured one of the sections of a series passing
through a morula stage, comprising as estimated 30 cells and
measuring 90 u, by 60 vu, by approximately 50 yu, in which the
very beginning of the formation of the segmentation cavity is
shown. Near one pole the outermost cells have separated slightly
from the more deeply placed cells, so that an irregularly shaped

48 G. CARL HUBER

cavity, eecentrically placed and passing clearly through two
of aseries of five sections of 10 « thickness, is evident. The small
cleftlike cavity is bounded by the surrounding cells, the outline
of which is distinct. So far as may be judged from the appear-
ance noted as presented in the two sections in which this cavity
is found, this arose as a single space and as a result of the separa-
tion of the enclosing cells.

In C, of figure 20, there is presented a slightly older stage
showing the blastodermic vesicle formation and measuring 80 u,
by 50 uw, by approximately 50 uw, comprising as is estimated, 34
to 386 cells. Unfortunately, the lower part of this vesicle is
slightly folded as is shown in the lower left of the figure. The
appearances presented in the sections are reproduced as faith-
fully as could be. Owing to the folding, a portion of the thin
wall is cut tangentially. The more darkly colored curved line
represents in reality the outer boundary of this portion of the
vesicle. The segmentation cavity in this vesicle is distinctly
larger than that shown in B of this figure. In the section re-
produced the segmentation cavity is bounded for the greater
part by four somewhat flattened cells, the increase in the size
of the cavity being accompanied, it would seem, by a flattening
of the enclosing cells.

In these three closely approximated stages, which, since
they are taken from the same uterus are probably separated in
time of development by only short intervals, the cells though
varying in size and shape, show no essential or fundamental
difference in structure, neither in cytoplasm nor nuclei; nor do
they show any regularity in arrangement. Only few mitotic
figures are to be observed; none in the morula mass shown in A,
and but two in each of the other two stages, shown in B and C.
Judging from these preparations, one would be led to con-
clude that segmentation cavity formation in the albino rat is
not associated nor accompanied by active cell proliferation. This
point will be referred to again after the presentation of further
material at hand. In slightly older stages of the blastodermic
vesicle than here considered, the thicker portion of the vesicle
is designated by Sobotta and others as its floor, which is directed

DEVELOPMENT OF THE ALBINO RAT 49

toward the mesometrial border of the uterine horn, while its
thinner portion is designated as its roof, directed toward the
antimesometrial border. Therefore, in slightly older stages
than thus far figured, the vesicle lies with its long axis approxi-
mately at right angle to the long axis of the uterine horn. In
further description of the blastodermic vesicle, I shall use the
term ‘floor’ and ‘roof’ as here specified. Jn D and EF of figure 20,
there are reproduced typical sections of the two blastodermic
vesicles taken from the uterus of rat No. 68, killed 4 days, 16
hours after insemination. Vesicle D measures 90 » by 30 u
by approximately 60 uw, and is of distinetly ovoid form and
slightly compressed. This vesicle is found lying free in a long
but narrow fold of the mucosa, both sides of which are slightly
molded in conformity with the form of the vesicle. The long
axis of the vesicle is still parallel to the long axis of the uterine
horn. The roof of the vesicle appears as if slightly contracted,
though when traced through the series of six sections it does
not appear folded. The roof is composed of only a few cells,
perhaps seven in all. The segmentation cavity presents a regu-
lar outline. This vesicle supports the contention of Widakowich,
that the form of the blastodermic vesicle of the rat is dependent
in a measure on the form of the space in which itis found. Vesi-
cle E, of figure 20, measuring 85 » by 45 » by approximately
40 uw, presents a roof that is slightly folded and shows an early
stage in segmentation cavity formation. <A figure of the vesicle
is included since it represents more clearly than any other blas-
todermic vesicle of the albino rat in my possession, a differentia-
tion of a layer of surface cells in the mass constituting its floor.
This is a question to be more fully considered in further discussion.

In all the measurements of blastodermic vesicles thus far
given, even in those given for the morula mass shown in A, figure
20, it is evident that one of the short diameters is appreciably
shorter than the other. The vesicles are not only of ovoid form,
but slightly flattened, so that even when not folded, the form of
the vesicle as seen in section, even when cut parallel to the long
axis of the respective vesicles, is dependent in a measure on the
plane of the section, whether parallel to the longer or the shorter

50 G. CARL HUBER

of the two cross diameters. This may be seen from the series of
drawings made of a blastodermic vesicle cut cross-wise, taken
from the uterus of rat No. 68, from which were also taken the
two vesicles shown in D and E of figure 20. This series of figures
is shown in figure 21, in which are reproduced in serial order
the seven successive cross sections into which the vesicle was
cut. It measures 65 uw by 38 uw by approximately 70 u, and is
found at the bottom of a mucosal fold, found at the mesometrial
border, and is resting with one side on the epithelial lining of
a shallow pit, the other wall of this mucosal fold, also showing
a shallow pit, is slightly retracted. From a study of this series

E F G

Fig. 21 A complete series of cross-sections of an early stage of blastodermic
vesicle of the albino rat. X 200. Rat No. 68, 4 days and 16 hours. A to C,
sections through roof of vesicle, showing segmentation cavity; D to G, sections
through floor of vesicle.

of sections, I feel certain that the plane of section is cross and not
oblique to the long axis of the vesicle. The roof of this vesicle
passes through three sections, A, B and C. The segmentation
cavity has thus a depth of less than 30 uw. The overlapping of
the cells surrounding the segmentation cavity is to be noted,
especially as seen in B of this figure. This arrangement of the
cells may explain how the cavity may be enlarged without a
material increase in the number of the enclosing cells—in part,
by a flattening out of the cells, in part by a rearrangement of the
relations of the cells. In the figures of the sections passing
through the floor of this vesicle, D to G, attention is drawn to
the size, form and relations of the cells and to the fact that there
is no distinct covering layer. In this series of sections, there are

DEVELOPMENT OF THE ALBINO RAT 51

shown in all 35 nuclei, two of which are in a late diaster phase.
By excluding the nuclei that appear to be cut in two, appearing
thus in two successive sections, I estimate that the blastodermic
vesicle is made up of only about 30 to 32 cells.

In figure 22, there are reproduced typical sections of four
blastodermic vesicles taken from the uterus of rat No. 53, killed
five days after the beginning of insemination. This uterus con-
tained in all, eight vesicles, one of which was distinctly pathologie.
The four vesicles selected for reproduction and discussion pre-
sent each certain characteristics worthy of consideration. - Vesi-
cle A, which presents an early stage of segmentation cavity forma-

Fig. 22 Sections of early stages of blastodermic vesicle of the albino rat,
< 200. Rat No. 53, 5 days.

tion is of interest owing to the number of mitotic divisions it
contains. As a rule, I have noticed only a few mitoses at this
stage. In this vesicle, which measures 90 uw by 55 uw by approxi-
mately 40 u, there are no less than five mitoses to be noted, three
of which are in cells forming the roof of the small segmentation
cavity, and are included in the section figured. This is the only
vesicle in my possession in which an increase in the size of the
segmentation cavity is accompanied by active mitoses in the
cells bounding it. The vesicle lies free in the uterine lumen,
one wall of which is only slightly pitted. In B of figure 22 is
reproduced a section of a blastodermic vesicle, measuring 90 4
by 55 » by approximately 45 yu. It is evident that this vesicle

je G. CARL HUBER

passes through five sections of 10 » thickness, though one of the
end sections, the fifth section of the series, seems to have fallen
out during manipulations of staining and mounting, since the
preceding, or fourth section does not quite complete the series.
This vesicle hes free in the lumen of the uterus, and there is evi-
dent only a shallow pit in the mucosa juxtaposed. In this
vesicle the cells forming the roof of the segmentation cavity are
relatively numerous, and are not markedly flattened, and in one
an early mitotic phase is recognized. Here again cell prolifer-
ation appears to have accompanied increase in size of segmenta-
tion cavity.

The vesicle shown in C of figure 22, measuring 130 » by 30 u
by approximately 40 u, lies free in a long, narrow fold of the
uterine mucosa, in close proximity to a shallow mucosal pit,
lined by cubie epithelium; the pit conforming in shape and
extent to the form of the side of the vesicle presented to it.
Therefore, it would seem that the form of the vesicle as seen in
sections of the fixed material is essentially the same as that
obtained in vivo. The two vesicles, typical sections of which
are shown in B and C of this figure, are almost in identically the
same phase of development, although their form as seen in sec-
tions differs markedly. The plasticity of the living blastodermic
vesicles is no doubt such that their form is in a great measure
dependent on the shape of the mucosal fold in which they are
lodged. In D of figure 22, there is reproduced a section of a
blastodermic vesicle which points to a stage of development
which is slightly more advanced than that shown in previous
figures. The vesicle measures 100 « by 70 u by approximately
50 uw. The roof enclosing the segmentation cavity is slightly
folded; a portion of its wall is thus cut tangentially, as shown
in the lower left of the figure. The segmentation cavity is
distinctly larger than that shown in the preceding figures, and
is bounded by a relatively large number of cells, fourteen in that
portion of the roof sketched in this figure, one of which is in a
mitotic phase. The mass of cells constituting the floor appears
as slightly compressed, in consequence of a slight intravesicular
pressure which aided in the enlarging of the segmentation cavity.

DEVELOPMENT OF THE ALBINO RAT 53

The cells forming the roof of the segmentation cavity do not
appear so distinctly flattened as is the case in certain of the
vesicles figured in figures 20 and 21. It would appear, there-
fore, that at least two factors are operative in the increase of
size of the segmentation cavity after its anlage—a flattening out
and consequent increase of the exposed surfaces of the enclosing
cells, and secondly, a cell proliferation; and it would appear that
both of these factors may be operative from the time of the
beginning of segmentation cavity formation.

Early stages in the blastodermic vesicle formation in the
albino rat have been previously described by Robinson, Christi-
ani, Duval, and Widakowich; Selenka’s youngest stage is slightly
older than any discussed by me. My own observations are
wholly in accord with those of Widakowich in so far as his account
covers early stages of blastodermic vesicle formation. He dis-
cusses and figures, however, only two vesicles, obtained four
days after fertilization—‘Befruchtung,’ in each of which the
segmentation cavity presents a smooth and regular outline and
is of appreciable size. The observations of the other observers
who have considered these stages will be discussed in connection
with a very brief presentation of much more comprehensive
observations on the mouse in similar stages of development. Of
these latter, those of Sobotta (’03) are based on abundant and
apparently well fixed material. Sobotta begins his discussion
with the consideration of three ova taken from the same mouse,
the second half of the fourth day after fertilization, each of which
shows beginning of segmentation cavity formation, one of which
was cut in longitudinal axis and is figured in his figure 1. This
ovum is interpreted as showing that the segmentation cavity
arises not as a single space, but as a number of disconnected
spaces, which later become confluent and form a single space.
A similar observation was made by Van Beneden on the bat,
a fact which Sobotta uses to support his contention that the
mouse ova studied by him were of normal structure. Melis-
sinos gives a number of figures showing early stages in the forma-
tion of the segmentation cavity in the mouse. His figures 21
and 22 (66 hours) are not unlike my own figures shown in B of

54 G. CARL HUBER

figure 20 and A of figure 22. According to this observer, the
segmentation cavity arises as a single space, due to an accumula-
tion of fluid secreted by the cells of the morula. This secretion
is evidenced by globule-like droplets which are shown in his
drawings as adhering to certain of the cells bounding the seg-
mentation cavity. In my own preparations of the albino rat,
I find no evidence which would lead to the supposition that the
segmentation cavity does not begin as a single space nor do I
find any evidence of secretory globules as described by Melis-
sinos. Selenka has described quite fully two blastodermic
vesicles of the mouse, lying free in the uterine lumen. His
account of their structure, supported by two somewhat dia-
grammatic figures, is as follows: The wall of the vesicle is formed
by a layer of covering cells—‘ Deckzellen’—constituting a cover-
ing layer—‘‘Deckschicht or Rauber’s layer.’’ The space en-
closed by this layer, for about one-third to one-half of its extent,
contains the ‘formative cells,’ for the remainder it contains
fluid. The covering cells and formative cells are said to be sep-
arated by a sharp line. The formative cells are in all parts
separable into two fundamental germ layers. An inner layer,
bordering the cavity and constituting the entoderm, is said to
be composed of cells possessing more deeply staining nuclei,
irregular outline, with tongue-like processes which extend into
the cavity, and a granular protoplasm; further, of cells which
are more clear, more peripherally placed, and which constitute
the ectoderm. Each of these fundamental germ layers con-
sists of a single layer of irregularly formed cells. According to
the observations of Selenka, therefore, the floor of the vesicle
consists of three layers of cells; an outer covering layer—‘Deck-
schicht’ or ‘Rauber’s layer’—an inner layer of entodermal cells,
and an intermediate layer of ectodermal cells. Jenkinson’s
account reads as follows: ‘‘There are present (1) an outer layer,
one cell deep, of trophoblast, which is continuous over (2) an
inner mass which becomes differentiated into the embryonic
epiblast and the hypoblast, and which is quite distinct from
the overlying trophoblast, as my specimens invariably show.”
In Jenkinson’s figures 1 and 2, giving early stages of the blasto-
dermic vesicle, there is not shown a differentiation of the inner

jt
t

-
-.
~

DEVELOPMENT OF THE ALBINO RAT

mass into ectodermal and entodermal cells; the outer layer,
covering layer, Rauber’s cells, or trophoblast, is clearly differ-
entiated from the inner mass by a distinct space. Duval has
recognized in early stages of blastodermic vesicle formation of
the mouse and rat, in the thicker part of the vesicle, entodermal
and ectodermal cells. The former are of irregular form, pos-
sess granular protoplasm and are said to possess the property
of ameboid movement. The remaining cells are recognized
as ectoderm; a distinct covering layer is not recognized. In
Christiani’s figures (rat), which are, however, so diagrammatic
as to be of little value and are evidently drawn from poorly
fixed material, entodermal cells, ectodermal cells, and covering
cells may be recognized as per legends. Melissinos (mouse),
while not describing definitely a peripheral or covering layer,
states that outer cells of the thicker pole, like the cells enclosing
the segmentation cavity, stain less deeply than do the more
centrally placed cells. In earlier stages of vesicle formation,
neither in figures nor in text as given by this observer, do I find
reference to a differentiation into ectodermal and entodermal
cells. The observations of Selenka, Duval, Robinson, Jenkin-
son, and others, bearing on the structure of the blastodermic
vesicle of the mouse and the rat in early stages of development
have been so thoroughly and critically reviewed by Sobotta
that an extended discussion has here been deemed unnecessary.
It may here suffice to say that while Sobotta’s observations were
made and his discussions based on ova obtained from the mouse,
my own observations made on the albino rat are in agreement
with his and support the contention that in early stages of blas-
todermic vesicle formation a differentiation of the thicker part
or the floor of the vesicle into a covering, Rauber’s cell, or tropho-
blast layer, and a further differentiation into ectodermal and
entodermal cells, is not to be made: we differ in our accounts of
the beginning of the formation of the segmentation cavity.
An outer or covering layer is suggested in certain of my own prep-
arations, most clearly in that sketched in E of figure 20. How-
ever, a uniform difference in structure and reaction to staining
reagents of the outer layer of cells is not present in my own
preparations. None of my own preparations gives evidence of

56 G. CARL HUBER

such an early differentiation of ectoderm and entoderm as given
by Selenka, Duval, Christiani, and others. Cells of irregular
outline with tongue-like projections, such as figured by Selenka
and Duval I have not observed. The cells constituting the
floor or the thick part of the vesicle all present essentially the
same structure, while the segmentation cavity, as soon as it
presents appreciable size, shows a smooth and regular outline.
In figures 1 and 2, of Widakowich’s communication, excellent
figures of early stages of blastodermie vesicles of the albino rat,
there is presented no evidence of a trophoblast layer nor a dif-
ferentiation of ectodermal and entodermal cells. My own
figures, 20 to 22, were drawn with the aid of camera lucida at a
magnification of 1000 diameters and with the use of an intense
Welsbach light. They are reduced five times in reproduction.
With the exceptions of cell outlines, which as sketched do not
in the preparations fall in the same optical plane, andaresketched
more sharply than is perhaps warranted, the figures portray
quite accurately the structural appearances presented, so far
as may be with the use of a single color.

BLASTODERMIC VESICLE, BLASTOCYST, OR GERMINAL VESICLE

The material on hand is listed in table 6.

TABLE 6
RECORD NUMBER AGE NUMBER OF VESICLES
75 5 days, 15 hours 6
91 5 days, 16 hours 2
88 5 days, 21 hours 7
89 5 days, 21 hours 6
73 6 days 10
74 6 days 5
99 6 days 6
106 6 days 10
104 6 days 6 Total 58

During the sixth day, the blastodermie vesicle of the albino rat
increases in size relatively rapidly. The greater portion of its wall
is, at this stage, composed of a single layer of flattened cells. The
vesicles are not as yet attached to the uterine wall, though the uter-

DEVELOPMENT OF THE ALBINO RAT 57

ine mucosa shows a distinet reaction to their presence. Localized
thickenings of the uterine mucosa, sufficient to cause localized
swellings of the uterine tube, indicating the position of the ova,
are evident. I have experienced more difficulty in successfully
fixing the vesicles during this stage than any of the earlier or
later stages studied. Although my material contains 58 vesicles
of the stage under consideration, none of them may be regarded
as being well fixed, and the majority of them are so folded as a
result of contraction during fixation that they are of little value
as objects for especial study. That the vesicles are still un-
attached to the uterine wall is readily determined by the fact
that the shrivelled vesicles are found lying free in the depres-
sions of the uterine mucosa, lined by a low cubic epithelium,
intact throughout, and retaining its normal relation to the
mucosa. The molding in these mucosal depressions no doubt
gives the size of the respective vesicles as in vivo.

It is not my purpose at this time to consider more than super-
ficially the changes affecting the uterine mucosa during ovum
implantation in the albino rat. It is hoped that this may be
the subject of a future communication. It is the purpose in the
present communication to confine consideration to the develop-
ment of the ovum itself. Many of the observations recorded
by Burekhard on the implantation of the ovum of the mouse
and the formation of the decidua, I find equally adapted to
similar phenomena in the albino rat. Differences are to be
observed in certain details which it is not the purpose to enter
into here. Grosser gives a number of excellent figures (67 to
70, and 112 to 116) showing implantation and decidua formation
in the albino rat; to these the interested reader is referred for
the present. The thickening of the mucosa affects primarily
its antimesometrial portion. During this process of thickening,
the mucosal fold in which the ovum primarily finds lodgment,
becomes deepened and converted into a funnel shaped crypt
communicating with the uterine lumen, and surrounded by the
‘Hibuckel,’ or oval fold. Burekhard’s schematic figures (text
figures 2 to 4) may be consulted to make the phenomenon

_

intelligible.

58 G. CARL HUBER

In figure 23, there are reproduced representative sections of
five blastodermic vesicles falling to the end of the sixth day after
insemination. None of these five vesicles can be regarded as
well fixed. All show a certain amount of distortion, much more
evident were the entire series of each of the respective vesicles
shown. The form of the blastodermie vesicle of the albino
rat at this stage of development, as indicated by the molding
of the uterine mucosa, is ellipsoid. Their size as in vivo, when
distended and of regular outline, again as indicated by the
molding of the uterine mucosa, is slightly larger than would be

Fig. 23 Sections of blastodermic vesicles or blastocysts of the albino rat.
xX 200. A and C, rat No. 99, 6 days; B, D, E, rat No. 100, 6 days. y.ent., yolk
entoderm; p.ent., parietal layer of entoderm; p.ect., parietal or transitory
ectoderm.

supposed from the drawings presented. By reason of this dis-
tortion, exact measurements of size cannot be given.

In A of figure 23, there is reproduced that portion of one of the
sections of a blastocyst (rat No. 99, 6 days) which passes through
its floor; the thin roof of this vesicle was so folded that its inclu-
sion in the drawing was deemed undesirable. However, its
floor or the germinal disc, seems to have retained its normal
form and structure, presenting when traced through the series
a regular concavo-convex, discoidal form. It consists in the
main of three layers of cells of polyhedral type; toward the
border of the disc, of two layers of somewhat flattened cells, the
peripheral layer being continuous with the single layer of cells

DEVELOPMENT OF THE ALBINO RAT 59

forming the roof of the vesicle, not shown in the figure, and
known as the parietal or transitory ectoderm. In the floor or
germ disc, there is evident a single layer of cells bordering the
segmentation cavity or blastocele and possessing a more gran-
ular protoplasm, which stains a little more intensely in Congo
red. Their differentiation and characteristic reaction to stain-
ing agents is at this stage of development not quite so distinct
as in slightly older stages. This layer of cells, similar to that
described by Sobotta for the blastodermic vesicle of the mouse
in essentially the same stage of development, he has termed the
yolk entoderm, ‘Dotter entoderm,’ a designation which is here
followed. In the more superficial layer or layers of cells no
characteristic differentiation is observed. In no portion of the
floor of this vesicle was a distinct covering or trophoblast layer
recognized.

In the vesicle, a section of which is reproduced in B of this
figure (rat No. 100, 6 days), the floor or germ dise presents
essentially the same structure as that shown in A. The vesicle
shown under B, was also folded, especially its roof, which was
drawn to one side and was thus not cut through its entire length
in the section figured. Furthermore, the section chosen for
drawing does not pass quite through the center of the germ disc,
but a little nearer to one of its edges, which probably accounts
for the fact that there is recognized for the greater part only a
single layer of cells, superimposed over the yolk endoderm,
which layer is continuous with the parietal or transitory ecto-
derm forming the roof of the vesicle. The cells forming the
yolk entoderm constitute a single layer and are quite distinctly
differentiated; one of the cells shows a mitotic phase. The roof
of the vesicle formed by the parietal or transitory ectoderm, is
composed of a single layer of flattened cells with flattened nuclei,
the form and structure of which is more correctly shown in the .
right half of the roof wall, which in the section is cut transversely,
while the left half, owing to the folding, is shown as cut obliquely.

In C of figure 23 (rat No. 99, 6 days), there is shown a greatly
compressed blastodermic vesicle, taken from a series of cross
sections of the uterine horn. In this figure there is reproduced
the fifth of a series of 10 sections of 10 « thickness; therefore,

60 G. CARL HUBER

the third dimension of the vesicle is approximately 100 ». It
is evident that had this vesicle been cut in a favorable plane at
right angles to the present series, or parallel to the mesometrial
plane, its form would have approached that of a circle. I have in
my possession one vesicle of this stage of devlopment, similarly
compressed, cut parallel to the plane of compression, in which
almost the entire roof falls within a single section of 10 u thick-
ness. The structure of the vesicle shown in C is very similar
to that shown in A and B of this figure. The normal form of this
vesicle is quite readily reconstructed from a study of the series
of sections into which it has been cut. The cells of the volk
entoderm are evident. The parietal or transitory ectoderm
constituting the roof consists of a single layer of much flattened
cells, with relatively few nuclei, having, as seen in cross section,
a long ovoid form, which, when seen in surface view present a
regular, nearly circular outline (see lowermost nucleus in the
figure). In similarly compressed vesicles cut parallel to the
plane of compression, the germ disc may appear as consisting
of three to four layers of cells. In an imaginary section passing
in a plane at right angles to that figured in C, and having perhaps
a slight obliquity, the germ dise would appear as if much thicker
than that shown in A and B of this figure. Such sections may
readily lead to false conclusions.

It seems evident from a study of the material at my disposal
that during the sixth day after the beginning of insemination
in the albino rat, the blastodermic vesicle or blastocyst, which
has its anlage in the latter part of the fifth day, enlarges relatively
rapidly; this largely owing to a distension of the segmentation
cavity or blastocele. This enlargement is accompanied by a
flattening and extension of the enclosing roof cells and by a re-
arrangement of the cells of the floor, which is reduced in thick-
ness to a discoidal area, the germinal dise or germ area, forming
about one-fifth to one-sixth of the wall of the vesicle and con-
sisting of two or three layers of cells. During the rearrangement
of the cells which constitute the floor of the vesicle, those adjacent
to the segmentation cavity or blastocele differentiate to form the
anlage of the yolk entoderm. The remaining cells of the ger-

DEVELOPMENT OF THE ALBINO RAT 61

minal disc, having all essentially the same structure, are of
irregular polyhedral form and are mutually compressed. To
designate them as a distinct germ layer at this stage seems
inappropriate. <A differentiation into a layer of covering cells
and a layer of formative ectoderm (Selenka) is not to be made.
Active cell proliferation as evidenced by mitotic figures does not
appear to accompany this enlargement of the vesicle. This
phenomenon seems rather to be accomplished by a rearrange-
ment of the cells constituting its floor, however, primarily by
an extension and consequent flattening of the cells forming
the roof of the vesicle. A similar stage is shown for the mouse
by Sobotta (’03) in his figures 3, 4, and perhaps 5, of mouse vesi-
cles from the fifth day after fertilization—Befruchtung’. So-
botta had at his disposal much more perfectly fixed vesicles than
my material contains. The structure of these vesicles as given
by this observer, both as depicted in figures and text, is very
similar to the presentation given by me. He also recognizes
in this stage the anlage of the yolk entoderm. Figure 30, ac-
companying the account of Melissinos (mouse, 84 hours) presents
a similar stage, although he figures fairly distinctly a layer of
covering cells, which if I read him correctly, however, is of only
transitory existence. None of the figures given by Robinson
and Jenkinson is comparable with figures A, B, C, of figure 23
of this account.

In D, of figure 23 (rat No. 100, 6 days) there is reproduced a
section of a blastodermic vesicle which on superficial study
presents a somewhat later stage of development than those
shown in A to C, of this figure. It is, however, only very slightly
older than the three vesicles discussed. Vesicle D, cut in good
longitudinal direction, is in reality much more folded than ap-
pears from the section figured. Its floor or germ disc is com-
pressed in a plane parallel to that of the plane of section, so that
the germinal dise is cut obliquely and not transversely, and
thus appears thicker in the section than it in reality is. A dis-
tinct layer of covering cells, continuous with the cells of the
parietal ectoderm, is evident. Such a layer of covering cells is
figured by Selenka, Jenkinson, and Duval. The yolk entoderm
has differentiated and extends by perhaps three cells, in the

62 G. CARL HUBER

section figured, onto the layer of parietal ectoderm. Selenka
and Duval, who regard the cells of the primary entoderm as
having ameboid properties, are disposed to regard the entodermal
cells found lining the parietal ectoderm as having wandered
from their seat of origin to the side wall of the vesicle. Sobotta
sees no evidence of such wandering of the primary or yolk ento-
dermal cells, but suggests that they are drawn to their position
on the wall of the vesicle during its increase in size; their wander-
ing, therefore, is more relative than absolute. Certain cells
nearer the edge of the yolk entoderm, having attachment to the
parietal ectoderm, which attachment they retain as the vesicle
enlarges, are thought to be drawn from their close relation to
the yolk entoderm and to appear as scattered cells lining the
parietal ectoderm. Now and then, such cells may divide, re-
sulting in further distribution. Sobotta’s suggestion seems to
me to be more in accord with the observed facts. In vesicle
D, the roof, consisting of a single layer of flattened, parietal
ectodermal cells, presents several major folds as well as minor
folds. The latter particularly account for the variation in
thickness of the wall of the vesicle as seen in sections. At the
lower left of the figure is seen a portion of the wall as seen cut
on the flat, the shape of the two nuclei here shown as seen in
surface view may be compared with the long ovoid form of
similar nuclei when seen in cross section.

Vesicle E of figure 23 (rat No. 100, 6 days) presents a stage
that is slightly older than the other four vesicles shown in this
figure. The floor of this vesicle, the germinal disc, as seen in
cross section, presents the form of a triangle with its base rest-
ing on the cavity, the blastocele. When compared with the
shghtly vounger stages this portion of the vesicle presents an
increase in the number of constituent cells, arranged in irregular
layers to the number of five in its thickest portion. The thicken-
ing is no doubt in part due to the slight lateral compression of the
vesicle, but this does not wholly account for it. The cells con-
stituting this thickened germinal disc are for the main of irregular
polyhedral form with relatively large nuclei rich in chromatin.
A distinct covering layer is not evident. On its under sur-
face there is found a single layer of cells of yolk entoderm. The

DEVELOPMENT OF THE ALBINO RAT 63

thin-walled roof of this vesicle, the parietal or transitory ecto-
derm, deserves no special consideration, except to state that its
variation in thickness, as seen in the section figured, is due to
the plane of section—cross or oblique—of different portions of
the wall, owing to slight folding. This vesicle I believe to be
in stage of development and structure very similar to that
shown by Sobotta (’03) in his figure 6, mouse vesicle of the first
half of the sixth day, and perhaps also figure 31, of the account
of Melissinos, mouse vesicle, end of fourth day, also figure 7
of Jenkinson’s article who, however, describes and figures a
distinct covering or trophoblast layer.

The cell rearrangement and proliferation resulting in the
thickening of the floor or the germinal dise as noted in E, of
figure 23, marks the beginning of a much more distinct thick-
ening of this portion of the vesicle, partly due to cell proliferation,
in part also due to the rearrangement and enlargement of the
constituent cells, during which thickening process this portion
of the vesicle grows outward as well as into the cavity of the
vesicle, initiating the phenomenon known as the ‘inversion of
the germ layers’ or as ‘entypy’ of the germ layers, to be discussed
as to its anlage in the following section.

LATE STAGES OF BLASTODERMIC VESICLE, BEGINNING OF
ENTYPY OF GERM LAYERS

The material at hand is listed in table 7.

TABLE 7
RECORD NUMBER AGE NUMBER OF VESICLES
46 6 days, 14 hours 10
54 6 days, 16 hours 9
67 6 days, 16 hours if
24 6 days, 17 hours 3
90 6 days, 17 hours 6
72 7 days 9
80 7 days 9
92 7 days 6 Total 59

The fixation of the blastocysts of the albino rat obtained
during the seventh day after insemination was much more

64 G. CARL HUBER

readily accomplished than in those obtained during the preceding
day. Of the 59 vesicles of this stage obtained, many show ex-
cellent fixation. The thin wall of the vesicle is no longer so prone
to fold as in the preceding stage, and does not readily retract
from the uterine epithelium or mucosa, no doubt owing to a
distinct adhesion of vesicle wall to the maternal tissue. It is
difficult, however, so to orient the vesicles as to obtain sections
of a desired plane. The general position of a given vesicle is
readily determined, since the enlargement of the uterus marking
its location is very evident. The vesicles are located in approx-
imately cylindrical cavities, known as decidual erypts, which
are directed toward the antimesometrial border.

These decidual crypts communicate with the lumen of the
uterus, which lies eccentric and nearer the mesometrial border,
by means of funnel-shaped openings. The decidual crypts or
cavities are still lined with uterine epithelium, though this is
now much flattened in the immediate vicinity of the vesicle
and may be found in part separated from the mucosa of this
region. The vesicles are now so placed that in all of them, the
thicker portion, the floor of the blastodermic vesicles of younger
stages or region of the germinal disc, is directed toward the
mesometrial border, thus toward the still patent lumen of the
uterus, while the roof of the vesicles is directed toward the an-
timesometrial border, thus toward the bottoms of the decidual
crypts. The general direction of the decidual crypts is in the
main at right angle to the long axis of the uterine horn, and
directed from the mesometrial to the antimesometrial border.
They may deviate, however, from the general direction at
various angles and in almost any direction. The decidual crypts
as seen in cross section do not as a rule present a circular outline,
but appear as slightly compressed from side to side, having thus
an oval outline as seen in cross section, with the long axis of this
oval space as seen in cross section approximately parallel to the
long axis of the uterine horn. Since the direction of the decidual
crypts can in uncut material be only approximated, the obtaining
of sections cut in a desired plane becomes largely a matter of
chance. In a large number of my preparations the contained

DEVELOPMENT OF THE ALBINO RAT 65

vesicles are cut in an oblique plane, which may deviate only
a little from the longitudinal or may approach a cross axis,
while only a relatively small number of vesicles were cut favor-
ably in the longitudinal plane, and the majority of these are in
series cut parallel to the plane of the mesometrium. The vesi-
cles on which the special consideration of this stage is based are
reproduced as seen in sections, in figure 24.

Vesicle A, of figure 24 (rat No. 46, 6 days, 14 hours), is drawn
from two successive sections. The upper portion of the figure

Fig. 24 Sections of blastodermic vesicles or blastocysts of the albino rat
showing the early stages of entypy of the germ disc. > 200. A and B, rat No.
46, 6 days, 14 hours; C, rat No. 54, 6 days, 16 hours; ect.pl., ectoplacental cone or
Trager; ect.n., ectodermal node; p.ect., parietal or transitory ectoderm; v. ent.,
visceral layer of entoderm; p.ent., parietal entoderm.

was drawn under camera lucida from one section, then by super-
imposing certain of the cells so as to give proper orientation, the
lower half of the figure was added from the succeeding section.
The slightly oblique plane in which this vesicle was cut made this
procedure desirable. This relatively small vesicle seems in
excellent state of fixation, as is evident from the symmetrical
outline shown by the successive sections of the series. When
compared with vesicle E of figure 23, though the two are sepa-
rated in time of development by only afew hours, it is evident

66 G. CARL HUBER

that a distinct advance in development has taken place. The
so-called floor of vesicle A, the region of the germinal dise of
former stages, directed toward the mesometrial border, is mark-
edly thickened, resulting in an outgrowth toward the mesometrial
border and an ingrowth into the cavity of the vesicle. The out-
growth forms the anlage of the ‘Triiger’ (Selenka) or the ‘ecto-
placental cone’ (Duval), and appears to have developed largely
as a result of an increase in size of the more superficially placed
cells, since cell proliferation is not marked in this region. It is
admitted that the critical stages are here lacking in my material.
These stages appear to fall to the early hours of the seventh
day, the material for which is lacking.

As may be seen from the figure, the cells constituting the an-
lage of the ectoplacental cone are of relatively large size with
large vesicular nuclei, and are continuous at the base with the
parietal ectodermal cells which form the roof of the vesicle or
its antimesometrial portion. In the cell mass which extends
into the cavity of the blastodermie vesicle or blastocyst in which
there is recognized the anlage of the ‘egg-plug’—‘Eizapfen,’ or
‘egg cylinder’—‘Eicylinder’ (Sobotta) there is evident a fairly
clearly circumscribed compact mass of cells, which stain some-
what more deeply than the surrounding cells and which may be
designated as the ectodermal node. It represents the anlage
of the true ectoderm of the embryo, as may here be stated in
anticipation of further description. In all of the vesicles of this
stage of development, even when cut obliquely or in cross section,
this small nodule of compactly arranged cells is evident. It is
circumscribed both from the cells of the ectoplacental cone as
also from the cells lining the blastocele. The metamorphosis
leading to the formation of the ectodermal node will receive con-
sideration in a brief general discussion of this stage. The cells
covering the egg-plug, and surrounding the ectodermal node,
so far as it extends into the blastocele, are arranged in a single
layer, forming a dome-shaped membrane, which appears as forced
into the cavity of the vesicle consequent on development of the
ectodermal node. This layer of cells constitutes the yolk ento-
derm, the anlage and differentiation of which has been previously

DEVELOPMENT OF THE ALBINO RAT 67

considered. The antimesometrial portion of this vesicle, its roof,
consists of a single layer of somewhat flattened cells, the parietal
or transitory ectoderm. The parietal ectoderm presents on its
inner surface a few—four in the section figured—entodermal
cells of irregular outline. These may be designated, after So-
botta, as cells of the parietal entoderm.

Vesicle B, of figure 24, taken from the same rat as was vesicle
A (rat No. 46, 6 days, 14 hours) presents a very favorably cut
vesicle, which, however, is slightly compressed from side to side,
so that its form appears more nearly circular in the sections cut
in the plane of the figure, than were they cut at right angles to
this plane. This is especially true of the ectoplacental cone,
which for the greater part appears in only two sections of 10 u
thickness, while in the plane of the figure it measures nearly 90 wu.
Cognizance of this is to be taken in considering the relative
size of the ectoplacental cone as shown in this figure. This
vesicle is only very slightly older than that shown in A of this
figure. Its ectoplacental cone is made up of a core of relatively
large cells, bordered by more flattened cells, which in this prepara-
tion stain somewhat more deeply than do the more centrally
placed cells. These covering cells are continuous with the cells
of the parietal ectoderm. The cell mass projecting into the
blastocele is more definitely circumscribed than in the slightly
younger stage shown in A of this figure. The ectodermal node
appears as an oval mass composed of compactly arranged cells,
and is separable on all sides from the surrounding cells. The
yolk entoderm, which may now be known as the visceral layer
of the entoderm (Sobotta) passes as a single layer of cells of quite
regularly cubic or short columnar form, nearly about the ecto-
dermal node to reach the base of the ectoplacental cone, extend-
ing over on the parietal ectoderm at one side (see right side of
figure). A few of the cells of the parietal entoderm, three in
the figure, are evident. The parietal ectoderm forming the roof
or antimesometrial portion of this vesicle consists of a single layer
of flattened cells, which rest on, and are adherent to the decidual
tissue; the uterine epithelium lining the decidual erypt in which
the vesicle is lodged having in part disappeared in the immediate
region of the vesicle.

68 G. CARL HUBER

Vesicle C of figure 24 (rat No. 54, 6 days, 16 hours) presents a
stage which is almost identical in development with that shown
in B of this figure, though in shape these two vesicles, as seen in
sections, appear quite different. The vesicle shown in C is less
compressed than the one shown in B, and probably presents more
correctly the form of the blastodermic vesicle or blastocyst of
the albino rat at this stage of development. The ectoplacental
cone presents a cylindrical outline and contains two cells showing
mitotic phases, both included in the section figured. Its cells,
more particularly the ones bordering the periphery, present a
vacuolated protoplasm, the vacuoles containing lightly colored
globules which from reaction to the stain are to be regarded as
blood cells or fragments of such, which blood cells are regarded
as of maternal origin. In this preparation, the decidual crypt
contains a small amount of extravasated maternal blood, found
in part surrounding the ectoplacental cone; also in the antimes-
ometrial portion of the erypt in relation with the roof of this
vesicle. ‘These findings will receive further consideration in the
succeeding pages. The cell mass projecting into the cavity of
the vesicle, consisting of the ectodermal node and the layer of
visceral entoderm is slightly larger than in the preceding stage
but presents no special features deserving discussion. The vesi-
cle in the section sketched presents very few cells of the parietal
entoderm. The parietal ectoderm forming the roof of this
vesicle consists of a single layer of flattened cells in the proto-
plasm of certain of which vacuolization is evident. Certain of
the cells show inclusions of lightly staining globules of a color
similar to those found in the cells of the ectoplacenta, particularly
evident in the lower right of the figure in which they are repre-
sented as uncolored circumscribed areas. The color reaction
of these globules is like that of the maternal blood cells and frag-
ments of blood cells found in the decidual erypt in the immediate
vicinity of the vesicle, and they are regarded as blood cells or
fragments of such, taken up by the cells of the parietal ectoderm
at this stage in the development of the vesicle.

The blastodermic vesicles or blastocysts figured in figure 24,
represent an important stage in the development of the albino

DEVELOPMENT OF THE ALBINO RAT 69

rat, as also in a number of other rodents, in that they show the
anlage of the phenomenon known as the inversion of the germ lay-
ers or entypy of the germ layers. ‘‘Inversion of the germ layers

Blatterumkehrung’—in the ova of rodents was probably first
recognized by Reichert in the guinea-pig, mouse, and rat, though
it was much more fully and correctly described by Bischoff
as observed in the guinea-pig and a little later by Hensen, also
in the guinea-pig. Further observations on this phenomenon
were recorded by Kupffer in his study of the development of the
field mouse, Arvicola arvalis, and by Fraser on the gray and
white rat and the mouse. Selenka gave this question special
study, and in a number of monographic communications deals
with the phenomenon of Blatterumkehrung as observed in three
varieties of the mouse, the white rat, and the guinea-pig. Selen-
ka’s observations have formed the basis for future work on this
problem. They have been widely accepted and extensively
quoted. It was he who introduced the term ‘Triiger’ to denote
the cell mass which results from proliferation of the covering
cells. His own words concerning this point read as follows:

Wahrend bei dem Kaninchenei, nach erfolgter Sonderung der forma-
tiven Furchungszellen in fiusseres Ektoderm und inneres Entoderm,
die gesammste Lage der fusseren Deckzellen zu einer diinnen resisten-
ten Membran zusammenschrumpft, verdickt sich bei den Nagern
mit invertirten Keimblittern der mit den formativen Zellen in Con-
tact befindliche Abschnitt der Deckschicht unter lebhafter Zellver-
mehrung zu einem sphirischen oder konischen Gebilde, welches ich als
‘Triger’ bezeichne; * * * * Die Einwucherung dieses Triigers
ins Innere der Keimblase hat zur Folge, dass die scheibenférmigen
Grundblitter (Ektoderm und Entoderm) sich nicht wie beim Kanin-
chen zu zwei concentrischer Hohlkugeln erweitern, sondern, ehe sie
noch zu dieser Gestalt gelangten, ins Centrum der Keimblase vor-
geschofen, vorgestiilpt und damit invertirt werden.

In a later publication, this observer also suggested the name
‘Entypie des Keimfeldes’ as a more comprehensive term than
‘Blitterumkehrung’ under which may be included types with
inversion of the germ field without actual inversion of the germ
layers. In later years Duval, Christiani, Robinson, Jenkinson,
Sobotta, Kolster, D’Erchia, Spee, Burckhard, Melissinos, Wida-
kowich, Lee and others have studied the earlier developmental

70 G. CARL HUBER

stages of rodents presenting the so-called inversions of the germ
layers. O. Hertwig in his chapter ‘‘Die Lehre der Keimblitter”’
gives a brief résumé of our knowledge of the inversion of the
germ layers as observed in certain rodents, noting that three main
modifications are to be observed. The first and simplest, as found
in the field mouse; the second or intermediate as found in the
rat and mouse; the third and most complex as observed in the
guinea-pig. Hertwig’s account is based largely on the observa-
tions of Selenka, the accuracy of which is now questioned from
many sides.

My own conclusions concerning the early stages of the entypy
of the germ layers in the albino rat are made on stages which
do not portray the very beginning of this process. The vesicles
shown in figure 24, in which this process is well initiated, however,
present appearances, on the basis of which certain conclusions
may be drawn. It is the contention of Selenka that the Trager
or ectoplacental cone is developed as a result of proliferation of
covering or Rauber’s cells, superimposed on the formative cells
of the germ disc. He is followed in this view by Jenkinson,
who states that “At a certain stage this proximal trophoblast
(the so-called Rauber’s cells of the rabbit) certainly becomes very
thin, but it never wholly disappears, and soon thickens again to
form the Traiger, or, to use a modern expression, trophoblastic
syncytium, which is destined to play an all-important part in
the formation of the placenta.’’ The account of Melissinos is
difficult to follow, owing to his application of the term ‘Rauber-
sche Schicht.’ The outer layer of the blastocyst in the region
of the germinal disc is said to have a transitory existence and to
disappear almost completely in the earlier stages of blastocyst
formation. Ina later paragraph he states, ‘“‘dass nur die Rauber-
sche Schicht existiert und sogar in den folgenden Stadien mit
zahlreicheren Kernteilungsfiguren, und dass sie den Placentar-
conus liefert.’’ Attention has previously and on a number of
occasions been called to the fact that in the albino rat I have not
been able to differentiate a distinct covering layer—Deckschicht
or Rauber’s Schicht (Selenka); trophoblast layer (Jenkinson)—
and have expressed myself as wholly in accord with Sobotta’s

DEVELOPMENT OF THE ALBINO RAT 71

observations on the mouse egg as concerns this point. He has
critically reviewed Selenka’s and Jenkinson’s contentions as
to the participation of the covering layer in the formation of
the Triger or ectoplacental cone, reaching the conclusion that
there is no evidence in support of this. In accord with Duval
and in this I coneur—he states: ‘“‘Die mesometrale Spitze des
‘Trigers Selenkds’ ist, wie auch Duval richtig bemerkt, sogar
ganz auffillig arm an Mitosen.”’ The anlage of the ectoplacental
cone or Triger, it would appear to me, is primarily the result
of enlargement of its constituent cells, this enlargement of cells
involving the more peripherally placed cells of the somewhat
thickened germinal disc. In none of my preparations showing
early stages in the formation of this structure are mitotic figures
evident. Grosser in his figures 67 and 113, shows a germinal
vesicle of the albino rat of 65 days in its normal position in the
decidual crypt. The vesicle there figured is about identical in
time and stage of development to those figured by me in figure
24. In his figures, the Triger (7’r.) is represented as consist-
ing of relatively few cells in which no mitoses are evident. In
slightly older stages after the means of nutrition of the vesicles
is improved through ingestion of maternal blood cells (Sobotta)
mitotic figures may be observed in the ectoplacental cone, as
shown in C of figure 24. In the rat as in Mus sylvaticus and
the guinea-pig (Selenka) the ectoplacental cone arises as a
solid mass of cells; in Arvicola arvalis (Kupffer) it is at first a
hollow structure and is in part formed by invagination; in the
white mouse (Sobotta) the form of this cell mass may vary greatly
and may be solid or penetrated by a mere slit or again by a more
extensive cavity.

The earlier stages in the formation of the egg-plug or egg-
eylinder I have not been able to follow. In the youngest stage
showing this, at my disposal, A of figure 24, it consists of a cen-
tral node of compactly grouped cells, of polyhedral form, quite
definitely demarked from the surrounding cells, and very generally
of oval form. This mass of cells I have designated the ectoder-
mal node. In Grosser’s figures (67 and 113, e, Hc) an identical
structure may be observed, designated as ‘Ectoderm der Em-

2, G. CARL HUBER

bryonanlage.’ The same may perhaps be observed in figure
26, plate 14, of Selenka’s account. In figures 26, 28, 31, and 33
of Christiani’s contribution this may be postulated, though his
figures are useless for a close comparison. Duval does not figure
this stage. Sobotta’s (03) figure 7, and figure 33 of the con-
tribution of Melissinos, appear to give a corresponding stage for
the mouse, but in neither of these figures is the ‘ectodermal
node’ so clearly depicted as in Grosser’s and my own figures,
at least not until a somewhat older stage. Figure 6 of Sobotta
(03) may very probably be regarded as representing an inter-
mediate stage between that shown in E of figure 23 and in A
of figure 24. By a proliferation of the cells of the germinal area
as shown in the former figure a stage resembling that shown in
Sobotta’s figure 6, is readily postulated. That the formation
of the ectodermal cells is in part due to rearrangement of the
cells of the germinal area I believe to be the case, since cell pro-
liferation is not marked in this stage. The enlargement of the
more peripheral cells of the germinal area, leading to the anlage
of the ectoplacental cone, would of necessity cause the forming
ectodermal node to foree the yolk entoderm into the cavity of
the vesicle, and thus form the anlage of the egg-plug and initiate
the phenomenon of entypy of the germ layers. O. Hertwig, in
describing the inversion as observed in the mouse and rat, after
considering the formation of the Traiger through proliferation of
the cells of the Deckschicht, following here Selenka’s account,
states, referring to the Trager, ‘“Durch ihn wird der formative
Teil des Ektoblasts nach dem Centrum der Blase vorgetrieben,
wobei er sich in eine allseits abgegrenzte Epithelkugel umwan-
delt.”’ And again, in referring to the development of the guinea-
pig, he states: ‘‘Wie bei Maus und Ratte zieht sich das forma-
tive Ektoderm zu einer Epithelkugel zusammen.” Hertwig
thus appears to regard the formation of the ‘Epithelkugel,’
the ectodermal node, as in part at least developed owing to a
rearrangement of the cells of the germinal disc. After the
formation of the egg-plug or egg-cylinder that portion of the yolk
entoderm which covers it is designated by Sobotta as the visceral
layer of the entoderm. The scattered entodermal cells, attached

DEVELOPMENT OF THE ALBINO RAT 73

here and there to the inner surface of the parietal ectoderm, in
the albino rat at no time forming a continuous layer, he has desig-
nated as the parietal entoderm. He is followed in this by Wida-
kowich. This nomenclature has been used by me in the sense
employed by Sobotta. The parietal or transitory ectoderm
(Kolster’s ‘feinfasserige Haut’) forming the roof or antimesome-
trial portion of the vesicles, is constituted of a single layer of
flattened cells, which in the rat show no regional differentiation.
The resorption of maternal blood, incidentally noted with
reference to celis of the ectoplacental cone and certain of the
cells of the parietal ectoderm in connection with vesicle C of
figure 24, to which phenomenon attention has been drawn by
Sobotta and Kolster for the mouse, will receive further consider-
ation in the discussion of older stages.

DEVELOPMENT AND DIFFERENTIATION OF THE
EGG-CYLINDER

The material at hand is listed in table 8.

TABLE 8
RECORD NUMBER AGE NUMBER OF OVA
17 8 days, 17 hours (?) | 2 (not all cut)
35 8 days, 18 hours (?) 6
21 7 days, 16 hours 10
66 7 days, 16 hours Wows
27 7 days, 17 hours 7
89 7 days, 20 hours 5
81 7 days, 22 hours 7
94 8 days 7
95 8 days 9
5

96 8 days

For the stages showing the development and differentiation
of the egg-cylinder in the albino rat I am able to present a series
of stages which follow one another in close succession. The
figures presented are in themselves so elucidative that an extended
description is obviated. The stages under consideration fall
within the eighth day after the beginning of insemination,
judging from the great majority of the specimens at my dis-
posal, although two rats (Nos. 17 and 35) killed in the latter

74 G. CARL HUBER

half of the ninth day, contained stages which are younger than
nearly all of those obtained the latter half of the eighth day.
I am unable to state whether this is owing to a retardation in
the rate of development of the ova in rats Nos. 17 and 35, or
due to an error of record. The record gives date and hour of
insemination and of killing, and J have no reason to doubt its
accuracy. However, the two rats in question give the only
instances of marked deviation from what appears as a normal
rate of development as presented by the bulk of my material.
Sobotta (11) has called attention to the difficulty of obtaining
successively staged material in the mouse, and cites Kolster as
contending: ““Man kénne auf die Altersbestimmung gar nichts
geben.” During this stage of development the decidual crypts
lodging the ova are deeper than in the preceding stage, their
mesometrial portion being narrower, though they are not as
yet separated from the uterine lumen. The orientation of
the decidual crypts and the contained egg-cylinders is perhaps
more readily made than in slightly younger stages, though not
definitely enough to insure the cutting of sections in a given
plane. Sections of the egg-cylinder cut in the longitudinal
plane may be obtained by cutting parallel to the plane of the
mesometrium or at right angles to the same. However, it is
still largely a matter of chance as to whether the sections ob-
tained pass through the midplane or at an angle thereto.

In figure 25, there are reproduced representative sections of
three germinal vesicles taken from the same uterus (rat No. 35,
8 days, 18 hours) which show three closely approximated early
stages in the development of the egg-cylinder. Noneof these three
vesicles is cut in exactly the mid-longitudinal plane; especially is
this true of the ends of the vesicles. Furthermore, the antimes-
ometrial portion of each, lower part of the figure, composed of
the thin-walled parietal ectoderm, shows a certain amount of
folding, so that a portion of each wall is cut en face instead of
en profile. The appearances here presented by the antimesome-
trial portion of these vesicles is not to be confused with a ‘giant
cell’ formation of this portion of the roof of the vesicle, described
by Sobotta in his earlier publications, but corrected and retracted

DEVELOPMENT OF THE ALBINO RAT 75

in his later communications. Vesicle A, figure 25, when com-
pared with vesicle C of figure 24, shows only a slight difference in
degree of development. Vesicle A is of more elongated and of
more distinctly cylindrical form. Its thin-walled portion (an-

ect pl

Fig. 25 Longitudinal sections of blastodermic vesicles of the albino rat, show-
ing entypy or inversion of germ Jayers with early stages in egg-cylinder formation.
The ectoplacental cone of each is not cut through its entire length and the lower
portion of each vesicle is slightly folded. X 200. A, B, and C, rat No. 35, 8
days, 18 hours, after insemination. To fit properly into the entire series these
three vesicles should be from the early hours of the seventh day after insemina-
tion. ect.pl., ectoplacental cone or Triiger; ect.n., ectodermal node; ez. ect.,
extraembryonic ectoderm, early stage of its ingrowth shown in vesicle A; p. ect.,
parietal or transitory ectoderm; v.ent., visceral layer of entoderm; p.ent., cells
of parietal entoderm.

timesometrial portion) is longer, its cavity more extensive;
this is owing to a further flattening of the cells of the parietal
or transitory ectoderm. In vesicle A in the section preceding
the one figured, the ectoplacental cone is thicker by about two

76 G. CARL HUBER

rows of cells than in the one figured; the section figured not pass-
ing through the center of this structure. In vesicle A, the ecto-
dermal node, which is distinctly demarked, no longer rests against
the base of the ectoplacental cone, as in C of figure 24, but has
been forced farther into the cavity of the vesicle by reason of
proliferation of the cells at the base of the ectoplacental cone,
resulting in the formation of a nearly cylindrically formed column
of compactly arranged, polyhedral-shaped cells interposed be-
tween the ectodermal node and the base of the ectoplacental
cone, but merging into the latter without sharp demarcation.
To this mass of cells the name of extraembryonic ectoderm has
been given by Widakowich. However, under this term this
author includes also the cells of the ectoplacental cone. The
ectodermal node is of larger size than in the slightly younger
stage, C of figure 24, the result of cell proliferation. In the
section sketched, three mitotic figures are evident in this struc-
ture. Its cells are of polyhedral shape, and show no definite
arrangement. The ectodermal node and the extraembryonic
ectoderm, to the base of the ectoplacental cone, together form a
cylindric structure enclosed within a layer of visceral entoderm,
which in the section figured is in part cut tangentially, and
thus simulates an epithelium consisting of two layers of cells,
but consisting in reality of a single layer of cells. Eetodermal
node, extraembryonic ectoderm, and the layer of visceral ento-
derm together form a structure of cylindric shape which ex-
tends into the cavity of the vesicle for a distance about one-half
its extent, forming the anlage of the egg-cylinder (Sobotta).
Very few parietal entodermal cells are to be found on the inner
surface of the parietal ectoderm. Vesicles B and C of figure 25
differ from that discussed under A, only to the extent to which
the ectodermal node has been forced into the cavity of the vesicle
owing to further growth of the extraembryonic ectoderm, to
the extent that in C, the elongated egg-cylinder approaches
the antimesometrial end of the cavity of the respective vesicle.
Eetodermal node and extraembryonic ectoderm are at this stage
distinctly demarked, though in close apposition. An indenture
from the surface at the region of the union of these structures

DEVELOPMENT OF THE ALBINO RAT oe

with a consequent infolding of the layer of visceral entoderm is
not as a rule evident, if so, only very slightly, as to the left in B;
such infolding of the visceral entoderm is not regarded as having
special significance. These structures, ectodermal node and extra-
embryonic ectoderm, are appropriately referred to as ectodermal
eylinder by Widakowich, and with the visceral entoderm, as
constituting the egg-cylinder of Sobotta.

Under A of figure 26 (rat No. 17, 8 days, 17 hours), there is
shown a representative section of a vesicle which is only very
slightly older than that shown under C, figure 25. This vesicle
was exposed, by teasing away, after fixation, the decidual tis-
tue forming one side of the decidual crypt; this being done before
embedding, so as to admit of orientation of its long axis. This
accounts for the collapsed state of the thin wall of the vesicle
and its slight folding, also for the fact that the ectoplacental cone
is reflected upon itself. The egg-cylinder is cut in a very favor-
able longitudinal plane. In its antimesometrial portion, lower
part of the figure, the cells of the ectodermal node now show
definite arrangement in practically a single layer, with alter-
nating nuclei. The beginning of a central cavity is evident with
reference to which the cells are arranged. This cavity is the
anlage of the ‘Markamnionhohle’ of Selenka, more appropriately
known as the antimesometrial portion of the proamniotic cavity.
The cells forming the wall of the ectodermal vesicle (Ektoderm-
blase, Selenka), derived from the ectodermal node, may now be
known as the primary embryonic ectoderm (Widakowich).
The extraembryonic ectoderm in the mesometrial portion of the
egg cylinder has differentiated to form a relatively long irregu-
larly cylindric structure, continuous with the base of the ecto-
placental cone, composed of irregular polyhedral cells, com-
pactly arranged and showing as yet no definite orientation. In
these cells active proliferation is evidenced by numerous mitoses.
The egg-cylinder is covered by a single layer of cells of the
visceral entoderm. Over the antimesometrial end of the egg-
cylinder, the entodermal cells now present a cubic or thick pave-
ment form, while along the sides of the egg-cylinder they are of
columnar form, especially long in the region where the primary

Fig. 26 Longitudinal sections of egg-cylinders of the albino rat, showing
the anlage of the antimesometrial and mesometrial portions of the proamniotic
cavity. X 200. A, rat No. 17, 8 days, 17 hours; B and C, rat No. 81, 7 days,
22 hours, after insemination. A, shows the very beginning of the development
of the antimesometrial portion of the proamniotic cavity developing within the
ectodermal node; C shows the beginning of the proamniotic cavity develop-
ing in the extraembryonic ectoderm; ect.pl., ectoplacental cone or Triger; p.ect.,
parietal or transitory ectoderm; ez.ect., extraembryonic ectoderm; v.ent., vis-
ceral entoderm in B and C, the cells of this layer showing the anlage of the three
zones showing absorption of maternal hemoglobin; a.met.pr., antimesometrial
portion of proamniotic cavity, developing in the ectodermal node; pr.emb.ect.,
primary embryonic ectoderm; ect.ves., ectodermal vesicle; met.pr., mesometrial
portion of the proamniotic cavity, developing in the extramebryonic ectoderm.

78

DEVELOPMENT OF THE ALBINO RAT 79

embryonic ectoderm and the extraembryonic ectoderm meet.
The special eytomorphosis undergone by the columnar cells
of the sides of the egg-cylinder, in contradistinction to those of
the antimesometrial end, will be considered in later pages. The
visceral layer of the entoderm extends to the base of the ecto-
placental cone, in part passing over onto the layer of parietal
ectoderm. In the section figured, cells of the parietal layer of
the entoderm are not evident. The ectoplacental cone has
grown in length in the direction of the lumen of the uterus or the
mesometrial border. In the great majority of my preparations
this structure is slightly compressed from side to side, so as to
be broader in a plane parallel to the long axis of the uterus. In
vesicle A, it is cut at right angles to the long axis of the uterus,
thus appears as much narrower than in the other two vesicles
of figure 26, which were cut in a plane parallel to the plane of
the mesometrium. The increase in size of the ectoplacental
cone is the result of active cell proliferation. Mitotie figures
to the number of one, two or three, may now be observed in
nearly every section of this structure. The parietal or transitory
ectoderm, continuous with the base of the ectoplacental cone,
has been reduced by this stage to a thin, practically homogeneous
membrane, presenting scattered, flattened nucleated cells on its
inner surface. This thin membrane is now quite firmly adherent
to the wall of the decidual erypt, throughout nearly its whole
extent.

Under B of figure 26 (rat No. 81, 7 days, 22 hours) there is
shown a representative section of a vesicle which is slightly more
advanced in development than that shown in A of this figure.
The antimesometrial portion of the proamniotic cavity, the
anlage of which was shown in the preceding stage, is well estab-
lished. Its wall, consisting of primary embryonic ectoderm
is composed of a single layer of cells with nuclei in essentially
the same plane. The primary embryonic ectoderm forms a
closed vesicle (EKctodermblase, Selenka) distinctly demarked
from the extraembryonic ectoderm. In this as in the preceding
stage the extraembryonic ectoderm forms a long cylindrical
structure continuous at its mesometrial end with the base of the

£0 G. CARL HUBER

ectoplacental cone. The cells are of irregular polyhedral form,
compactly grouped, showing as yet no definite arrangement.
Cell proliferation as evidenced by mitoses is active, amply ac-
counting for the increase in length of this structure. The vis-
ceral entoderm encloses the long egg-cylinder as a single layer
of cells and is continuous at its base with the parietal entoderm,
well shown at the left of the figure. The ectoplacental cone of
this vesicle is very favorably cut in a plane parallel to the long
axis of the uterus. This vesicle was unusually well fixed and
may be regarded as showing normal relations of the thin mem-
branous wall, derived from the parietal ectoderm, and of the
egg-cylinder, which reaches quite to the antimesometrial end
of the vesicle.

Vesicle C of figure 26, obtained from the same uterus as was
vesicle B (rat No. 81, 7 days, 22 hours), differs from that shown
under B, in that it presents the anlage of a mesometrial portion
of the proamniotic cavity. In the extraembryonic ectoderm,
near its junction with the base of the ectoplacental cone, two
irregular spaces may be observed. These are distinctly evident,
passing through the entire section, only in the section figured.
The antimesometrial portion of the egg-cylinder is not cut
quite through its center, so that the primary embryonic ectoderm
of the ectodermal vesicle appears as a stratified epithelium, and
the antimesometrial portion of the proamniotic cavity appears
as relatively small, this owing to a slight curvature shown by
this egg-cylinder. The other features presented by this vesicle
are sufficiently well portrayed in the figure to obviate the neces-
sity of further description.

In figure 27, there are shown three further stages of egg-cylinder
differentiation, showing progressively older stages than shown
in the preceding figure. Under A of this figure, there is re-
produced a representative section of a vesicle taken from the same
uterus as were vesicles B and C of figure 26 (rat No. 81, 7 days,
22 hours). The figure is not of a single section, but is com-
bined from two sections, superimposed so as to give correct
dimensions and relations. The egg-cylinder of A of this figure
differs from that shown in C of figure 26, in that the mesometrial

: Wee ect &

eS

rae

i)

shee

we

pr.emb, ect.~ NN k

Fig. 27 Longitudinal sections of egg-cylinders of the albino rat showing
fusion of the antimesometrial and the mesometrial portions of the proamniotic
cavities. XX 200. A, rat No. 81, 7 days, 22 hours; B, rat No. 96, 8 days; C, rat
No. 94, 8 days, after insemination; ect.pl., ectoplacental cone or Triger; p.ect.,
parietal or transitory ectoderm; exr.ect., extraembryonic ectoderm; ect.ves., ecto-
dermal vesicle, with wall composed of primary embryonic ectoderm, at + junc-
tion with the extraembryonic ectoderm; a.met.pr., antimesometrial portion of
proamniotic cavity; mel.pr., mesometrial portion of proamniotie cavity; pr.c.,
proamniotic cavity; v.ent., visceral entoderm; pr.emb.ent., primary embryonic

entoderm.
81

82 G. CARL HUBER

portion of the proamniotic cavity, developing in the extra-
embryonic ectoderm, is of greater dimension. Two relatively
large spaces, bordered by a single layer of cells of the extra-
embryonic ectoderm, are to be observed. At the junction of
the extraembryonic ectoderm and the ectodermal vesicle of
primary embryonic ectoderm a further space of triangular out-
line may be seen. The primary embryonic ectoderm is ar-
ranged in the form of an oval-shaped vesicle, forming the anti-
mesometrial end of the egg-cylinder. Its wall is relatively thin
at the region of its apposition to the extraembryonic ectoderm,
just below the triangular space above mentioned. This ecto-
dermal vesicle is peculiar in that its cavity contains the re-
mains of four cells. A study of the series of sections shows
that these cells do not represent the crest of a fold of the wall
of this vesicle, since they are not nearly so distinct in preceding
and succeeding sections. It may only be conjectured that
during the rearrangement of the cells of the ectodermal node,
resulting in the formation of the ectodermal vesicle, certain of
the cells became separated from the wall and remained free in
the cavity. The primary embryonic ectoderm, forming the
wall of the ectodermal vesicle is readily differentiated from the
extraembryonic ectoderm, both by the fairly sharp definition of
the ectodermal vesicle and by reason of the fact that its cells
stain somewhat more deeply than do the cells of the extraem-
bryonic ectoderm, as also the cells of the visceral entoderm.
In the egg-cylinder shown under B of figure 27 (rat No. 96, 8
days) the antimesometrial portion of the proamniotic cavity,
developing in the ectodermal node, and the mesometrial portion
of the proamniotic cavity, developing as several discrete spaces
in the extraembryonic ectoderm, have in part joined to form
a single proamniotic cavity. The mesometrial portion of this
cavity is still bridged by a septum of extraembryonic ectodermal
cells, closing off a relatively large space found in its mesometrial
portion. With the junction of the antimesometrial and the
mesometrial portions of the proamniotic cavity, the primary
embryonic ectoderm and the extraembryonic ectoderm become
a continuous layer, the line of union of the two portions, however,
remains evident and is readily recognized in all the egg-cylinders

DEVELOPMENT OF THE ALBINO RAT 83

of this and older stages, a question which will receive further
consideration in following pages.

In C of figure 27 (rat No. 94, 8 days) the proamniotie cavity
forms a continuous, single space. The figure presented is drawn
from two sections; its greater portion, to the base of the ecto-
placental cone from one section, the ectoplacental cone from
another section. The junction of the membranous wall of the
vesicle to the base of the ectoplacental cone, in the two sections
used for the figure, was superimposed under camera lucida in
joining the portions drawn from the two sections. It is be-
lieved that the drawing as presented gives correctly dimen-
sions and relations of the different parts of this vesicle. The
wall of the antimesometrial portion of the single proamniotic
cavity is formed by the primary embryonic ectoderm, the cells
of which are for the main of irregular columnar shape, with
alternately placed nuclei. These cells are in active proliferation,
as is evidenced by numerous mitoses. The wall of the meso-
metrial end of the proamniotic cavity is formed of a single layer
of cells of the extraembryonic ectoderm; these cells are of quite
regular shape with nuclei placed in about the same plane. They
stain less deeply than do the cells of the primary embryonic
ectoderm. In this egg-cylinder (C, fig. 27) the proamniotic
‘avity does not extend so near the base of the ectoplacental
cone as in a number of other preparations in my possession,
showing about the same stage of development; in certain of these,
the proamniotic cavity extends to near the mesometrial end
of the egg-cylinder.

A more definite characterization of the different parts of the
ege vesicle of the albino rat at the stage of development shown
in C, figure 27, end of the 8th day, seems desirable, and in doing
so I shall use the terminology used by Sobotta and Widakowich.
The vesicle under consideration has reached a length of 0.65
mm., and a width of 0.12 mm. Somewhat more than one-
fourth of its length consists of ectoplacental cone or Trager.
The cavity enclosed is derived from the cavity of the blasto-
dermic vesicle with germ disc, the blastocele, and is termed by
Sobotta and Widakowich the ‘Dottersackhéhle’ or yolk-sae
cavity. This cavity is bounded by a thin structureless mem-

84 G. CARL HUBER

brane derived from the parietal or transitory ectoderm and the
seattered cells forming the parietal layer of entoderm. This
membrane is continuous with the base of the ectoplacental cone
and presents scattered flattened cells on its inner surface. I have
designated this thin membrane with cells on the inner surface
as the parietal or transitory ectoderm (Kolster’s feinfaserige
Haut). The egg-cylinder which extends to the antimesometrial
end of the yolk-sae cavity, encloses the proamniotic cavity, the
antimesometrial portion of which is walled by primary embryonic
ectoderm, its mesometrial portion by extraembryonic ecto-
derm, the two forming a continuous layer, with line of union
of the two types of ectoderm evident. The uncleaved extra-
embryonic ectoderm is continuous with the base of the ecto-
placental cone. The egg-cylinder is surrounded by a single
layer of cells of the visceral entoderm, differentiated so as to
consist of a portion which surrounds the antimesometrial end
of the egg-cylinder in relation with the primary embryonic ec-
toderm; the cells of this portion being of a rather thick pavement
type, constituting the primary embryonic entoderm, and fur-
ther a portion which covers the sides of the egg-cylinder, with
cells of a columnar type, showing special cytomorphosis. The
egg-vesicles and egg-cylinders of the stage of development under
consideration and for somewhat older stages show no bilateral
symmetry so far as can be discerned by study under the micro-
scope. In longitudinal sections of egg-cylinders, cut respectively
in two different planes, at right angles to each other, no differ-
ence in form, relation and structure of different parts can be
observed. Selenka, Kupffer, Duval, and Sobotta have pre-
viously called attention to this fact and shown that longitudinal
sections of egg-cylinders may be obtained no matter whether
the sections are cut parallel to the plane of the mesometrium,
thus parallel to the long axis of the uterus, or at right angles to
this plane. The want of bilateral symmetry is also evident in
cross sections of the egg-cylinder, as may be seen from the
series of sections presented in figure 28 (rat No. 27, 7 days, 17
hours). The cross-cut egg-cylinder, from several sections of

which these figures were drawn, represents a stage of develop-

DEVELOPMENT OF THE ALBINO RAT 85

ment very similar to that of the egg-cylinders shown in longitudi-
nal section in figure 26.

Widakowich, after discussing very briefly the mode of develop-
ment of the egg-cylinder, discusses and figures an egg-cylinder
of the albino rat, obtained 6? days after the last coitus. His
figure 3 corresponds in stage of development very closely to
that shown by me in A of figure 27. In his figures, there is pre-
sented an egg-cylinder showing the anlage of the mesometrial

Fig. 28 A series of cross sections at different levels of an egg-cylinder of the
albino rat after the anlage of the antimesometrial portion of the proamniotic
cavity. X 200. Rat No. 27, 7 days, 17 hours, after insemination. The sec-
tions selected for the several levels drawn, A to D, are as follows: A, middle
of ectoplacental cone; B and C, through extraembryonic ectodermal portion of
egg-cylinder, just below junction with ectoplacental cone (B), and just above
ectodermal vesicle (C); D, through middle of ectodermal vesicle. Compare
with B, figure 26, a longitudinal section of an egg-cylinder of the same stage of
development; p.ect., parietal or transitory ectoderm; ez.ect., extraembryonic
ectoderm; pr.emb.ect., primary embryonic ectoderm of the ectodermal vesicle;
v.ent., visceral entoderm; pr.emb.ent., primary embryonic entoderm; a.met.pr.,
antimesometrial portion of proamniotic cavity.

portion of the proamniotic cavity. Emphasis is given to the
fact that in the antimesometrial portion of the egg-cylinder,
there may be recognized the primary embryonic ectoderm.
His own words with reference to this point read as follows:

Der Schnitt zeigt nun sehr deutlich, dass sich die Zellen, die die
antimesometrale Hohle so begrenzen, dass die alte Kugel-oder Eiform
dieses Teiles noch zu erkennen ist—das primire embryonale Ectoderm
—intensiver fiarben wie die Zellen des mesometralen Abschnittes oder die
des Ectoplacentarconus—das extraembryonale Ectoderm. Die Kerne

zeigen keinerlei Unterschied in der Farbung, wohl aber das Plasma,
dass im antimesometralen Teile von dichterer Structur zu sein scheint.

This description corresponds very closely to that given by
me for a similar stage. The differentiation of these two kinds
of ectoderm was also recognized by¥ Robinson, who states:

S6 G. CARL HUBER

(

The epiblastic cylinder is closed at its distal end, the trophoblastic
at its proximal, and the open ends of the two cylinders are in close apposi-
tion, but not indistinguishably fused, for the character of each por-
tion of the ectoderm, after treatment with carmine, is still quite dis-
tinctive; the protoplasm of the trophoblast being tinged much more
faintly than that of the epiblast.

Selenka, on the other hand, who has recognized in his ‘Ekto-
dermblase’ with ‘Markamnionhohle’ a distinctive structure,
believes this to blend completely with the Trager. Since his
account with reference to this point has influenced later workers,
I may be permitted to quote him in the original. Referring to
the ‘Ektodermblase’ with ‘Markamnionhohle,’ he states:

Dieser Ektodermkeim, welcher von dem vorriickenden Tragerzap-
fen anfinglich sehr wohl abgegrenzt ist, indem beiderlei Gebilde sich
in Folge der convexen Kriimmung ihrer einander zugekehrten Flachen
sozusagen nur in einem Punkte beriihren, fliesst endlich mit dem
Traiger vollstandig zusammen, und zwar bei der Waldmaus bevor,
bei der Ratte und Hausmaus aber nachdem die Markamnionhéhle
enstanden war.

That the proamniotic cavity of the egg-cylinder of the albino
rat has its anlage in two distinct cavities, the one developing in
the ectodermal node in the antimesometrial portion of the egg-
cylinder, which is the first to develop; the other in the meso-
metrial portion in the extraembryonic ectoderm, was recognized
by Selenka. (fig. 30, plate 14, E, Markamnionhohle, E’, falsche
Amnionhohle), Duval (fig. 100,) Robinson, and Widakowich
(fig. 3). Corresponding stages of egg-cylinder development as
presented by me in figures 26 and 27, for the albino rat, are
shown by Sobotta (’02), for the mouse in his figures 12 to 14 and
text figures a to f. On comparison of my figures with Sobotta’s,
it becomes evident that the egg-cylinder of the rat is much longer
and more slender than that of the mouse. According to the
account of Sobotta, the egg-cylinder of the mouse, soon after its
anlage, shows by reason of a distinct transverse furrow a division
into two parts, an antimesometrial portion of globular form,
surrounded by a visceral layer of entoderm, corresponding to
what I have designated as the ectodermal node; and a meso-
metrial portion which early,shows the anlage of a proamniotic

DEVELOPMENT OF THE ALBINO RAT 87

‘cavity. A lumen is obtained in the antimesometrial portion later
than in the mesometrial portion. As development proceeds, this
sharp demarkation of antimesometrial and mesometrial portion
is gradually lost. This, as stated in his own words, reads:

Sehen wir von dem die (der Keimhéhle zugekehrte) Oberfliche
des Cylinders tiberziehenden Dotterentoderm zuniichst ab, so sieht
man, dass die Furche, welche die oben erwiihnten mesometralen und
antimesometralen Abschnitte in Stadium der Fig. 11 u. 12 trennte,
jetzt wieder wenig deutlich ist. Es bahnt sich eine Verschmelzung
beider Abschnitte wiederum an, was man am leichtesten daraus er-
sieht, dass bald (Fig. 14) beide Abschnitte ein gemeinsames Lumen
erhalten,

With the formation of a continuous proamniotic cavity,
this is bordered by a single layer of ‘ectodermal cells,’ with al-
ternately placed nuclei. The cells are described as being the
same throughout; neither in text nor figure does Sobotta differ-
entiate between ectodermal cells derived from the antimesome-
trial portion of the egg-cylinder and those derived from the
mesometrial portion. Melissinos also recognizes antimesome-
trial and mesometrial portions in the development of the egg-
cylinder of the mouse, in his figure 34. According to this ob-
server, the antimesometrial portion of the proamniotic cavity
is the first to appear; later it appears in the mesometrial por-
tion, the two cavities joining as development proceeds. The
parts of the ectoderm derived from these two portions may be
recognized, however, after a single proamniotic cavity has
developed. ‘This Melissinos states in the following words: ‘‘Trotz
aller Vereinigung der beiden Héhlungen bleibt die Unterscheidung
des normals abgesonderten antimesomtralen Abschnittes von
dem mesometralen immer leicht zu machen, sei es durch eine
klare Grenzlinie oder durch eine an der Peripherie des visceralen
Dotterblattes befindliche Furche.”” The account of Melissinos
is more in agreement with the presentations as observed in the
albino rat than is that of Sobotta.

Selenka, Sobotta, and Melissinos recognize three different
regions of constriction to which significance is given, in the
egg-cylinder of the mouse. As stated by Sobotta, the first con-

88 G. CARL HUBER

striction is in the region of the original furrow which demarks
the antimesometrial and the mesometrial portions of the egg-
cylinder, the region of the primary amniotic fold; the second
where the mesometrial cavity ends; and the third where the
original blastodermic cavity reaches its mesometrial end. The
three folds recognized by Melissinos, are characterized by the
specificity of the ectoderm. Since his statement concerning this
point is somewhat involved, I find it necessary to use his own
words; they read as follows, referring to these folds he states:

Der eine derselben a liegt antimesometral und ist der bekannte
erste kugelférmige Buckel (Ektoderm) mit den langlichen, cylinder-
pyramidalen oder polygonal-pyramidalen Zellen; der zweite 6 liegt
in der Mitte und besteht aus kubisch-polygonalen Zellen, und der
dritte Buckel c, aus polygonalen Zellen bestehend, legt mesometral
und ist von dem mittleren durch Einschniirung, von der Basis des
Ectoplacentarconus aber durch die bekannte Urfurche des Eicylinders

getrennt, in der sich das viscerale Dotterblatt zum parietalen Dotter-
blatt umbiegt.

So far as I am able to determine, the account of Melissinos
agrees with that given by Sobotta, as concerns the folds of the
ege-cylinder of the mouse. Selenka’s account need not receive
special consideration.

In well-fixed egg-cylinders of the albino rat no such folds are
recognized. At the line of junction of the primary embryonic
ectoderm and the extraembryonic ectoderm, a slight infolding
of the layers, variable in degree, is recognized. Other foldings
of the wall of the egg-cylinder I have regarded as accidental and
not of special significance. Therefore, I am wholly in accord
with Widakowich, who has also discussed this question with
reference to the albino rat and has described the low fold in the
region of the junction of the primary embryonic ectoderm and
extraembryonic ectoderm. Referring to that fold, he states:
“Dass war die einzige konstante, bald stiirker, bald schwiicher
ausgeprigte Einschniirung der Proamnionhohle.”’

Sobotta deserves credit for having described fully the differ-
entiation and cytomorphosis of the cells of the visceral entoderm
of the egg-cylinder, and since his observations on this point apply
in the main to the albino rat, they may at this time be given

DEVELOPMENT OF THE ALBINO RAT 89

consideration. During the early stages of egg-cylinder differ-
entiation and anlage of the proamniotic cavity, the layer of viscer-
al entoderm differentiates into a portion which is in relation
with the primary embryonic ectoderm of the antimesometrial
portion of the egg-cylinder, in which region the cells of the en-
toderm are first of short cubic shape, later of the pavement type;
this portion may be regarded as forming the primary embryonic
entoderm, since it forms the greater part of the entoderm of the
embryo. The greater part of the visceral entoderm, that which
surrounds the sides of the mesometrial portions of the egg-cylin-
der, consisting of extraembryonic ectoderm, differentiates into
cells of the columnar type. In this latter portion, with the
formation of a continuous proamniotic cavity, the entodermal
cells undergo characteristic cytomorphosis. In them, as stated
by Sobotta, there may be recognized three main zones: (1) a
basal zone with denser protoplasm containing the nucleus;
(2) a middle zone with markedly vacuolated protoplasm; (3)
an outer zone in which hemoglobin granules are recognized, the
latter zone staining deeply in eosin. These three zones in the
cells of the visceral entoderm in the region of the extraembryonic
ectoderm of the egg-cylinder may be recognized in figures 26 and
27, not so clearly as in Sobotta’s colored figures, particularly his
figure 17 (03) and figure 8 (’11).. However, I am able to follow
closely his description in my own preparations of a somewhat older
stage than thus far figured. It is Sobotta’s contention that in
the extravasated blood surrounding the egg vesicle, in close
apposition to its thin outer wall, there may be observed many
red blood cells which, though presenting normal form, show
a distinctly granular content. These granules stain deeply in
eosin and are in shape, size, and reaction to stain very similar
to granules found in the peripheral part of the cells of the visceral
entoderm. On the outer surface of the thin wall of the vesicle;
on its inner surface; in the cells lining this; in the yolk sae cavity;
and on the outer surface of the cells of the visceral entoderm,
similar granules are found. These appearances are interpreted
as showing an absorption of maternal hemoglobin by the ento-
dermal cells of the mesometrial portion of the egg-cylinder.

90 G. CARL HUBER

Sobotta’s statement concerning this point, which, owing to its
importance, | quote in full, reads as follows:

Man wird diese mikroskopisch erkennbaren Verhaltnisse nicht an-
ders deuten kénnen als in folgender Weise: Die Hamoglobinschollen,
die durch die fussere Wand des Dottersackes in die Dottersackhoéhle
gelangt sind, werden von der Oberfliche des zylindrischen, die ganze
Seitenfliche des Eizylinders iiberziehenden visceralen Dottersack-
epithels aus resorbiert und zwar geschieht das in der Weise, dass die
Himoglobinschollen ziinachst als solche in der Zelle selbst eintreten,
dann aber im vacuolisierten Teil der Zelle gleichsam verdaut werden,
wobei die einzelnen kleinen Schollen vorher zu grésseren Tropfen zusam-
men-fliessen scheinen.

My own observations on the albino rat as concerns this phe-
nomenon, more particularly as concerns the structure of the
cells of the visceral entoderm in the region of the extraembryonic
ectoderm, corroborate Sobotta in many particulars. This
question will beagain and more fully considered in a contemplated
later publication dealing with the implantation and decidua
formation in the albino rat. It could not be considered now
without a discussion of the changes involved in the development
of the decidua, a question which I am not prepared to consider
fully now. It may be stated, however, that judging from my
own preparations and the figures of Grosser, the extravasation
of blood into the egg chamber is not nearly so extensive in. the
albino rat as is shown in the figures of Sobotta for the mouse.

The thin membrane which surrounds the yolk-sac cavity,
which I have designated as the parietal or transitory ectoderm,
is derived in development from the parietal or transitory ecto-
derm, and the relatively few parietal entodermal cells, as de-
scribed and figured for younger stages. At the stage of egg-cylin-
der development under consideration—with ccntinuous pro-
amniotic cavity—this structure appears as a thin, practically
homogeneous membrane with scattered, flattened nucleated
cells on its inner surface. Sobotta regards these cells as derived
from the parietal entoderm, the cells of the parietal ectoderm
having disappeared. As concerns this, I am unable to speak
with certainty, since the Congo red solution used as a double
stain is not particularly favorable in differentially coloring these

DEVELOPMENT OF THE ALBINO RAT 9]

cells. However, I am disposed to regard these flattened cells
as derived from the parietal ectoderm. The parietal entodermal
cells are never numerous in the rat, and mitotic figures are sel-
dom observed in them. With the extension of the vesicle with
the enlargement of the blastocele, the cells of the parietal or
transitory ectoderm become attenuated until they appear for
the greater part as a thin cuticular membrane, and I am dis-
posed to regard the flattened nucleated masses of protoplasm
lining the inner surface of this membrane as derived from the
cells of the parietal ectoderm.

Much attention has been given to certain large cells which are
found in close relation with the outer surface of this thin mem-
brane. These cells, generally referred to as giant cells (Riesen-
zellen) were, by Duval, Sobotta (earlier publications) and Gros-
ser thought to be of embryonic origin and derived from the
cells of the parietal ectoderm. Selenka, Disse, Kolster, Melis-
sinos, Pujiula, Widakowich, and later Sobotta (11) regard them
as derived from the maternal tissue and as representing differ-
entiated decidual cells. It is not my purpose to consider more
fully these cells in the present communication, since they are
by me not regarded as of embryonic origin. My own observa-
tions as concerns them agree in the main with those of Wida-
kowich, who, in the albino rat has followed their origin from
decidual cells. Since not of embryonic origin, they have been
disregarded in making the figures.

I have previously, in connection with a discussion of the
structure of vesicle C, figure 24, alluded to the fact that the
cells of the ectoplacental cone as also the cells of the parietal
or transitory ectoderm have a phagocytic action for maternal
blood cells. This Sobotta has also observed for the mouse, in
which he is confirmed by Kolster who has further shown that the
cells of the ectoplacental cone also take up fat particles. Withthe
ingestion of maternal blood cells by the cells of the ectoplacental
cone, more particularly, with the absorption of hemoglobin by
the entodermal cells of the mesometrial portion of the egg-
cylinder, a period of rapid growth of the egg vesicle is initiated.
To this Sobotta has called attention for the mouse; the same

92 G. CARL HUBER

is evident in the albino rat. Indeed, Sobotta presents the
far-reaching conclusion that the explanation of the phenomenon
of germ layer inversion or entypy of the germ layers is to be
found in the dearth of food supply of the ovum in the stages
preceding the formation of more definite relations between the
ova or germ vesicles with the decidua. It is thought by this
observer that the inversion of the germ disc has for its purpose
the increase of the absorptive surface of the visceral or yolk sac
entodermal epithelium, which as a differentiated layer comes
to surround nearly the whole of the egg-cylinder on comple-
tion of the inversion, and is thus increased in extent and brought
in relatively close relation with the maternal blood lacunae
surrounding the egg vesicle.

LATE STAGES IN EGG-CYLINDER DIFFERENTIATION AND
THE ANLAGE OF THE MESODERM

In the rat series there are found 24 egg cylinders showing the
stages of development considered in this section; certain of them
are cut longitudinally and others cross-wise.

For the special consideration of egg-cylinder formation just
prior to the anlage of the mesoderm, I present two egg-cylinders
obtained during the latter half of the ninth day after insemination;
one of these was cut longitudinally, the other in favorable cross-
section. The egg-cylinder shown in figure 29, rat No. 40, 8
days, 17 hours after insemination, seems unusually well fixed,
as evidenced by its symmetrical outline, and is cut in a very
favorable plane. The sections are from a series cut at right an-
gles to the long axis of the uterine horn. The decidual crypts
lodging the egg-cylinders of this stage are by this time nearly
completely separated from the lumen of the uterus, and are
surrounded by a well-developed decidua. Extravasated mater-
nal blood nearly surrounds such egg-cylinders.

Fig. 29 Longitudinal, sagittal section of egg-cylinder of the albino rat show-
ing the final mesoderm-free stage. > 200. Rat No. 40, 8 days, 17 hours, after
insemination; ect.pl., ectoplacental cone or Triger; p.ect., parietal or transitory
ectoderm; pr.emb.ect., primary embryonic ectoderm; ez.ect., extraembryonic
ectoderm; pr.c., proamniotic cavity; v.ent., visceral entoderm, absorptive for
maternal hemoglobin, cells showing the three zones described by Sobotta;
pr.emb.ent., primary embryonic entoderm.

a
c
o

a
E
o
=
a

94 G. CARL HUBER

The egg-cylinder shown in figure 29 presents a total length
of 1.15 mm., a width of approximately 0.18 mm. The ectopla-
cental cone presents a length of 0.4 mm. and of the proamniotic
cavity, 0.5 mm., of which 0.2 mm. falls to the antimesometrial
portion lined by primary embryonic ectoderm. This egg-cylin-
der differs only in shape and size from that shown in C of figure
27, obtained 8 days after insemination. The primary embryonic
and extraembryonic ectoderm lining or enclosing the proam-
niotic cavity are readily differentiated. The primary embryonic
ectoderm, derived from the ectodermal nede, constitutes a pseu-
dostratified epithelium, composed of relatively long columnar
cells, with nuclei radially placed with reference to the lumen of
the proamniotic cavity, and shows active cell division, no less
than 12 mitotic figures occurring in the section figured. The
protoplasm of its cells stains distinctly deeper than does that
of the cells of the extraembryonic ectoderm. The cells of the
latter are of cubic, short columnar, or polyhedral shape, ar-
ranged in a single or double layer, with no definite arrangement
of the long axes of its nuclei. It is, therefore, possible readily
to distinguish—by reason of shape and size of cells, relative posi-
tion of nuclei, reaction to stain of protoplasm—between the
cells of the primary embryonic and extraembryonic ectoderm,
and to determine the sharp line of junction at which the two
types of cells form a continuous layer, a fact which will receive
further consideration in dealing with the anlage of the mesoderm
as observed in slightly more advanced stages. At the meso-
metrial end of the proammiotic cavity, the cells of the extraem-
bryonic ectoderm become continuous with the cells at the base
of the ectoplacental cone; in the region of this Junction, active
mitosis are often to be observed. In this egg-cylinder the visceral
entoderm may readily be differentiated into two portions. The
portion which surrounds the primary embryonic ectoderm to
nearly the region of its junction with the extraembryonic ecto-
derm, consists of a single layer of bread, flattened cells which
assume a cubic or short columnar shape as the mesometrial
border of the primary embryonic ectoderm is approached.
This portion of the visceral entoderm we have designated as

DEVELOPMENT OF THE ALBINO RAT 95

the primary embryonic entoderm. The portion of the visceral
entoderm surrounding the sides of the egg-cylinder in the region
of the extraembryonic ectoderm, to near the base of the ecto-
placental cone, consists of a single layer of columnar cells, regu-
larly arranged and presenting the three zones described by So-
botta. In this stage of egg-cylinder development of the albino
rat, the absorption of hemoglobin granules derived from maternal
blood cells, first shown for the mouse by Sobotta and Kolster,
may be readily made out. In preparations stained in hematoxylin
and Congo red, in and on the outer zone of the visceral entodermal
cells there may be observed granules staining deeply in the Congo
red, presenting the color reaction of hemoglobin. In the mid-
dle zone of these cells the protoplasm is distinctly vacuolated,
while the inner zone, containing the nuclei, presents a denser
protoplasm. The transitory or parietal ectoderm consists of a
homogeneous membrane, closely adherent to the maternal de-
cidua, especially along the sides of the egg-cylinder. This layer
presents scattered nucleated protoplasmic masses of spindle or
dome shape on its inner surface, the relations and distribution
of which may be clearly seen in the figure. Attention needs
yet be drawn to the ectoplacental cone of the egg-cylinder. Its
relation to the maternal decidua is very intimate, so that in
places, owing to blood extravasations, it is difficult to differentiate
between embryonic and maternal tissue. Many of the cells
of the ectoplacental cone present a vacuolated protoplasm, the
vacuoles enciosing maternal blood cells. Therefore, they are
distinctly phagocytic. Sobotta has also observed and described
this for the mouse. Referring to a slightly older stage after the
anlage of the mesoderm, his own words read as follows:

Weiterhin sehen wir im Stadium der Fig. 5 auch eine starke Ver-
langerung und Vergrésserung des Ectoplacentarconus, an dem im meso-
metralen Teile jetzt Vacuolen auftreten, die in spaiteren Stadien regel-
missig gefunden werden und zwar erfillt mit miitterlichen Blutex-
travasaten. Die Ebrnaihrung des Embryo mit miitterlichem Hamo-
globin * * * * ist jetzt im vollen Gang.

Absorption of maternal hemoglobin by the cells of the ecto-
placental cone appears to be established at a relatively earlier
period in the rat than in the mouse.

96 G. CARL HUBER

The egg-cylinder presented in figure 29 constitutes the final
mesoderm-free stage, the final stage in which no distinct bilateral-
ity may be determined. I assume that the egg-cylinder pre-
sented in the figure is cut in the sagittal plane. This assumption
is based on the fact that the primary embryonic ectoderm ex-
tends slightly farther toward the mesometrial pole on the one
side than on the other. In good frontal sections one side of the
egg-cylinder in this stage of development should present a mir-
ror picture of the other side. The side on which the primary
embryonic ectoderm extends farther toward the mesometrial
pole, the left in the figure, is regarded as containing the caudal
end of the future embryo. In the primary embryonic ectoderm
of this region, it is believed, will develop the primitive streak and
groove, and thus the anlage of the mesoderm. Not in all the
egg-cylinders of this stage of development found in my series can
the caudal end of the future embryonic area be postulated prior
to the anlage of the mesoderm, and in cross-sections no such
differentiation can be made. The proamniotic cavity of the
egg-cylinder shown in figure 29 presents a regular and nearly
smooth contour, not divisible into regions such as described
for a similar stage for the mouse by Selenka, Melissinos, and
Sobotta. A very slight constriction is to be observed only in
the region where the primary embryonic and extraembryonic
ectoderm are joined in a continuous layer. I am thus wholly
in accord with Widakowich, who in describing a similar stage in
one of his preparations, states: ‘‘Das war die einzige konstante,
bald stirker, bald schwiicher ausgeprigte Einschniirung der
Proamnionhéhle,” as previously quoted.

A series of figures of critical regions taken from a series of
cross-sections of an egg-cylinder of a stage nearly identical with
that shown in figure 29, though of a slightly smaller egg-cylinder,
is given in figure 30, rat No. 42, 8 days, 16 hours, after insemina-
tion. The sections chosen for the several drawings, A to D, are
from the following regions, as may be ascertained by compari-
son with figure 29; A, through about the middle of the ectoplacen-
tal cone; B, through the proamniotic cavity just below its meso-
metrial end; C, through the proamniotie cavity just above the

DEVELOPMENT OF THE ALBINO RAT 97

region of the junction of the primary embryonic and extraem-
bryonie ectoderm; D, a little above the middle of the antimeso-
metrial portion of the proamniotic cavity. The levels of the

Fig. 30 Four figures from a series of cross sections of an egg-cylinder of the
albino rat in the stage of development shown in figure 29. > 200. Rat No. 42,
8 days, 16 hours after insemination.

The levels at which the several sections drawn were taken is approximately
indicated by the several crosses found to the left of figure 29. A, middle of
ectoplacental cone; B, ectoplacental end of the proamniotic cavity; C, just above
level of junction of the primary embryonic and extraembryonic ectoderm; a
little above the middle of primary embryonic ectoderm. The want of any definite
bilateral symmetry of albino rat egg-cylinders of this stage of development is
shown by this series of sections; p.ect., parietal or transitory ectoderm; ex.ect.,
extraembryonic ectoderm, surrounding mesometrial portion of proamniotic cay-
ity; pr.emb.ect., primary embryonic ectoderm; v.ent., visceral entoderm; p.emb.
ent., primary embryonic entoderm; pr.c., proamniotic cavity.

98 G. CARL HUBER

several sections drawn in figure 30 is approximately indicated
by the several crosses found to the left of the egg cylinder drawn
in figure 29.

In A of figure 30, there may be observed a vacuolization of the
protoplasm of the more peripherally placed cells of the ecto-
placental cone, the vacuoles enclosing maternal blood cells. The
more centrally placed cells of this ectoplacental cone show a
tendency to concentric arrangement. Figures B and C present
structural appearances nearly identical. The egg-cylinder is
bounded by the thin layer of parietal or transitory ectoderm having
scattered masses of nucleated protoplasm on its inner surface.
This membrane of apparently homogeneous structure stains
sharply in well fixed preparations and may be readily discerned.
The cells of the visceral entoderm, somewhat taller in the section
taken nearer the antimesometrial pole (C), present clearly the
three zones to which attention has been drawn. The cells of
the extraembryonic ectoderm bounding the mesometrial portion
of the proamniotic cavity, are of cubic, short columnar, or poly-
hedral form disposed in single or double layer, presenting relative-
ly lightly staining protoplasm. In D of figure 30, the cells form-
ing the primary embryonic ectoderm are of distinct columnar
shape, with relatively deeply staining protoplasm and nuclei
arranged nearly in a single layer except for such as show mitotic
phases. The cells of the primary embryonic entoderm are of
a broad, pavement type for a greater part of the circumference,
and may be contrasted with the cells of the visceral entoderm
shown in B and C of the figure; the latter are absorptive cells, the
former not. This series of figures, more especially B, C, and D,
show clearly the absence of bilaterality in the egg-cylinders of
the albino rat at this stage of development. The slight com-
pression observed in this egg-cylinder, as shown in the figures,
I regard as not of moment.

Fig. 31 Longitudinal sagittal section of egg-cylinder of the albino rat show-
ing anlage of the mesoderm. X 200. Rat No. 34, 8 days, 18 hours, after insem-
ination; ect.pl., ectoplacental cone or Triger; p.ect., parietal or transitory
ectoderm; pr.emb.ect., primary embryonic ectoderm; ez.cct., extraembryonic

ectoderm; pr.emb.ent., primary embryonic entoderm; mes., mesoderm in anlage;
pr.c., proamniotie cavity; v.ent., visceral entoderm.

Vent.

pr emb.ect\_

pr emb. ent__

100 G. CARL HUBER

Grosser has figured in his figures 68 and 114, an egg-cylinder
of the albino rat which measures nearly 2 mm. in length. The
age of this is given as 83 days. So far as may be determined from
his figures, the preparation is not described in his text, the age,
size, form, and structure of the egg cylinder shown in figure 29
and Grosser’s figures 68 and 114, are very similar. In Grosser’s
figures, I see no evidence of his having differentiated between
primary embryonic and extraembryonic ectoderm, while the
reference letters for ectoderm and entoderm are reversed. Selen-
ka’s figure 31, plate 45, may be of a similar stage. This figure is,
however, too diagrammatic to admit of close study. No differ-
ence is shown in the shape and structure of the cells bounding
the two parts of the proamniotic cavity. Christiani’s figure
39 may be of the same stage, but is too schematically drawn.
Figure 4 of the article of Widakowich is of a slightly older stage
and presents only a part of the egg-cylinder; it is recorded as
about 63 days old. The stage under consideration is not figured
by Widakowich, although his text description corresponds closely
with what has been here presented.

The next stage and the one with which this communication
is to be completed is one of importance since it is characterized
by the anlage of the mesoderm. My own observations may be
introduced with the consideration of an egg-cylinder, a section
of which is presented in figure 31, rat No. 34, 8 days, 17 hours,
after insemination. This was cut in the sagittal plane and
measures 1.1 mm. by 0.2 mm., of which 0.4 mm. fall to the
ectoplacental cone. This egg-cylinder is almost an exact dupli-
cate, both in size and form, of that figured in figure 29 of the
same age. In the egg-cylinder shown in figure 31, however,
there may be observed, to one side, in the region of the junction
of the primary embryonic and extraembryonic ectoderm, and
between primary embryonic ectoderm and entoderm, a small
group of cells which lie in close relation to the ectoderm and
constitute early mesodermal cells. The sections of this series
pass not exactly parallel to the mid-sagittal plane throughout the
whole extent of the egg-cylinder; especially is this true of its an-
timesometrial portion, in the region of the primary embryonic

DEVELOPMENT OF THE ALBINO RAT 101

ectoderm. This portion in the section figured, passes a little
to one side of the mid-sagittal plane. The two sections preced-
ing the one figured enclose the mid-sagittal plane, and in them,
the group of cells found between primary embryonic ectoderm
and entoderm are in closer relation to the ectodermal layer and
at all points distinctly separated from the entoderm. They
are regarded as having wandered from the primary embryonic
ectoderm to the place they occupy, a fact which is more easily
ascertained in cross sections of a similar stage, as will appear
from further discussion. From a study of very slightly older
stages it can be determined that this region constitutes the
primitive streak region of the future embryonic area. It is not
my purpose at this time and in this communication to give es-
pecial consideration to the much discussed question of the
origin of the mesoderm in Mammalia. In the rat, this question
is complicated by the question of the anlage of the amniotic
fold, which separates the proamniotic cavity into amniotic
cavity proper and the ectoplacental cavity, the development
of which will be considered in a projected contribution. In
anticipation of this second publication, however, the following
facts may here receive consideration. Widakowich presents
in his figure 4, giving only the antimesometrial end of an egg-
cylinder obtained the latter part of the 7th day, the anlage of the
mesoderm as observed by him. This figure and my own figure
31 present almost identical relations, his figure showing only
three mesodermal cells between primary embryonic ectoderm
and entoderm. His own words concerning the anlage of the
mesoderm in the albino rat, with which I find myself in full
accord, except as to the age of the egg-cylinder, read as follows:

Das erste auftreten des Mesoderms beobachtete ich an Keimen
vom Ende des 7 Tages. Die ersten Mesodermzellen liegen im Bereiche
der vom mesometralen Ende des stiirker fiirbbaren primiren embryo-
nalen Eetoderm gebildeten Falte. Es kommt hier eine ganz bestimmte

Stelle in Betracht, die dort liegt, wo sich spiter das hintere Ende des
Primitivstreifens befindet.

There is, however, wide divergence of the views of authors
as concerns the anlage of the mesoderm in the rat and mouse.

102 G. CARL HUBER

Selenka, it would seem, in part at least, interpreted correctly
the development of the mesoderm in the rat, although a stage
showing its anlage was not observed. Duval believes that the
mesoderm has origin from a thickened part of the entoderm,
probably in the region of the anterior portion of the future em-
bryonic area; the primitive streak was not recognized. Christi-
ani’s figures 45 and 47, transverse sections of the egg-cylinder
from the eighth day, give correctly the relative position of the
mesoderm with reference to the primitive streak; however, they
show stages some little time after the anlage of the mesoderm.
According to Robinson, in the early part of the eighth day the
cavities of the epiblast (primary embryonic ectoderm) and of
the trophoblast (extraembryonic ectoderm) meet and fuse to
form a hollow cylinder, the proamniotic cavity. He states
that ‘‘For a time the united cavities of the epiblast and tropho-
blast increase in size, together with the general growth of the
ovum, and this increase continues until in the latter part of the
eighth day the mesoblast appears around the margin of the
epiblast where it is in apposition with the trophoblast.’’ Robin-
son was able to differentiate between the primary embryonic
ectoderm (epiblast) and the extraembryonic ectoderm (tropho-
blast) and his figure 14 (plate 23-24), though schematic, shows
that he recognized the positions of the anlage of the mesoderm
correctly, as also its derivation from the primary embryonic
ectoderm. The observations of Melissinos, bearing on the an-
lage of the mesoderm have been critically reviewed by both
Widakowich and Sobotta, and I am wholly in accord with their
views when they state that no credence can be given these ob-
servations since it is clear that Melissinos has confused sagittal
and frontal sections in such a way as to make his observations
of no value. According to Melissinos, the mesoderm arises
from the outer surface of the middle fold of the egg cylinder,
in the region of its union with the antimesometrial ectodermal
fold; it is certain that it does not arise from the part of the egg-
cylinder that has differentiated from the primary embryonic
ectoderm; but, if I interpret him correctly, from the extra-
embryonic portion of the ectoderm. That Melissinos did not

DEVELOPMENT OF THE ALBINO RAT 103

have before him the stages showing the anlage of the mesoderm
seems clear. Sobotta’s (11) observations, mouse material,
deserve fuller consideration. In interpreting his results, I am
mindful of the fact that he was unable to locate the line of union
between primary embryonic and extraembryonic ectoderm, as
can readily be done in suitable rat material, as has previously
been shown by Robinson and Widakowich, and to which atten-
tion has constantly been drawn in this communication. I am
unable to state from personal observation whether in the white
mouse these two types of ectoderm which form the lining of the
proamniotic cavity, can be differentiated on ascertaining the
right technical method. Sobotta’s material seems well fixed.
If not, it would seem to me difficult to determine definitely
the exact place of origin of the mesodermal cells, whether extra-
embryonic or embryonic. Sobotta recognized the anlage of
the mesoderm in the mouse during the last hours of the seventh
day or first hours of the eighth day. This is said to appear at
the caudal end of the future embryo as a group of loosely ar-
ranged cells lying between the inner and outer layers of the
ege-cylinder. At the place where the mesodermal cells arise
from the inner layer of the egg-cylinder, there is developed a
fold, recognized as the caudal amniotic fold (‘“‘Schwanzfalte
des Amnios’’). After discussing these observations at length,
Sobotta concludes as follows:

Was die Deutung dieser friihen Stadien der Mesodermbildung in
der Keimblase der Maus anlangt, so handelt es sich hier nicht um die
Bildung des embryonalen Mesoderms, die erst mit der eigentlichen
Gastrulation spéter emsetzt, sondern um Entstehung ausserembryo-
nalen Mesoderms, besonder des Teils des mittleren Keimblattes, dass
bei der Bildung der primiren Eihaiute, Amnios und Chorion in Betracht
kommt und des den ausserembryonalen Teil der Leibeshdhle, das
Exocoelom auskliedet, der Héhle, die eben Amnios und Chorion von-

einander trennt. Es erfolgt also, um einen kurzen Ausdruck zu gebrau-
chen, die Bildung des Amniosmesoderms.

An embryonic anlage is said not to exist at this stage; this is
recognized only after the development of the primitive streak.
It is not my purpose to enter fully into a discussion of this im-
portant question in this communication. This would involve

104 G. CARL HUBER

consideration of older stages, and the making of a number of
reconstructions, which it is not contemplated to consider now.
It must suffice to state at this time that in the albino rat, as
shown by Widakowich and here shown by me, it is possible to
delineate clearly the primary embryonic ectoderm and to show
that the first evidence of the mesoderm is found antimesometrial
to the future amniotic fold and in the region of the future primi-
tive streak; therefore is mesoderm which I would regard as
peristomal mesoderm in the sense of C. Rabl, reference to which
is made by Sobotta in his discussion of this question. It may
be that the rat offers more suitable material for the elucidation
of this question than is to be found in the mouse. In the albino
rat, the anlage of the mescderm is from the sagittal portion of
the caudal region of the primary embryonic ectoderm, the caudal
part of the future primitive streak and antimesometrial to the
amniotic fold. Sobotta gives very favorable consideration to
the observations of Widakowich, touching this question, which
he regards as ‘‘Bei weitem die beste Darstellung des Gegenstan-
des.’ My own observations fully confirm those of Widakowich.
These questions will receive fuller consideration in a later pub-
lication dealing with the embryology of the albino rat, carrying
the development from the time of the anlage of the amniotic fold
to the stage of embryo form, the material for which is at hand.
In figure 32 are shown cross-sections of the antimesometrial
portion of three egg-cylinders in the region of the developing
mesoderm. Sections drawn in A and B, were taken respectively
from egg-cylinders cbtained from the same uterus as was the
one shown in sagittal section in figure 31, rat No. 34, 8 days, 17
hours, after insemination; C, from rat No. 41, 8 days, 16 hours,
after insemination. It is very probable that the series from
which A of this figure was drawn, is net cut in exactly the cross
plane. A study of the series shows, however, that the deviation
from this plane is not marked. The sections from which this
figure was drawn pass a little below (antimesometrial) to the
region of junction of the primary embryonic and extraembry-
onic ectoderm. To one side, the lower in the figure, the primary
embryonic ectoderm shows a slight thickening and evidence of

DEVELOPMENT OF THE ALBINO RAT 105

pr.emb.cct.

Fig. 32. Three cross sections from egg-cylinders of the albino rat, show-
ing early stages in the development of the mesoderm. X 200. A and B, rat
No. 34, 8 days, 17 hours; C, rat No. 41, 8 days, 16 hours, after insemination.

These sections taken from three egg-cylinders are through the primary embry-
onic ectoderm, near its junction with the extraembryonic ectoderm, thus through
the antimesometrial portion of the proamniotic cavity. A, early stage, in
anlage of the mesoderm; B, anlage of the primitive streak and groove; C, well
developed primitive streak and groove, with lateral wings of mesoderm; pr.emb.
ect., primary embryonic ectoderm; pr.emb.ent., primary embryonic entoderm;
mes., mesoderm; pr.str., primitive streak; pr.gr., primitive groove; p.ect., parie-
tal or transitory ectoderm; pr.c., proanmiotic cavity.

cell proliferation. The cells of this region have not the form
of tall columnar cells, such as seen in the greater part of the
remaining primary embryonic ectoderm, but are of polyhedral

106 G. CARL HUBER

form and are continuous, in the mid sagittal plane, with cells
that have wandered between the primary embryonic ectoderm
and entoderm, cells regarded as constituting the mesoderm.
In all of the sections of this series, so far as the mesoderm ex-
tends, this is distinctly separable from the entoderm, and is
continuous with the primary embryonic ectoderm only along a
narrow region of thickened primary embrycnic ectoderm, situ-
ated in the mid-sagittal plane, and which may in this series be
regarded as the anlage of the primitive streak. From the sides
of this region of slightly thickened primary embryonic ectoderm,
the extent of which is evidenced by the absence of an external
limiting membrane, cells wander laterally to form the mesoderm.
B, of figure 32, presents essentially the same appearance, although
representing a slightly older stage. The sections of this series
I regard as cut fairly well in a plane at right angles to the long
axis of the respective egg-cylinder. The section taken for the
sketch is. situated a very little further away from the line of
junction of the primary embryonic and extraembryonic ecto-
derm, than is the section the drawing of which is shown in A
of this figure, as may be judged from the more uniformly pave-
ment type of the entodermal cells. The triangular form of the
proamniotic cavity is regarded as normal, and as indicating an
early stage in the anlage of the primitive groove. In this figure,
in its lower portion, the region of the primitive streak is readily
discernible by reason of the fact that there is wanting here an
external limiting membrane, and further by reason of the form
of the cells and the form and relative position of their nuclei;
certain of these cells indicating, both by their form and their
position, the source and the direction of the wandering of the
cells which constitute the anlage of the mesoderm. The wan-
dering of the mesodermal cells between the primary embryonic
ectoderm and entoderm, to form the lateral mesodermal wings,
is clearly shown in this figure, especially to the left. The anti-
mesometrial ends of the egg-cylinders, sections of which are
shown in A and B of this figure, are as yet free from the invading
mesoderm, as is also the part of the egg-cylinders lying opposite
the region of the primitive streak, the upper portions of the

DEVELOPMENT OF THE ALBINO RAT 107

respective figures, these forming the region of the future anterior
ends of the respective embryos. In © of figure 32 is shown a
drawing of one of the sections of a series of cross-sections of an
egg-cylinder taken from rat No. 41, 8 days, 16 hours, after in-
semination, presenting a stage in which the primitive groove
may be definitely made out. This figure is not unlike figure 6
of the article of Widakowich, obtained from an egg-cylinder
secured on the eighth day. Concerning this figure he states:
“Das Eectoderm steht in direktem Zusammenhange mit zwei
Mesodermzungen die gegen die der Primitivrinne gegeniiberlie-
gende Seite zu auswachsen.” The section drawn in C© of this
figure is taken from the region very near the junction of the
primary embryonic and the extraembryonic ectoderm, as may
be observed from the character of the entodermal cells, in the
lower part of the figure. The increase in the thickness of the
mesodermal wings, the result, in part at least, of proliferation
of mesodermal cells, as evidenced by the presence of mitotic
figures, is clearly shown in this figure. The mesoderm is dis-
tinctly separable from the entoderm as also from the primary
embryonic ectoderm except in the region of the primitive streak
and groove. The growth of the mesoderm after its anlage has
been correctly shown for the albino rat by Selenka, Robinson,
and Widakowich; the latter especially giving excellent figures.
His figure 5 is especially instructive. In this, he represents the
appearances shown by two views of an isolated egg-cylinder, with
the primitive groove in anlage, showing the lateral extensions
of the mesoderm. Sobotta (11) has given the best and most
comprehensive account of the anlage and growth of the meso-
derm in the mouse. An excellent cross-section of a mouse egg-
cylinder in the primitive streak stage is presented in his figure
6, which presents very similar appearances to my C of figure 32.
None of the figures of cross-sections of egg-cylinders included
by me show the very beginning of the anlage of the mesoderm,
though A of figure 32 approaches this very closely, as does also
figure 31, presenting a sagittal section. The evidence at hand
warrants the conclusion that in the albino rat, the mesoderm
has its anlage in the caudal region of the primary embryonic

108 G. CARL HUBER

ectoderm, from a narrow zone of cells situated in the region of
the future primitive streak. From this region there is an out-
wandering of cells which invade the potential cleft between pri-
mary embryonic ectoderm and entoderm, spreading laterally
in wing-like sheets. This I would regard as prostomial mesoderm
in the sense of C. Rabl. The anlage of the mesoderm in the
albino rat, and the early stages of its lateral extension, with the
anlage of the primitive streak and groove, falls to the latter
part of the ninth day after insemination.

Beginning with the pronuclear stage, found at the end of the
first day, 8 days are required for the completion of the process
of segmentation, blastodermic vesicle formation and the forma-
tion of the primary germ layers—ectoderm, mesoderm, and
entoderm—in all, 9 days out of a possible 21 to 23 days, the
normal gestation period of the albino rat.

CONCLUSIONS

Early stages of mammalan development may readily be
obtained from the albino rat (Mus norvegicus albinus). When
care is exercised, mating may be observed and the age of the
embryo, reckoned from the time of mating (insemination),
determined with a fair degree of accuracy. Ovulations occur
about the time of parturition and again 29 to 30 days post partem.
This latter period is more favorable for obtaining insemination
and semination, thus fertilized ova. The process of fertilization
probably takes place during the latter half of the first day after
insemination.

The pronuclear stage, a stage which extends through a period
of perhaps 12 to 15 hours, in the middle phase, is observed at
the end of the first day after insemination; the fertilized ova
having wandered about one-fourth of the length of the oviduct
by that time. Of the two pronuclei, the female pronucleus is
slightly the larger. The two pronuclei le near the center of
the ovum, are distinctly membraned, and do not fuse prior
to the formation of the first segmentation spindle.

The formation of the first segmentation spindle and the first
segmentation occur during the early part of the second day after

DEVELOPMENT OF THE ALBINO RAT 109

insemination. The resulting 2-cell stage extends for a period
of about 24 hours and is found in about the middle of the oviduct.
The first two blastomeres are equivalent cells. One of these
segments before the other, resulting in a 3-cell stage, present
for each ovum for only a relatively short period.

The 4-cell stage is observed at the end of the third day after
insemination. The ova have by this time traversed about nine-
tenths of the length of the oviduct.

The 8-cell stage is observed the latter half of the fourth day
after insemination and at the end of the fourth day the ova
pass from the oviduct to the uterus in the 12-cell to 16-cell stage.
The oolemma is lost usually in the 4-cell stage, the segmenting
ova conforming in shape to the general form of that portion of
the oviduct in which they are found.

Three successive segmentation stages, spaced at intervals
of about 18 hours, resulting in 2-, 4-, and 8-cell stages oceur dur-
ing transit through the oviduct. During the fourth segmenta-
tion the ova pass from the oviducts to the uterine horns, at the
end of the fourth day.

The mass increase of the ova during the first three segmenta-
tions 1s approximately from 0.15 c.mm. in the pronuclear stage
to 0.18 c.mm. in the 8-cell stage. The slow rate of segmentation
and the relatively small mass increase may be attributed to the
relative searcity of the embryotroph during transit through
the oviducts.

During the early hours of the fifth day after insemination, all
of the segmenting ova are found lying free in the lumen of the
uterus, spaced about as in the later stages of development, the
fifth series of segmentations having been completed by this
time, the resulting morula masses having ovoid form, measuring
approximately 80 u by 50 uw and consisting of from 24 to 32 cells.
The mechanism operative in spacing the ova in the uterine horns
has not been determined.

The early stages of blastodermic vesicle formation are observed
during the middle and latter half of the fifth day. The seg-
mentation cavity begins as a single, irregularly crescentic space,
eccentric in position, and arising between the cells of the morula.

110 G. CARL HUBER

By the end of the fifth day after insemination, all fertilized,
normal ova are found in the blastodermic vesicle stage. One
pole of each vesicle, its floor, consists of a relatively thick mass
of cells, in which there is no differentiation in layers and no
evidence of ectodermal and entodermal cells. The other pole
of each vesicle, its roof, consists of a single layer of flattened
cells, bordering the segmentation cavity.

During the sixth day, the blastodermic vesicles which still
lie free in the lumen of the uterus, increase in size, partly as a
result of extension of the roof cells, partly owing to rearrange-
ment and flattening of the cells of the floor. This portion of
the vesicle now presents the form of a concavo-convex disc,
forming about one-sixth of the vesicle wall and consisting, as a
rule, of three layers of cells, the inner of which is now differen-
tiated to form the yolk entoderm.

During the seventh day after insemination the blastodermic
vesicles become definitely oriented in a decidual crypt, the
thicker portion, its floor, being directed toward the mesometrial
border. The phenomenon of the ‘‘inversion of the germ layers’’
or ‘‘entypy of the germ layers” is initiated, the result of cell
rearrangement and cell enlargement in the germinal disc, mani-
fested as an outgrowth to form the ectoplacental cone or Trager
and an ingrowth into the vesicle, the anlage of the egg-plug or
ege-cylinder. In the egg-plug there is recognized a circum-
scribed, compact mass of cells, staining more deeply than sur-
rounding cells, which constitute the ectodermal node, the anlage
of the primary embryonic ectoderm of the future embryo. This
ectodermal node, so far as it extends into the cavity of the
blastodermic vesicle, is surrounded by yolk entoderm.

During the eighth day after insemination, the egg-cylinder
comes in definite relation with the maternal decidua and re-
celves as embryotroph maternal hemoglobin, partly through
phagocytic action of the cells of the ectoplacental cone, partly
through absorption of maternal hemoglobin by the cells of the
entoderm, initiating a period of very active growth as evidenced
by active mitosis. The egg-cylinder increases in length, and
entypy is completed. A cavity develops in the ectodermal

DEVELOPMENT OF THE ALBINO RAT 1h

node, the antimesometrial portion of the proamniotic cavity.
A little later a second cavity develops in the extraembryonic
ectoderm, the mesometrial portion of the proamniotic cavity,
the two cavities fusing by the end of the eighth day to form a
single proamniotic cavity, lined in its antimesometrial portion
by primary embryonic ectoderm, and in its mesometrial portion
by extraembryonic ectoderm, the two types of ectoderm forming
a continuous layer with the line of junction readily distinguish-
able. No evidence of bilateral symmetry is at this stage ob-
served in the egg-cylinder.

During the ninth day after insemination there is observed
the anlage and the early developmental stage of the mesoderm
and the anlage of the primitive streak and groove. The meso-
derm has its anlage in the caudal portion of the primary em-
bryonic ectoderm in the sagittal region and is of the nature of
prostomial mesoderm, extending laterally in wing-like exten-
sions between the ectoderm and entoderm.

Lt, G. CARL HUBER

LITERATURE CITED

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vergleichenden und experimentellen Entwickelungslehre der Wir-
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DEVELOPMENT OF THE ALBINO RAT EUS

Husrecut, A. A. W. 1895 Die Phylogenese des Amnios und die Bedeutung
des Trophoblast. Verh. Kon. Akad. Wetensch. Amsterdam, Ser. 2
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114 G. CARL HUBER

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der weissen Ratte. Anat. Hefte, Bd. 42.

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THE DEVELOPMENT OF THE ALBINO RAT,
MUS NORVEGICUS ALBINUS

II. ABNORMAL OVA; END OF THE FIRST TO THE END OF THE

NINTH DAY

CONTENTS

LEONG IDAHO eee eee ae OAS oltrcn cen doe dap ros Moc cfacdsneionotcadcr sco aomeabic 115
vantnembr ys iim): Wismarnelliay cram eye alee ate xs apoyenetaiiste: son's ciana ste aw cee nansperevnouere of aes 117
Degeneration of ova at the end of segmentation.....................+-.-- 120
Incomplete. or retarded Segmentation: th, fees se ance os ele oe were ots ee 121
Abnormal secmentatlonecaivainys td OLIMaulOMs se sr este sees tem cris eit rsee yet: 126
Degeneration of ova as a result of pathologic mucosa..................... 129
Imperfect development of ectodermal vesicle.....................00000ees 132
iworees-cylindersimone decidual crypt... acess: acts one. seelnee etal 138
(Ciayine NUEMONANS Sac 6 WeaRNATH SG aa ES Gemoics cae Berk alc cuee hoe PRIA ee reaena oRe I & trey 5 cl ctr cee 140
Gat HieeH OURS, CSMEELG |G eS cS cx ovoettacs h SacI CCIE Oar EEE trent AcicearS ott Aattrco rai 142
INTRODUCTION

In the course of my study of the normal development of the
albino rat, from the end of the first to the end of the ninth day
after insemination, as recorded in Part I of this series of contri-
butions, there were encountered from time to time ova which
appeared to deviate both in rate and type of development from
what, as a result of extended study, came to be regarded as the
normal developmental cycle of the albino rat. When taken col-
lectively, the number of these abnormal ova is not large, al-
though they embrace nearly all of the developmental stages
studied. When taken. singly, it may be stated that while it is
comparatively easy to record the points of deviation from the
normal, it must be admitted that the probable fate of the respec-

tive stages can only be conjectured. Nevertheless, a record of
115

116 G. CARL HUBER

the abnormal stages met with seems warranted, especially in
view of the fact that the literature is very meager in its ac-
count of early stages of mammalian ova presenting abnormal
development.

The excellent and comprehensive studies of Mall on pathologie
human ova, extending over many years, may be interpreted as
leading to the general conclusion that pathologic ova and mon-
sters ‘‘are produced from normal eggs by conditions which either
interfere with their nutrition or poison them.” There is evi-
dence to show that defective implantation, using the term in its
broadest sense so as to include relation to the embryotroph or
pabulum, is directly associated with abnormal development.
Comparative experimental teratology so successfully followed by
a number of European and American experimental embryolo-
gists warrants the conclusion that all of the abnormalities or
malformations observed in the human embryo may be brought
forth by the application of suitable mechanical interference or
chemical solutions. Experimental teratology possesses the very
great advantage of enabling the observer to follow the pathologic
process from step to step, admitting more readily of their inter-
pretation, than when single stages are obtained from nature.
The evidence appears to be accumulating that the primary causes
which produce pathologic ova lie not in the germ cells, but are
rather to be sought in the environs of the germ cells in the course
of their development.

I am cognizant of the fact that the interpretation of the chance
findings of abnormal stages of mammalian ova is much more
difficult than of abnormal ova produced experimentally. The
fact, however, that nearly all of the abnormal ova observed by
me in my albino rat material were found in tubes and uteri con-
taining normal ova also, tubes and uteri which so far as observ-
able appear in most instances to be normal, and the further fact
that certain of the abnormal ova are of stages prior to what may
be regarded as showing implantation, stages concerning which
we possess no data as far as human ova are concerned, has lead
to the tentative conclusion that certain of the abnormal ova
may be the resultant of abnormal germ cells, perhaps of an
abnormality which may not show a structural expression.

PATHOLOGIC OVA, ALBINO RAT a

It is my primary purpose to make records of the abnormal ova
observed in the material at hand; and to follow these records with
a brief consideration of the observations made. There is no
literature dealing with the problem immediately at hand—abnor-
mal rat ova. It is not my purpose at this time to enter into the
extensive literature of comparative experimental teratology.
This has been critically summarized relatively recently by O.
and R. Hertwig, and by Mall, in his several contributions deal-
ing with human pathologie ova.

HALF EMBRYOS IN MAMMALIA

The first preparation to which attention is called is one taken
from the oviduct of rat No. 60, 1 day, 18 hours, after insemina-
tion. The two oviducts of this rat contained seven ova in the 2-
cell stage, to one of which especial attention was drawn in Part I
(page 271). As there recorded, in one of the 2-cell stages, the
first two blastomeres were separated by an appreciable distance.
There is loss of oolemma. The possibility of half embryos in
Mammalia was suggested. The preparation under consideration
is figured in figure 1, A and B. In A of this figure there is pre-
sented a portion of the wall of the oviduct, its epithelial lining
and the immediately adjacent mucosa, including the fourth of a
series of six sections (10 «) passing through the two blastomeres.
In this region, the cilia of the epithelium are clearly observable,
as may be seen from the figure. In B of this figure there are
sketched in approximately relative position the several sections
of the series passing through the two blastomeres, the relative
position of which, with reference to the walls of the tube, is shown
in A of the figure. The six drawings were made from a well
ribboned series; the slide was moved from section to section by
means of a mechanical stage, and the perpendicular indicated on
each drawing as made. The relative position of the several
drawings, therefore, is quite correct. It may be observed that
throughout the series the two blastomeres are separated by an
appreciable space, and that one of the cells has rotated slightly
on its axis. If these two blastomeres had remained in close appo-
sition, they would present the appearance of a normal 2-cell stage

118 G. CARL HUBER

A

Fig 1 Oviduct and ovum of albino rat, in 2-cell stage, with first two blasto-
meres separated. Rat No. 60, 1 day, 18 hours, after the beginning of insemina-
tion. X 200. A, epithelial wall of oviduct with adjacent mucosa, and the fourth
of a series of six sections of the 2-cell stage with separated blastomeres, showing
them in their relation to the epithelium. B, the series of six sections which
pass through the separated blastomeres, the fourth of which is shown in A.
The series reads from right to left.

as shown in B and © of figure 1, Part I. There is here clearly
a separation of the first two blastomeres and not a close approxi-
mation of two unfertilized ova. In all of the unfertilized ova
met with in the oviducts in the series at my disposal, these pre-
sent the second maturation spindle and oolemma and are not to
be confused with the blastomeres of the 2-cell stage, either as
to size or structure. Both of the blastomeres in the prepara-
tion under consideration present normal protoplasmic structure,
having a finely granular protoplasm. Their nuclei, as may be
seen from the figures, are of normal size and structure. They pre-
sent regular form, are distinctly membranated, have large chro-
matoid nucleoli, and chromatin seattered in fine granules and
threads. However, attention needs to be drawn to the presence
of two micro-nuclei, one in each of the two blastomeres, showing
in the third and fourth section of the series respectively (B,
fig. 1). These micro-nuclei are nearly free from chromatin,
each presenting a small chromatoid nucleolus. They are not to
be regarded as cell inclusions, as perhaps representing phagocytic
leucocytes. It may be conjectured that they were formed by
amitotic division, by budding and constriction from the parent

PATHOLOGIC OVA, ALBINO RAT 119

nuclei, perhaps indicating altered metabolism in the two blasto-
meres. I am inclined to think that both of these cells would
have degenerated in the course of further development; however,
their fate can only be guessed and not predicted. The possibility
of their developing into half embryos is suggested. Half embryos
developing as a result of a separation of the first two blastomeres
has not been observed in the Mammalia, and an experimental
test of the question is for the present not a probability.

As a result of experimental embryology it has been clearly
shown that through mechanical interference polysomatous mon-
sters may be produced from normal ova. The first two blasto-
meres are totipotent as expressed by Driesch. Driesch was
able to produce polysomatous forms by mechanical separation
of the first two blastomeres in sea urchin eggs; Wilson, by sepa-
rating through shaking of 2- and 4-cell stages in Amphioxus; O.
Hertwig, Herlitzka and Spemann, by separating the first two
cells in amphibian eggs; O. Schultze and others, by use of gravity
and compression; and Loeb and others by use of chemical agents.
By various means, then, when suitably applied and at the right
time, hemiembryos have been produced by separating or poten-
tially separating the first two blastomeres in certain forms. O.
Hertwig states:

Bei den kleinen, mit geringen Mengen von Dotter ausgestatteten
Kiern der Wirbeltiere sind spontan entstandene, das heisst, ohne ex-
perimentelle Eingriffe veranlasste Mehrfachbildungen ausserordentlich
selten, bei manchen Klassen tiberhaupt noch nie beobachtet worden,
dagegen sind sie relativ hiufige Befunde bei manchen untersuchten

Arten von Knochenfischen und V6geln, besonders bei der Forelle und
beim Hiihnechen.

So far as I am aware, the possibility of hemiembryos in Mam-
malia has not been shown. In the albino rat, the oolemma
may be lost as early as the 2-cell stage. In forms with early
loss of oolemma, the separation of the two first blastomeres does
not appear to me as an impossibility. The probable fate of sepa-
rated mammalian blastomeres can only be conjectured, since it
is manifestly impossible, for the present, to follow them in
further development.

120 G. CARL HUBER

DEGENERATION AND DEATH OF OVA AT THE END OF
THE SEGMENTATION STAGES

In figure 2, A and B, are presented drawings of typical sec-
tions of two morula masses showing complete degeneration and
death. The degenerated ovum shown in A, of this figure was
obtained from rat No. 52, 4 days, 15 hours, after insemination.
In all, eight normal ova were found in the uterus of this rat,

Fig 2 Ova of the albino rat in late segmentation stages, showing death and
dissolution of the constituent cells. X 200. A, rat No. 52, 4 days, 15 hours,
after the beginning of insemination. B, rat No. 68, 4 days, 16 hours, after the
beginning of the insemination. This figure shows an imperfectly developed
morula with probable retention of oolemma.

these showing late morula stages and stages of early blastodermie
vesicle formation, three of which were sketched and are shown in
A, B, and C of figure 20, Part I. The degenerated ovum here
under consideration lies in very close proximity to the normal
blastodermic vesicle shown in C of figure 20, Part I. The
shallow mucosal pits harboring the two ova are in contiguity.
The two contiguous pits resemble each other very much; the
mucosa underlying them is in every respect the same, indicating,
it would seem, that to a certain stage in development—to the
end of segmentation—the development of the degenerated ovum
proceeded normally. The degenerated egg-mass measured ap-
proximately 80 u by 50 u by 40 uw. In reaction to stains, it dif-
fers markedly from the adjacent normal vesicle. The staining
is very pale; cell boundaries are indistinct or lost, and the nuclei
searcely retain any coloring matter. Scattered through the pro-
toplasm are found small globular masses, perhaps of lipoid
character. Protoplasm and nuclei present evidences of cytolysis
and chromatolysis, and have the appearance presented by ne-
crotic tissue. Had normal development supervened, both ova

PATHOLOGIC OVA, ALBINO RAT 12]

(the pathologie and the adjacent normal one) would in all proba-
bility have been enclosed within the same decidual crypt, a con-
dition exceedingly rare, judging from the material at hand.
Whether the very close proximity of these two ova bears causal
relation to the death of one, by reason of the consequent lessen-
ing of the available pabulum or embryotroph, can only be con-
jectured. There is at this stage no question of faulty implanta-
tion, the ova, though presumably permanently lodged, lie free
in the lumen of the uterus. Whether on the other hand, the
death of this ovum was the result of some inherent nutritional
deficit must also remain unanswered. However, this prepara-
tion may serve to show that ova of the albino rat, after reaching
the uterine tube, and after apparently normal segmentation,
may undergo death and dissolution, for reasons which are not
structurally discernable.

B of figure 2, rat No. 68, 4 days, 16 hours, after insemination,
is from the uterus of a rat containing four ova in early stages of
blastodermic vesicle formation, three of which were sketched
under D and E of figure 20, and the series of figure 21, Part I.
The preparation here described lies free in the lumen of the
uterus, and appears to represent an uncompleted segmentation,
with cells and nuclei showing cytolysis and chromatolysis. The
mass is surrounded by a thin membrane regarded as an oolemma.
Normally the oolemma of the segmenting ova of the albino rat is
lost in the 4-cell stage, now and again in the 2-cell stage. Whether
the retention of the oolemma may be brought in causal relation
to the death and dissolution of the enclosed cells is problematic.
That such causal relation may exist for the ova of the albino rat,
appears to me as not impossible. This degenerated egg-mass
presents the only instance of the late retention of the oolemma
in the albino rat material at my disposal.

INCOMPLETE OR RETARDED SEGMENTATION

The blastodermic vesicles presented in figures 3 and 4 have
been interpreted as showing incomplete or retarded division of
certain of the cells of early stage morula masses. The probable
fate of such blastodermic vesicles in further development cannot

22 G. CARL HUBER

be projected with any degree of certainty. The most charac-
teristic vesicle showing this phenomenon is presented in figure 3,
and is taken from rat No 58, 5 days after insemination, the
uterus of which contained seven blastodermic vesicles showing
early stages of development, four of which are reproduced in
figure 22, Part I. In A and B of figure 3 are reproduced two
consecutive sections of a series of five sections of 10 » thickness,
includ ng this ovum. In the lower part of this ovum there is
found a small segmentation cavity, bounded by cells which
present normal appearances. The roof of this vesicle is slightly

Fig. 8 Early stages of the blastodermic vesicle of the albino rat, presenting
evidence of irregular or retarded segmentation. 200. Rat No. 53, 5 days after
the beginning of insemination.

Fig. 4 Three ova of the albino rat, showing early blastodermic vesicle stages,
in each of which certain of the cells suggest irregular or retarded segmentation.
« 200. A, rat No. 64, 4 days, 14 hours, after the beginning of insemination. B,
rat No. 68, 4 days, 15 hours, after the beginning of insemination. C, rat No.
54, 6 days, 16 hours, after the beginning of insemination.

folded and compressed, as a consequence of which the roof wall
in the sections figured is presented in part as seen in surface
view. In the floor of this vesicle there is to be observed, sur-
rounded by other smaller cells, one large cell, of nearly spherical
shape, having a diameter which is three or four times as great
as that of the majority of the surrounding cells. The protoplasm
of this large cell stains less deeply than does that of the majority
of the other cells constituting the floor of the vesicle. Its nu-
cleus is relatively large and slightly lobulated, so much so that in
the section of it shown in A of this figure, in the optical section
sketched, the nucleus appears as three separate nuclei, in reality,

PATHOLOGIC OVA, ALBINO RAT 123

lobules of the same nucleus. In A of this figure there is shown
to the lower left of the large cell another relatively large cell,
enclosing a globular inclusion, which stained faintly, and the
nature of which was not fully determined. In the upper part of
each of the two figures are seen cells which show cytolysis and
loss of nuclei; regarded as degenerating cells. When compared
with the normal blastodermic vesicles obtained from the same
uterus, the ovum here described presents a unique appearance,
and was readily recognized as showing development and structure
which deviated from the normal. At this stage of development,
the blastodermic vesicles of the albino rat are still found lying
free in the lumen of the uterus, showing no structural relation to
the uterine mucosa. This vesicle has been interpreted as show-
ing irregular or retarded segmentation. It is conjectured that
one of the cells, perhaps of the 8-cell stage, did not undergo
further cleavage. The large cell presents an appearance evidenc-
ing beginning stages of degeneration, and in further development,
would probably have undergone dissolution. The majority of
the smaller cells of the roof appear as if normal, as do also the
cells of the floor, certain of the smaller cells of the floor presenting
mitoses as evidence of further proliferation.

In figure 4, A, B, and C, there are presented typical sections
of three ova of the albino rat showing what has been regarded as
irregular segmentation. A of this figure represents an ovum
taken from rat No. 64, 4 days, 14 hours, after insemination, in
the uterus of which there were found five normal ova showing
early stages of blastodermic vesicle formation, four of which are
cut longitudinally, one in a series of cross-sections. In each of the
four longitudinally cut series the floor of the respective vesicles
is markedly folded, owing to fixation contractions; therefore,
fione were sketched as normal stages. In appearance, they re-
semble closely the vesicles sketched under C, D, and E of figure
20, Part I. In the pathologic ovum, shown in A of figure 4,
there is no evidence of segmentation cavity formation. How-
ever, the ovum cannot be regarded as presenting a late morula
stage such as is figured in A of figure 20, Part I, since it shows
distinct departure from the normal. The marked constriction

124 G. CARL HUBER

seen to the lower left of the figure passes through the series of
four 10 u sections including this ovum, and in part separates a
portion composed of relatively small cells from a larger portion
composed of larger cells. The rate of segmentation of certain
of the cells composing the upper larger portion of this cell mass
appears to have been retarded, thus retarding the development
of the whole mass. This pathologic ovum rests normally in a
shallow pit of the mucosa, very similar in form and structure to
the shallow pit lodging the five normal vesicles found in this
uterus.

The ovum shown in B of figure 4 was obtained from the uterus
of rat No. 68, 4 days, 16 hours, after insemination. with four
normal vesicles showing early stages of blastodermic vesicle for-
mation. From this uterus was also taken the completely de-
generated cell mass with persistent oolemma shown in B of figure
2. This vesicle on superficial observation does not appear to
depart markedly from the normal appearance for this stage. In
form and size it corresponds closely to the normal ova taken
from this uterus. The segmentation cavity seems to have de-
veloped normally. The slight folding of the roof seen to the left
of the figure is accidental, due to fixation shrinkage, and is
very similar to folding of the roof to be observed in many of the
normal preparations of the series. In the floor of the vesicle
there may be observed three relatively large cells, partly enclosed
by smaller cells of a size comparable to that of the cells forming
the floor of the normal blastodermie vesicles of this stage of de-
velopment. The three relatively large cells, clearly distinguished
in the figure, are interpreted as showing a retarded segmenta-
tion. So far as may be determined, their protoplasm and nuclei
present normal structure, the lowest of the three cells showing
an early mitotic phase. I am inclined to the opinion that this
ovum would have continued in development, perhaps in later
stages showing distinct arrest in development. This hypothesis
seems warranted on the basis of the study of a vesicle shown in
C of figure 4, taken from rat No. 54, 6 days, 16 hours, after in-
semination. Normal stages for the albino rat, taken about the
middle of the seventh day after insemination, are shown in figure

PATHOLOGIC OVA, ALBINO RAT 125

24, Part I. Reference to this figure may serve to show that dur-
ing the early hours of the seventh day after insemination, the
phenomenon of inversion or entypy of the germ layers is initiated
in the albino rat. The ova are, on reaching this stage of develop-
ment, enclosed within a well differentiated decidual erypt which
communicates as yet freely with the lumen of theuterus. These
erypts present a continuous lining of uterine epithelium; the con-
tained ova are thus not as yet in direct relation with the ma-
ternal decidua. In the normal blastodermic vesicle of this stage,
the ectoplacental cone is in anlage, and in the cell mass which
extends into the cavity of the vesicle—the egg-plug or egg-cylin-
der—there is evident a clearly circumscribed nodule of cells,
which has been designated the ectodermal node and recognized
as the anlage of the primary embryonic ectoderm; this node is in
part surrounded by the yolk entoderm. In the uterus of rat No.
54, there are contained nine blastodermie vesicles, one of which is
sketched in C of figure 24, Part I. Not nearly all of these ves-
icles are so favorably cut as that shown in this figure, the ma-
jority being cut in a plane which is oblique to the long axis of
the vesicle. However, in all of them the ectoplacental cone and
the ectodermal node may be determined except in the one shown
in C of figure 4. This vesicle was obtained from a series of sec-
tions passing at right angles to the plane of the mesometrium.
It lies free in a deep decidual crypt and passes through six sec-
tions of 10 » thickness; thus is compressed from side to side.
This vesicle is distinctly smaller than the normal ones taken
from this series, especially so as concerns its cavity. An ecto-
placental cone is not clearly differentiated, and it is not possible
to determine an ectodermal node, nor is it clear that the yolk
entoderm has differentiated. In the cell mass from which ecto-
placental cone and ectodermal node should have developed, the
upper portion of this figure, there are evident, in the sections
figured, four relatively large cells with relatively large nuclei,
cells which have been interpreted as evidencing retarded seg-
mentation with consequent retardation in the normal differen-
tiation of the vesicle. On tracing this vesicle through the series
of six sections it would seem that the direction of section is favor-

126 G. CARL HUBER

able. The uterine mucosa appears to have reacted normally;
the decidual crypt in which this vesicle is lodged presenting nor-
mal size and form, and the surrounding decidua normal structure.
The vesicle itself is retracted from the uterine epithelium, intact
throughout the crypt, thus, does not appear to have attained the
normal adhesions observed in normal vesicles of this stage.
The four ova depicted in figures 3 and 4, appear to present a
distinctive type of abnormal development, a type which is in-
terpreted as showing retarded segmentation in certain of the
cells of the 8-cell and perhaps 16-cell stage. All are found in

Fig. 5 Four consecutive sections of the ovum of the albino rat° showing
abnormal development of the segmentation cavity X 200. Rat No. 46, 6 days,
14 hours, after insemination.

uteri containing normal stages. The appearances presented, if
correctly interpreted, speak in favor of a structural or metabolic
defect inherent in the cells themselves and not primarily depend-
ent on environment, pabulum, or embryotroph.

ABNORMAL SEGMENTATION CAVITY FORMATION

The following three ova have been grouped as showing irregu-
larity in the formation of the segmentation cavity.

In figure 5 are reproduced four consecutive sections passing
through an abnormal ovum obtained from rat No. 46, 6 days, 14
hours, after insemination. There were obtained from the uterus

PATHOLOGIC OVA, ALBINO RAT 127

of this rat ten blastodermic vesicles, two of which are repro-
duced in A and B of figure 24, Part I, as showing typically early
stages of the anlage of the ectoplacental cone and entypy of the
germ layers. The ovum shown in figure 5 is found in a decidual
erypt which is in very close proximity to the one containing the
vesicle figured under B of figure 24, Part I, the two erypts being sep-
arated by a distance of approximately 1.3 mm., while the distance
between decidual crypts is normally 1 em. to 1.5 em. The de-
cidual crypt lodging the abnormal ovum presents a normal ap-
pearance, resembling very closely in form, depth and structure

Fig. 6 Two ova of the albino rat, interpreted as evidencing retarded or
irregular formation of the segmentation cavity. > 200. <A, rat No. 90, 6 days,
17 hours, after the beginning of insemination. B, rat- No. 90, 6 days, J7 hours,
after the beginning of insemination. p.ect., parietal or transitory ectoderm;
y.ent., yolk entoderm; p.ent., parietal entoderm.

of the surrounding decidua, the erypt and decidua enclosing the
adjacent normal vesicle figured in B of figure 24, Part I. The
abnormal ovum in question appeared to have proceeded nor-
mally in segmentation, its constituent cells being of about the
size and structure of the cells of normal vesicles taken the early
part of the seventh day after insemination. The cell-mass en-
closes a relatively small cavity which may be regarded as an ab-
normally placed segmentation cavity, in that its position is not
eccentric, and that it is surrounded on all sides by more than one
layer of cells. There is thus no differentiation of floor and roof
as in normal blastodermic vesicles, and no development of ecto-
placental cone and egg-cylinder as in the other ova obtained from

128 G. CARL HUBER

this uterus. I am for the present unable to offer any plausible
explanation or give reasons for such abnormal development of the
segmentation cavity. The fate of such a structure may perhaps
be conjectured from a study of the abnormal ovum shown in A
of figure 6, interpreted as showing a similar abnormality, but ob-
tained in early stages of degeneration. This ovum and that
shown in B of the same figure was obtained from the uterus of
rat No. 90, 6 days, 17 hours, after insemination. In the uterus
of this rat there are found six ova, only one of which was de-
veloped to a stage comparable to that shown in figure 24 (Part
I) of about the same age. Three other vesicles present a slightly
younger stage and may be compared with vesicles shown in
D and E of figure 23, Part I. None of these four vesicles is
favorably cut, but so far as may be determined, are of normal
structure for the respective stages represented. <A of figure 6
is also cut slightly obliquely, not sufficiently so, however, to
make difficult its interpretation. The figure drawn is that of
the third of a series of seven sections having 10 uw thickness, and
depicts what is regarded as representing an ovum with abnormal
segmentation cavity formation. In this ovum, the segmentation
cavity is slightly more eccentric than is that shown in figure 5,
and contains a granular detritus which in the preparations is
distinctly stained with Congo red. The roof of this vesicle is
composed almost throughout of more than one layer of cells.
There is no differentiation of ectoplacental cone and ectodermal
node, nor of yolk entoderm. Two cells regarded as phagocytic
leucocytes, staining much more deeply in Congo red than do the
cells of the ovum, have, in the section figured, penetrated the
egg-mass, indicating early degenerative changes.

The vesicle shown in B of figure 6, obtained from the same rat,
is favorably cut, and is readily followed through the series. The
structural appearance presented by this vesicle is not explained by
supposing it due to very oblique plane of section of a normal
vesicle, a plane of section which might include the roof of the
vesicle while avoiding its floor. The vesicle is abnormal in that
it presents a want of development of the thickened germ disc,
and a hyperdevelopment of the yolk entoderm. In none of the

PATHOLOGIC OVA, ALBINO RAT 129

sections of the series which includes this vesicle, which is cut
in very favorable longitudinal direction, and is thus readily ori-
ented with reference to mesometrial and antimesometrial por-
tion, is there seen any thickening of the outer layer of cells, to
form the part known as the floor of the vesicle, which at this stage
of development is uniformly directed toward the mesometrial
border. In A and B of figure 23, Part I, are shown vesicles with
which the ovum here discussed may be compared. In the
preparation under discussion, the yolk and parietal entoderm
form almost a continuous layer, one of the detached cells show-
ing a mitotic phase. In the normal vesicles of this stage of de-
velopment the parietal entoderm is represented by a few scat-
tered cells, as may be observed by a study of the figures to which
reference is above made. Whether this vesicle is to be regarded
as showing a later stage of an ovum in which there was irregu-
larity in the formation of the segmentation cavity, | must for the
present, leave as problematic. It has occurred to me that by
enlargement of the segmentation cavity of an ovum such as
shown in figure 5, with centrally placed segmentation cavity,
there might result in further development the formation of a
vesicle such as shown in B of figure 6.

It is freely admitted that the deductions here made, relative
to irregularity in the formation of segmentation cavity, are not
supported by conclusive evidence. It has seemed to me, however,
that the interpretations given to the appearances presented are
less open to criticism than others that might be suggested. These
abnormal ova also suggest an inherent defect in the ova, leading
to abnormal development, rather than abnormal development
resulting from defective environment.

DEGENERATION OF OVA AS RESULT OF PATHOLOGIC
UTERINE MUCOSA
In figure 7 are reproduced two ova which seem to me to show
the primary stages of degeneration owing to pathologic condition
of the uterine mucosa. Vesicle A was taken from the uterus of
rat No. 91, 5 days, 16 hours, after insemination. In the uterus
of this rat there were found only two ova. Vesicle B was taken

130 G. CARL HUBER

from rat No. 104, 6 days after insemination. In the uterus of
this rat there were found six ova. In both of these rats, the ova
present essentially the same stage of development, comparable to
that shown in A and B of figure 23, Part I. As may be observed
from the text of Part I (page 301) the stages obtained at the
end of the sixth day and early hours of the seventh day, were
found very difficult to fix. At this stage the ovum consists of a
relatively large, thin walled vesicle, very prone to fixation shrink-
age. <All of the ova or vesicles obtained from rats Nos. 91 and
104, are very badly folded in their roof portion. Those shown

lig. 7 Two ova of the albino rat partly surrounded by maternal blood with
many phagocytic leucocytes. The folding of the roof of the vesicles is due to
fixation shrinkage. X 200. A, rat No. 91, 5 days, 16 hours, after the beginning
of insemination. B, rat No. 104, 6 days after the beginning of insemination.

in A and B, figure 7, are representative. This folding, a result
of imperfect fixation, is present in all of the vesicles of this stage,
even though the respective vesicles present normal structure.
The ova here figured may be regarded as having fairly normal
structure, both as to rate of development and as to arrange-
ment, form, and structure of constituent cells. All of the eight
vesicles obtained from these two rats (No. 91, 2 ova; No. 104, 6
ova) are in part surrounded by exudated maternal blood, con-
taining numerous leucocytes. Small masses of blood with leu-
cocytes are found here and there in different parts of the uterine
lumen of both rats, lodged in mucosal folds other than the char-
acteristic decidual crypts enclosing the respective ova. These

PATHOLOGIC OVA, ALBINO RAT 131

decidual crypts are relatively shallow when compared with those
of normal uteri of similar stages with normal ova. The uterine
mucosa of the two rats under discussion does not appear to have
reacted in a normal manner. In these preparations, attention is
especially drawn to the presence of maternal blood with numerous
phagocytic leucocytes found in relation with the ova, a condition
never observed in normal development of ova and uterine mu-
cosa. In A and B, figure 7, the red and white blood cells with
granular detritus may be observed as found in relation with the
respective vesicles, these presenting essentially the same appear-
ances as do the other six ova obtained from these two rats; the
one figured having been more favorably cut than any of the
others. The appearances presented in these two rats are inter-
preted as showing a probable degeneration of the eight ova, and
probably complete dissolution and removal. The vesicles appear
to have developed normally to the stage at which they were ob-
tained. As a result, however, of pathologic condition of the
uterine mucosa, maternal blood, especially leucocytes, have
entered the lumen of the uterus, the leucocytes being destined
to play the role of phagocytes. In normal development of the
albino rat, maternal blood does not enter the lumen of the uterus
—decidual erypts—until after the uterine epithelium has become
detached from the mucosa of the wall of the decidual crypt, in
the region of lodgment of the enclosed ovum. Normally, very
few leucocytes are met with in the lumen of the uterus, even in
later stages of development, stages in which maternal red blood
cells are met with in the decidual crypts. After experience had
accumulated, uterine tubes supposed to contain developmental
stages aging from the fourth to the sixth day, which on examina-
tion revealed blood and especially leucocytes in the lumen of the
uterus, were regarded as not favorable specimens for finding ova.
In a number of such uteri, cut completely in serial sections, no
ova were found. It is possible that, owing to phagocytic action
of the leucocytes present, the ova may have been completely
removed prior to killing and fixing the tissues. In such condition,
it would seem to me as pertinent to speak of faulty implanta-
tion, due to abnormal uterine mucosa. It seems to me signifi-

132 G. CARL HUBER

cant that in the two rats in which the pathologie condition affects
primarily the maternal tissue, the uterine mpcosa, all of the con-
tained ova are prone to degeneration. In the abnormal ova
previously described, for which it was suggested that the causes
for the abnormality were to be sought in the ova themselves,
in the great majority of instances, only one abnormal ovum was
found in each uterus along with a variable number of ova which
are to be regarded as normal for the respective stage.

IMPERFECT DEVELOPMENT OF THE ECTODERMAL VESICLE

The series contains two ova, very favorably cut, ova in which
the ectodermal vesicle with the antimesometrial portion of the
proamniotic cavity does not seem to have developed normally.
Stages showing the differentiation of the egg-cylinder, the for-
mation of the ectodermal vesicle with the antimesometrial por-
tion of the proamniotic cavity, the formation of the mesometrial
portion of the proamniotic cavity in the extraembryonic ecto-
derm, the union of the two primary proamniotic cavities to form
a single space, are clearly shown in figures 26 and 27, Part I, in
the series of closely approximated stages there portrayed. Froma
study of these figures, it will be observed that the antimesometrial
portion of the proamniotic cavity develops within the ectodermal
node before the mesometrial portion of this cavity develops in
the extraembryonic ectodermal portion of the egg-cylinder. In
the egg-cylinder shown in figure 8, rat No. 94, 8 days after the
beginning of insemination, such is not the case. In the uterus
of this rat there were found seven egg-cylinders, one of which,
very favorably cut, is shown in C, figure 27, Part I. The other
ege-cylinders obtained from this uterus, except the abnormally
developed one to be discussed, though not favorably cut, present
essentially the same form and structure as that figured under C
of the figure above referred to. The egg-cylinder portrayed in
figure 8 compares in size and form with those regarded as normal
and taken from the same uterus. For the greater part it presents
normal structure and normal relations of cells. The ectopla-
cental cone, only in part included in the figure, and the parietal

PATHOLOGIC OVA, ALBINO RAT 133

Fig. 8 Egg-cylinder of albino rat showing retarded development of ecto-
dermal node and of the formation of the antimesometrial portion of the pro-
amniotic cavity. 200. Rat No. 94, 8 days after the beginning of insemina-
tion. ect.pl., ectoplacental cone or Triiger; v.ent., visceral entoderm; met.pr.,
mesometrial portion of the proamniotic cavity; p-.ect., parietal or transitory
ectoderm; pr.emb.ent., primary embryonic entoderm; ect.n., ectodermal node;
a.met.pr., imperfectly developed antimesometrial portion of proamniotic cavity;
ex.ect., extraembryonic ectoderm.

ectoderm, in structure and relation to decidual crypt, are to be
regarded as of normal development. The visceral entoderm,
surrounding the extraembryonic ectodermal portion of the egg-
cylinder, is of normal structure, showing the three zones evidenc-

134 G. CARL HUBER

ing its absorptive function. The extraembryonic ectoderm,
enclosing the mesometrial portion of the proamniotie cavity,
presents normal structure and relations of cells. The only ab-
normality observed is in the region of the ectodermal node, the
anlage of the ectodermal vesicle with the enclosed antimesometrial
portion of the proamniotic cavity. With this stage of develop-
ment of the egg-cylinder (see figs. 26 and 27, Part I) the ectoder-
mal node presents a well formed cavity, surrounded by the cells
of the primary embryonic ectoderm, radially arranged. In the
egg-cylinder under discussion (fig. 8) there is distinctly a retar-
dation in the development of the ectodermal vesicle with full
differentiation of the primary embrycnic ectoderm. An imper-
fectly developed antimesometrial portion of the proamniotic
cavity is evident. This small cavity, indistinctly bounded, ex-
tends obliquely through several sections of the ectodermal node,
and contains amorphous granular detritus, which in the prepa-
rations is stained by Congo red. The cells destined to form the
primary embryonic ectoderm show no definite arrangement,
especially as concerns the more centrally placed cells of the
node. Since the primary embryonic ectoderm is the anlage
for the ectoderm of the embryo, an arrest in its differentiation
would of necessity profoundly affect further development of
the embryo. Antimesometrial to the ectodermal node (just
above it in the figure) there is found a small vesicle the walls of
which are not distinctly delimited and composed of extraem-
bryonic ectodermal cells, surrounding a small, completely bounded
cavity. I am not prepared to say whether this small vesicle is
to be regarded as developing from cells of the extraembryonic
ectoderm, or from a displaced, accessory ectodermal node, in
which a discrete portion of the proamniotic cavity has developed.
If the latter, the possibility of a double anlage for the embryonic
ectoderm is to be considered. My interpretation of this egg-
cylinder as showing a retardation of the development of the
ectodermal node and differentiation of the primary embryonic
ectoderm, is confirmed from a study of a slightly older stage show-
ing essentially the same condition. This ovum is presented in
figure 9, and is taken from rat No. 41, 8 days, 16 hours, after the

Fig. 9 Egg-cylinder of albino
rat, in which the antimesometrial
and mesometrial portions of the
proamniotic cavity have failed to
unite to form a single or definite
proamniotic cavity. X 200. Rat
No. 41, 8 days, 16 hours, after the
beginning of insemination. ect.pl.,
ectoplacental cone or Triger;
p.ect., parietal or transitory ecto-
derm; v.ent., visceral entoderm;
met.pr., mesometrial portion of
the proamniotic cavity; ex.ect., ex-
traembryonic ectoderm; a.met.pr.,
antimesometrial portion of the
proamniotic cavity; pr.emb.ect.,
primary embryonic ectoderm; +,
region at which, in normal de-
velopment, by the end of the
eighth and beginning of the ninth
day, the two portions of the pro-
amniotie cavity would have united
to form a single space, the definite
proamniotic cavity.

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136 G. CARL HUBER

beginning of insemination. The uterus of this rat contains
eight egg-cylinders, all of which, except the one here figured,
show normal structure, though presenting quite different stages
of development. One of these, cut serially in cross-section, is
figured in C, figure 32, Part I, as showing anlage of mesoderm
with primitive streak and groove. Two of the other egg-cylinders
show the anlage of the mesoderm, two others show late pre-
mesoderm stages of the egg-cylinder, the remaining egg-cylinders
are less fully developed, one showing a development which may
be compared to B of figure 26, Part I, thus a much younger
stage. By the end of the eighth day and with the early hours
of the ninth day after the beginning of insemination in the
albino rat, the two parts of the proamniotice cavity, which de-
velop discretely, have joined to form a single space (C, fig. 27,
Part I). The egg-cylinder shown in figure 9, presents normal
development in all parts, except that there is as yet no union of
the two parts of the proamniotic cavity. This egg-cylinder is
most favorably cut, in longitudinal direction; the plane of sec-
tion being almost parallel to the mid-sagittal plane. This egg-
cylinder, therefore, is easily followed through the several sections
of the series into which it was cut. The irregularity of outline
of the ectodermal vesicle, lower right of figure, it is believed, is
not due to fixation shrinkage. Judging from size and structural
differentiation of this egg-cylinder, union of the antimesometrial
and mesometrial portions of the proamniotie cavity should have
been completed before this stage of development was reached,
with the primary embryonic ectoderm and the extraembryonic
ectoderm forming a continuous layer, as shown in figure 29, Part I.
The folding of the wall of the antimesometrial portion of the
egg-cylinder, lower right of figure, evident in nearly all of the
sections of the series, is regarded as indicating an abnormal
growth of the primary embryonic ectodermal cells composing the
wall of the ectodermal vesicle, as a result of retarded extension
of the antimesometrial portion of the proamniotic cavity, perhaps
an adjustment to meet the altered mechanical stress resulting
from abnormal development. The condition here seen, it would
seem, is foreshadowed in the egg-cylinder shown in figure 8.

pr.emb. ect. — P eu

pr.emb, ent. — ery

Tig. 10 Two egg cylinders of the albino rat found within the same decidual
erypt, with in part common ectoplacental cone. X 150. Rat No. 87, 9 days
after the beginning of insemination. ect.pl., ectoplacental cone or Triiger;
p.ect., parietal or transitory ectoderm; v.ent., visceral entoderm; ex.ect., extra-
embryonic ectoderm; pr.c., proamniotic cavity; pr.emb.ect., primary embryonic
ectoderm; pr.emb.ent., primary embryonic entoderm; mes., mesoderm.

137

138 G. CARL HUBER

The causes operative in this retardation of development and
differentiation of the ectodermal vesicle and primary embryonic
ectoderm, | have been unable to determine. They would ap-
pear to be inherent in the egg-cylinder, since ectoplacental cone
and visceral entoderm, so far as may be determined from a study
of sections, appear to have functioned normally, in furnishing the
necessary embryotroph in the form of maternal hemoglobin, as
is normal for egg-cylinders of the albino rat of this stage of
development.

TWO EGG-CYLINDERS IN ONE DECIDUAL CRYPT

The ova portrayed in figure 10 present a condition which must be
regarded as exceedingly rare, since it represents the only instance of
this condition observed in the extended series of preparations of
the various stages of the development of the albino rat from the
end of the first to the end of the ninth day after insemination,
in my possession. This preparation is from rat No. 87, 9 days
after the beginning of insemination. The uterus of this rat con-
tained, other than the preparation here considered, six egg-
eylinders of normal development, all showing a stage which is
slightly older than that shown in figure 31, Part I, in that the
mesoderm shows further development than is shown in that
figure. In the preparation here figured there are found two
ege-cylinders enclosed within the same decidual erypt. This
figure, which is drawn by combining the drawings made from two
sections, is reproduced at a magnification of 150 diameters, while
all. of the other figures portraying sections of ova, both in Part
I and in Part [If of this communication, are reproduced at a mag-
nification of 200 diameters. This should be borne in mind when
comparing this figure with the others. In figure 10, the lower
portion of the large egg-cylinder to the level of the lower end of
the smaller one was drawn from one section, while the remainder
of the figure was drawn from the fourth following one. The
adjustment was made by overlapping in the camera lucida
drawing (x 600) the sharp mesometrial border of the primary
embryonic ectoderm of the larger egg-cylinder. Scarcely any

PATHOLOGIC OVA, ALBINO RAT 139

adjustment was found necessary, none of the right wall of the
larger egg-cylinder, and only very slightly so of its left wall.
The slight deviation from the longitudinal axis of the larger egg-
cylinder made the procedure desirable. It is thought that the
figure as presented gives correctly the size of the respective egg-
cylinders, and in all essentials, their relations; the greater part
of the figure having been drawn from one section. Both of the
egg-cylinders reveal normal structure for the stages of develop-
ment attained. The larger one is cut in the coronal plane, as is
readily determined by the distribution of the mesoderm, one side
representing a mirror picture of the other. The direction of sec-
tion in the smaller egg-cylinder, except that it is longitudinal,
is not to be determined, since before the anlage of the mesoderm,
a bilateral symmetry cannot be recognized in sections. Since
these two egg-cylinders are in all essentials of normal form and
structure, and since their structure is clearly brought out in the
figure, an extended description of them at this place seems un-
called for. For respective stages the reader is referred to Part I.
Attention may be drawn, however, to the fact that the visceral
entoderm on the contiguous surfaces of the two egg-cylinders is
less fully differentiated, and shows less absorption of the ma-
ternal hemoglobin than is seen on the exposed or free surfaces,
this, no doubt, for mechanical reasons. Further, that in the
region where the two egg-cylinders are in contact, the parietal
ectoderm of each can be traced as a distinct layer to the bases
of the respective ectoplacental cones, showing that each developed
from a separate ovum. The ectoplacental cones are for a short
distance distinct. In tracing the sections through the series the
impression is gained that the ectoplacental cone of one of the egg-
cylinders overlaps that of the other in such a way that in the
plane of the sections obtained, one seems continuous with the
other, as represented in the figure. The boundary between the
two is not distinct, and it would seem that as a result of pres-
sure, partial fusion of the two had taken place. The presence
of two egg-cylinders, enclosed within a single decidual crypt, as
shown in this figure, with one of them having much smaller size
and representing a younger stage of development, I believe is

140 G. CARL HUBER

not to be explained on the supposition of superfecundation or
superfoetation. The record for this rat does not show insemi-
nation on successive days. At The Wistar Institute, after all
of the supposedly suecessful matings of albino rats, the females
rats are caged apart from the males. The smaller egg-cylinder,
though appreciably smaller, is in stage of development separated
from the other by a time interval of perhaps less than 24 hours.
It presents a stage of development which is comparable to C
of figure 27 (8 days) and except for size, to the one figured in
figure 29 (8 days, 17 hours) of Part I. It is believed that in this
case both ova were seminated at about the same time, and pro-
ceeded through normal segmentation and that on reaching the
lumen of the uterus during the fifth day they became lodged in
close proximity in the same mucosal fold. With the development
of the decidual crypts, both became enclosed within the same
crypt, at perhaps slightly different levels. In further develop-
ment one blastodermic vesicle dominated the other and from
about the seventh day on, one developed and differentiated
more rapidly than the other. Had development continued, two
distinct embryos, with separate amniotic cavities, attached to
the same placenta, would have been formed, with one embryo
large and more fully developed than the other. From mere
difference in size and of development of embryos in the same
litter it is not warranted to postulate superfecundation nor super-
foetation. I am of the opinion that usually when two morula
masses are lodged in close proximity in the same mucosal fold,
one or the other degenerates (fig. 2, A) and that the normal
development of both, as in the preparation shown in figure 10,
is of very rare occurrence.

CONCLUSIONS

A study of the abnormal or pathologic ova met with in the ex-
tended series of preparations covering the first ten days of the
development of the albino rat, enables grouping them in two
main classes:

a. Such in which all of the ova of a given rat show, or are
associated with, abnormal development.

PATHOLOGIC OVA, ALBINO RAT 14]

b. Such in which a single abnormal or pathologie ovum is
found in the same uterus along with an average number of
normally developed ova.

When all the ova in a given uterus show abnormality, the
presumption seems warranted that the underlying cause of the
abnormality is to be sought in an altered or pathologic condition
of the uterine mucosa. In the instances observed, the presence
of maternal blood with many phagocytic leucocytes was noted
in the lumen of the uterus, adhering to and surrounding the ova.
From the study of sections of the uteri of an appreciable number
of albino rats, in which insemination and supposedly semination
seemed normal, but in which on complete serial sectioning of the
uterine tubes no ova were found, but in the lumen of the uterine
tubes of which the presence of maternal blood and phagocytic
leucocytes was noted, the conclusion seems warranted that
death and complete absorption of ova, after a given stage of nor-
mal development has been reached, may occur. In such cases,
one may with propriety speak of faulty implantation, due to
altered or pathologic condition of the uterine mucosa, even in
cases where no actual implantation would have occurred in cor-
responding normal stages. In the two rats (Nos. 91 and 104)
in which this condition was observed, the decidual crypts were
shallow and not developed to the extent normal for the respec-
tive stages, evidencing the abnormal condition of the mucosa.

In cases in which a single abnormal or pathologic ovum is
found in the uterus along with several normal ova, the pre-
sumption seems justified that the underlying cause responsible
for the abnormal development is to be sought in the ovum itself,
and not in its environs.

Abnormal developmental stages, interpreted as due to irregu-
lar or retarded segmentation, irregular or abnormal segmenta-
tion cavity formation, and retarded development of the ecto-
dermal node and primary embryonic ectoderm, where only a
single ovum shows abnormal development in a uterus contain-
ing the average number of ova presenting normal development,
are difficult to explain on the assumption that extraneous in-
fluences affecting a single ovum are operative. Practically all

142 G. CARL HUBER

of the abnormal ova of the class described, and especially is this
true for older stages, present normal relations to the uterine
mucosa and the walls of the decidual crypt after implantation,
and so far as may be determined by structure, give evidence of
normal absorption of maternal hemoglobin in stages in which
such absorption is pertinent. It may be argued that a single
ovum may be less favorably placed in relation to embryotroph
or pabulum, and as a result of unfavorable nutrition, develop
abnormally. This is difficult to conceive for stages in which the
ova lie free in the lumen of the uterus, namely, to about the be-
ginning of the seventh day after the beginning of insemination,
when embryotroph or pabulum must be relatively evenly dis-
tributed. The presumption, it would seem to me, in such cases
is in favor of regarding the primary cause of the abnormal de-
velopment as inherent in the ovum.

Separation of the first two blastomeres and the presence of two
egg-cylinders in a single decidual erypt are regarded as chance
findings and as of rare occurrence, since each was met with only
once in the material at hand.

LITERATURE CITED

Literature on pathologic ova of the albino rat is lacking. For the literature
of all but the more recent work, dealing with comparative experimental tera-
tology, the bibliographies accompanying the chapters of O. and R. Hertwig
may be consulted; for that dealing with the pathology of human ova, the bib-
liographies accompanying the contributions of F. P. Mall may be consulted.

Hertwia, O. 1906 Missbildung und Mehrfachbildung, die durch Stérung des
ersten Entwicklungsprozesse hervorgerufen werden. Hertwig’s Hand-
buch der vergleichenden und experimentellen Entwickelungslehre der
Wirbeltiere, Bd. 1, Part 1; Fischer, Jena.

Hertwie, R. 1906 Der Furchungsprozess. Hertwig’s Handbuch, Bd. 1, Part 1.

Matt, F. P. 1900 Welch Festschrift, Johns Hopkins Hospital Reports, vol. 9.
1903 Vaughan Festschrift, Contributions to medical science, G.
Wahr, Ann Arbor.

1908 <A study of the causes underlying the origin of human monsters.
Jour. Morph., vol. 19.

1910 The pathology of the human ovum. Keibel and Mall, ‘‘Manual
of Human Embryology.”’ Lippincott Company, Philadelphia.

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