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THE AMERICAN JOURNAL

OF

ANATOMY

EDITORIAL BOARD

CHARLES R. BARDEEN G. Cart HUBER J. PuayrarR McMourricu
University of Wisconsin University of Michigan University of Toronto
Henry H. DoNaLpson GrorGE 8. HUNTINGTON CHARLES 8. MINotT
The Wistar Institute Columbia University Harvard University
Stmon H. Gage FRANKLIN P. Mau GrorGE A. PIERSOL
Cornell University Johns Hopkins University University of Pennsylvania

Henry McK. KNowerr, SECRETARY
University of Cincinnati

VOLUME 15
1915-1914

THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY
PHILADELPHIA, PA.

THE WAVERLY PRESS
BALTIMORE, U. S. A.

CONTENTS

1913-1914

Non 12 JULY; 191s

C. M,. Jackson. Postnatal growth and variability of the body and of the vari-
qus.organs inithealbmorat. Seveniigures... 3.00202. 56. ccs ede eee
J. A. BapertscHEeR. Muscle degeneration and its relation to the origin of
eosinophile leucocytes in amphibia (Salamandraatra). Seven figures. ...
SuHinkisHr Harar. On the weights of the abdominal and the thoracic vicera,
the sex glands, ductless glands and the eyeballs of the albino rat (Mus nor-
vegicus albinus) according to body weight. Twelve charts...............
Epwarp F. Matonre. The nucleus cardiacus nervi vagi and the three distinct
types of nerve cells which innervate the three different types of muscle.
BREST OUTES: .. Ur RUMMY A, cry agen ees aie cen = meu nea ahs

No. 2. SEPTEMBER

ApaM M. Mituter. Histogenesis and morphogenesis of the thoracic duct in
the chick; development of blood cells and their passage to the blood stream
via the thoracic duct. Twenty-eight figures (seventeen plates)...........

WixiiaM H. F. Appison AND Harotp W. How. On the prenatal and neonatal
line Hightitigiresaaeeee yes va eel er e ee Se a eee ee

Cuester H. Heuser. The development of the cerebral ventricles in the pig.
Twenty-six figures

No. 3. NOVEMBER

CHARLES R. Srockarp. An experimental study of the position of the optic
anlage in Amblystoma punctatum, with a discussion of certain eye de-
XAT UNGTHOVE TRE tb Retstatets cho: 6 o' od Bap SRA Reta ID MISO Sins Gina eels cla aots

W.B. KirkHam AND H.S. Burr. The breeding habits, maturation of eggs and
ovulation of the albino rat. THighteen figures...............-----+---:-

Ivan E. Watiixn. A human embryo of thirteen somites. Seven figures....

Water E. Danny. The nerve supply to the pituitary body. Three figures

B. F. Kinacspury. The morphogenesis of the mammalian ovary: Felis domes-
REcoN MELO O LUT CS am MenMees fs sic, yess ho Soe pete oe)’. +, Weve mes) days

ill

131

. 199

253

. 291

319
339

345

lv CONTENTS

No. 4. JANUARY, 1914

E. V. Cowpry. The development of the cytoplasmic constituents of the
nerve(\cells’of the chicks. biyesplates<. cpio. os. .- sone ee
J. F.GupERNAtTscH. Feeding experiments on tadpoles. II. A further contri-
bution to the knowledge of organs with internal secretion. Two double
TV BGOS pce ardor RT COE PN Aa de By gE oo so x ee
Caries H. Swirr. Origin and early history of the primordial germ-cells in

the chick... Mitfecmmicuressnte. se) o aly etek oes: « 2) ee ee

389

POSTNATAL GROWTH AND VARIABILITY OF THE

BODY AND OF THE VARIOUS ORGANS
IN THE ALBINO RAT

C. M. JACKSON

The Anatomical Laboratory of the University of Missouri

SEVEN FIGURES

CONTENTS

EEC TT. .. MM ete ce ead SERA ge Shige eure yee a
Mereral ang. meth odsmemmmmenya cc sculuce sleep tase oe was fe ekoteiel chai 8x ol oiayeialak ways a taraa ave
Growth and variability of the body
De rOw bin 1 Ty Owe MmMMER Gye 525 cad ers cocks apeey Hele elo cant mer Pim Conyecey Nanette ne
2. Variability in body weight
SMC RTCTOAL VALLAGIRME Hai. cis. a al ve cry deer o nee ca Select te ans gata deena
Growth and variability of the individual organs

. Central nervous system
JORG) SRM, 2! 1, CONIA A) Nie ee ea aT Od io TENE Ea ee

. Thyroid gland

cl iei.cf te he, (ers) Jn) wheliehs\usfelm) ©, ‘of olgiie.\e es, a \e'4e =) 9 sie! se) 0, &] opera le

a mile) a) 6) a) c's 6 0) «| alelsiiaMeMpMsi allel u\'e)s//e) ie) « \ei’s ei '9)la aie. «pie\'e) 0s) ilele eye ‘ahalie aiie (el 'e\e ,e) @ si avienee.=)/mue

—
oO
is)
Ur}
+

faa
—

. Suprarenal glands
. Kidneys
. Gonads

a
Co bo

SMEMEatbaal aie eel ula «| «.e°auvls) pile 0 60 o's «is 6 \« «4 6 aise 6 «6 e\0 9 (0's Be 2

Bibliography Mai gies. eve «6 lee miciwic.)4 wie © elm iqve
Tables

il

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 1
JULY, 1913

2 Cc. M. JACKSON

INTRODUCTION

The present paper includes statistical data upon certain phases
of the growth of the body as a whole and more especially the
relative growth and variability of the individual organs in the
albino rat. A study of this kind may be of value in two respects.
In the first place, it should give a better insight into the nature
of growth, a fundamental biological phenomenon worthy of more
study for its own sake. In the second place, a more complete
knowledge of the process of growth, including the limits of nor-
mal variation, should be of value for reference in experimental
work of various kinds, for which the rat is often used.

For both these purposes there is great need of a series of com-
plete growth norms, comparable to Keibel’s morphological ‘Nor-
mentafeln.’ A complete growth norni for any given species would
involve adequate data upon the prenatal and postnatal growth
of the body as a whole, and of its component parts, organs, tis-
sues and cells. It would include a determination of the extent
of variability due to intrinsic or hereditary factors and to extrin-
sic or environmental factors. It is evident that a single fixed or
absolute growth norm for a given species does not exist. We
can, however, discover the norm and limits of variation within a
group of animals homogeneous in constitution and in an environ-
ment as constant as possible. Furthermore, it is possible to
determine to what extent the norm and variability are changed
by varying the different factors involved.

Such a complete growth norm has not as yet been even approx-
imately determined for any species, although numerous observa-
tions have been made upon different phases of growth in various
animals. For the rat, extensive data on the growth of the body
and of the central nervous system are available in a series of
papers by Donaldson and Hatai. The relative growth of the
principal parts and systems of the rat has recently been studied
by Jackson and Lowrey (’12); and formulas for the growth of
the individual viscera have been determined by Hatai (’13) and
‘are published in the present number of this journal. Further
data upon the growth and especially the variability of the body
and of the various organs are included in the present paper.

POSTNATAL GROWTH IN THE ALBINO RAT 3

MATERIAL AND METHODS

The following data are utilized in the present paper. For the
weight of the whole body of the albino rat (Mus norvegicus
albinus) at various ages a series of 570 original observations is
given in table 1. For the individual organs, 344 animals were
killed and dissected at various ages, and the weights of the organs
were observed (tables 3 to 15).

Of data available for comparison, there should be mentioned
first the extensive series of observations by Donaldson on the
growth of the whole body (’06) and of the brain and spinal cord
(08) of the albino rat. I am furthermore indebted to Professor
Donaldson and Dr. Hatai, of The Wistar Institute of Anatomy,
for unpublished observations upon the weight of the principal
viscera in about 200 albino rats. With the exception of the
central nervous system, however, these data are not included
with my own, but were utilized merely for comparison.

The albino rats used by me (table 1) were reared from stock
obtained partly from M. Cattell, Garrison-on-Hudson, New York,
and partly from B. F. McCurdy, a dealer in Chicago. The litters
from each source were kept separate, but no constant difference
in growth and variability was noticeable between them. The 68
litters included in table 1 are for the most part not closely related
(although in a few cases observations upon the body weight of
the same litter are repeated at successive ages).

The albino rats were kept in stationary cages in an animal
house and were well cared for. A supply of chopped corn was
kept constantly in the cages. A liberal amount of wheat bread
soaked in whole milk was supplied daily, and fresh meat (beef)
once a week. Water in abundance was provided.

From the age of about six weeks onward, the sexes of each
litter were kept separate, and (with the exception of a part of
the females at one year) therefore represent unmated animals.
Watson (’05) has shown that weights of the body and of the
central nervous system are somewhat increased in the female
through bearing young. It is probable that other organs, also,
may be thereby more or less affected (especially the reproductive

4 Cc. M. JACKSON

system), although data are wanting to determine this point. The
external characters whereby the sexes may be distinguished in
young rats have been described by Jackson (712).

In general, the albino rat remains healthy and thrives in cap-
tivity. The chief exception noted is the frequent tendency to
lung disease, especially in the older rats and more rarely in the
younger. Two forms of disease were observed. The first is an
acute pneumonitis, which is usually fatal. This form appeared
but once in a period of three years. It occurred as an epidemic
which in a short time destroyed nearly the entire colony. This
epidemic was caused by a specific bacterium, bacillus muris, as
determined by Mitchell (12). The second form of lung disease
noted is a chronic disturbance which is common in the wild
Norway rat at Columbia as well as in the domesticated albino.
It appears to develop first in the form of multiple small clear
spots scattered over the surface of the lung. Later these spots
may form small abscesses, or lead to the consolidation of one or
more lobes. An associated catarrhal condition of the respiratory
mucosa causes an audible snuffling or wheezing, through which
the disease may usually be recognized when well developed. It
usually is not fatal, or at least not immediately so, and in some
cases does not appreciably affect the growth or the general state
of nutrition. Generally, however, it tends to produce more or
less emaciation. This is probably the same disease as the pneu-
monia mentioned by King (711) and by Currie (10). Mitchell
(712) failed in repeated attempts to find in this disease the spe-
cific bacterium causing the first form, and its etiology appears
uncertain.

Animals affected with lung disease were excluded from the
present data except in some cases in which the lesions were slight
and it appeared necessary to exclude only the lungs. This was
the case in 3 of the 48 albino rats dissected at the age of ten
weeks, 11 of 41 at five months, and 20 of 25 at one year. Even
though the lesions were slight, however, it is probable that the
average body weight at five months, and especially at one year,
is somewhat below the normal. ; :

POSTNATAL GROWTH IN THE ALBINO RAT 5)

Since the number of observations was somewhat limited, it was
thought better, especially for the study of variation with age, to
restrict them to certain definite ages, rather than to scatter them
over the entire period. Seven ages were chosen for this purpose,
namely, newborn (one day or less), seven days, twenty days, six
weeks, ten weeks, five months, and one year. ‘These ages were
selected for the following reasons. At seven days, the body
weight at birth has about doubled. At twenty days, the weight
has approximately doubled again, and at this age the albino is
usually weaned. At six weeks, the body weight has somewhat
more than doubled again, and the animal is well established upon
the new diet. At ten weeks, the rat has again about doubled
its weight, and sexual maturity is reached. At one year, the
weight has again approximately doubled, and at this age, accord-
ing to the observations of Slonaker (12), the albino rat (in sta-
tionary cages) has nearly reached its maximum body weight.
Five months was arbitrarily selected as a point intermediate
between ten weeks and one year.

A few observations upon the body weight at fourteen days and
at thirty days are also recorded -in table 1. The continuous
growth curves for the absolute weight of the various organs, in
terms of body weight, are given by Hatai (13). It is hoped that
the age periods selected for the present paper are sufficiently close
together so that no material change in variability, correlation,
and so forth, will be overlooked.

In some cases, aS previously mentioned, only the gross body
weight was observed. This weight was always taken in the fore-
noon, before feeding. In the cases where the animal was to be
dissected, it was killed by chloroform, and the gross body weight,
and lengths of trunk and tail recorded. The head was then
removed on a plane just posterior to the cranium and anterior
to the larynx, and was weighed; while the trunk was suspended
by the tail, allowing the blood (unmeasured) to escape. The
eyeballs and in some cases the brain were then removed and
placed in a closed jar upon moist filter paper. Next the trunk
was dissected and the following organs successively removed:
thyroid gland; thymus (dissected out of surrounding fat); heart

6 Cc. M. JACKSON

(cavities opened and blood-clots removed); lungs (right and left
separately) ; liver; spleen; stomach and intestines, including con-
tents, mesentery and pancreas; same, without contents; supra-
renal glands; kidneys; ovaries or testes (including epididymis) ;
spinal cord (in a few cases). The extremities, skin, skeleton and
musculature were also weighed in some cases, as described in a
separate paper by Jackson and Lowrey (’12).

The organs were weighed in closed containers, and loss by
evaporation was avoided so far as possible. The organs were
usually weighed to 0.1 mgm. (0.0001 gm.) excepting some of the
larger organs in the older rats, which were weighed to 1 mgm.
The observations were recorded on printed cards, and any unusual
conditions carefully noted. The calculations were made by the
aid of a Burroughs adding machine, Crelle’s Rechentafeln, and
the tables in Davenport’s ‘Statistical Methods.’ The calculations
were carefully checked independently to eliminate errors.

I am greatly indebted to Dr. 8. Hatai of The Wistar Institute,
for valuable aid and criticism, especially on the mathematical
phases of the work.

In calculating the various statistical constants—mean, stand-
ard deviation, coefficients of variation and of correlation and
probable errors—the usual formulas (Pearson’s) were employed
as given by Davenport (04). The ungrouped data were used in
all cases.

Since the present paper is concerned largely with variability
as measured by the coefficient of variation, a brief discussion of
this coefficient may be desirable. The coefficient of variation is
one hundred times the ratio which the standard deviation bears
to the mean; or the percentage of the mean which thestandard
deviation forms, as expressed by the formula:

= 7 100 (%)

For the standard deviation, the formula is as follows:

VEE
n

o

POSTNATAL GROWTH IN THE ALBINO RAT 7

where d represents the deviation from the mean, and n the num-
ber of variates. The standard deviation is therefore a concrete
number which serves to measure the dispersion from the mean.
If the variates tend to diverge greatly from the mean, the stand-
ard deviation will of course be large; while if they are concen-
trated closely around the mean, the standard deviation will be
small. It is further clear that the standard deviation is not
dependent upon the number of cases, but merely upon the
manner of their distribution.

Since the standard deviation is a concrete number, however,
it will in general vary with the absolute size of the variate.
It is therefore often difficult to judge the relative variability of
different objects by comparing their standard deviations, and
where different units of measurement are used the comparison is
meaningless. On this account the coefficient of variation is pref-
erable for measuring the relative magnitude of variations, since
it is independent of the size or character of the unit of measure-
ment.

It is of course obvious that from the statistical point of view
the number of observations is small and the results cannot be
considered final. Even although the probable errors are rela-
tively large, however, it will be found that certain conclusions of
importance may be drawn with a fair degree of certainty; and
others, more or less strongly indicated, may point the way to
further investigation with more adequate data.

To economize space, the individual data are not included in
the tables, but only the averages and ranges are given. The
cards containing the original individual data are, however,
deposited in The Wistar Institute of Anatomy in Philadelphia
whence they may be obtained if desired by anyone interested.

8 Cc. M. JACKSON

GROWTH AND VARIABILITY OF THE BODY
1. Growth in body weight

The data presented in this paper are inadequate for a complete
discussion of the growth of the body as a whole. Certain phases,
however, may be noted. Donaldson (06) has made numerous
observations upon the postnatal growth of the albino rat, on the
basis of which he has drawn certain conclusions, with which the
present data may be compared. Table A shows the average

TABLE A
Average gross body weight of albino rat at various ages, sexes separated
aie a JACKSON’S =) SG JACKSON’S SMALLER PERTDsON'S bea
aoe SERIES SERIES (1906)
grams °* grams grams c
fr (G3ane) 45-18 (44m.) 5.06 (40m.) 5.4
HewDOEN:--oe cal| Gok) pet Soe. 4. Saas
Ugh aaa { (56m.) 10.53 (30m.) 10.61 @ism:). 9:2
Mime nse Gs pce (64 f:)) 10.205) "@7#.) 10.48’ Mest.) 827
eae | (3m) 23:90) (24m.) 22:20 MiNI9'm.) 21,2
a a 69.) w2iv50Me abd.) 17.01 GU7 £0) 122"6
Bee os | ((455m.) 763972) 422m.) 52,890 G9m.) 46:3
Mee TRE aoe GO: ) SG neon.) 54. Samrat yt. jo 47.9
| (23 m.) 130.4 (20 m.) 121.9 (19 m.) 106.6
RR es sco a’ See { (25f.) 108.9 | (23f.) 103.3 | Q1f. ) 99.8
(20 m.) 167.5 (19 m.) 225.4
5 months... Be ho { (Q1f. ) 142.1 (11 f. ) 184.6
edad (5m.) 213.0 ( 6m.) 279.0
VGA as ve scrsare ip 4 (20 f. ) 163.7 (7£. ) 296.4

1 Jackson’s observations listed under ‘3 weeks’ were nearly all at 20 days, and
those of Donaldson under ‘6 weeks’ were at 43 days. The females were unmated,
excepting all of Donaldson’s series at 1 year, and a part of Jackson’s series at 1
year.

gross body weight in each sex at various ages in my larger series
(recorded in table 1), in my smaller series (dissected for the
organs), and in those observed by Donaldson at corresponding
ages.

From table A it is evident that the rats used in the present
study averaged somewhat smaller at birth for both sexes than
those of Donaldson, but show a more rapid growth during the
first week, so that at seven days their average weight is greater.
At three weeks the males in Donaldson’s series average somewhat

POSTNATAL GROWTH IN THE ALBINO RAT 9

smaller than in mine but the females are heavier. At six weeks
and ten weeks both sexes average smaller in Donaldson’s series,
but at five months and one year they are decidedly larger.

In general, therefore, it is evident that Donaldson’s rats re-
mained smaller than mine up to the age of six weeks, but sur-
passed them in the later stages. The probable errors were not
calculated for the gross body weights (except at six weeks, smaller
series); but as will be seen later the variation for the gross weight
is not materially different from that of the net body weight, the
probable errors of which, in the smaller series, are given in table
2. The differences between Donaldson’s results and mine are
greater than the probable errors and therefore appear significant.
The explanation of these differences will be considered later under
variability. !

The relative growth of the sexes of the albino rat was noted
by Donaldson (’06), who found that, beginning about the end of
the first week, the female grows more vigorously, overtaking and
usually exceeding the male in body weight from the fifteenth to
the fifty-fifth day of age. My data (table 1) show that in 16
newborn litters containing both sexes the males averaged greater
in every case; at seven days, the males exceed in 11 litters, the
females in 5; at fourteen days, the males exceed in 3, the females
in 1; at twenty days, the males exceed in 10, the females in 3;
at thirty days; the males exceed in 2, females in none; at six
weeks, the males exceed in 10, the females in 2; at ten weeks,
the males exceed in 6, the females in 1; at five months the males
exceed in 4, females in 1; at one year, the males exceed in 2,
the females in 1. According to litters, therefore, the excess of
average weight was invariably in favor of the male at birth, and
also in the majority of cases at all succeeding ages.

The ratio of the average gross body weight in the male to

that in the female at corresponding ages in my data is given in
table B.

1 Tt may be pointed out in this connection that whereas my rats represent for
the most part a ‘random sample’ of the general population at each age, those of
Dr. Donaldson were taken while young, and used throughout the period of obser-
vation.

10 Cc. M. JACKSON

According to table B, it is seen that the average body weight
of the male was greater at every age noted, except at six weeks,
when the female was slightly larger. At seven days, however,
the male was but slightly the greater.

The extent of the variation in the ratio of body weights between
males and females of the same litter may be noted from data

in table 1. Further information of interest is afforded by table
TABLE B
NUMBER OF | AVERAGE GROSS | AVERAGE GROSS |
AGE | EACH SEX BODY WEIGHT | BODY WEIGHT | RATIO OF MALE TO
| | FEMALE
Males Females) Male Female
| | | grams Pe ind
Mewborn.<'s..2 er eeel 63 «66 5.13 4.89 1.05
TALES bec 2 ee sO 0 NGA: 10.53 10.29) 1.02
OMG Ef PR ceo day an) 23.99 21. 50a ae
Grweeks.i (anual tao SoU 63.72 64.25° 0.99
10 weeks... .eteeaces|0 23) 4) 259 al 130.40 108.90 1.20
5 months. .ena ee 20 Wy 20 | 167.50 142. 100m 1.18
TSyears nes See an 20 213.00 163 7CRE lon: 1230
|
TABLE C

Ratio of average male to female gross body weight in individual litters at
successive ages

tee eee? ie NEWBORN 7 DAYS 14 Days 20 DAYS 30 DAYS | 6 WEEKS
AY32) (Samoa ae: a Veo 03 Sateeatts (03 |
IMO“ (5m, Stee. oe 1.06 1.06: | |
WON Gan ste ee 1.06 | 1.04 1.05
MM. 6) GansiSeeeee ane 1.04 1.08 1.03
NM 10m. 43 | 1.05 1.05 0.99 1.00 leat
M.S Gime bioees ae: That 1.06 1.06 1.05 1.09 1.222
M7 Canteen 0.98 0.868
E427: era) 4b ee | 1114 |) 100!
Met) (Asmaal es) eee 0.95 | 1.04
ML Oo (4 ansieto eee 1.08 | 1.10
A299 (on 4) 1.02 1.16 1.04 | 1.08
Avos (fimo) epee 1:08: || 0:99 1.01 0.90

|

1 Two males of this litter were killed at 7 days.

* In this litter, the ratio at 10 weeks was 1.39.
3 One male, a ‘dwarf,’ died before the age of 30 days.
4 One female a ‘dwarf’ at these ages.

POSTNATAL GROWTH IN THE ALBINO RAT 11

C showing the ratios in a few cases where the gross body weight
was observed in the same litters at successive ages.

It is clear that there is considerable variation among individual
litters, and evidently observations upon a much larger number of
litters would be necessary to determine the typical growth rela-
tions by sexes. The available data seem in general to confirm
the conclusion of Donaldson (’06) that during the first two months
of postnatal life the growth of the body of the albino rat is more
vigorous in the female. In the majority of litters, however, the
average body weight of the females does not appear to reach
that of the males, at least at the ages observed.

2. Variability in body weight

It is evident from the foregoing that there is in some respects
considerable difference between my data and those of Donaldson
on the growth of the body of the albino rat. The question
naturally arises as to the significance of this difference, as both
series are presumably normal (excepting the possible effect of
lung disease in the older rats). This brings up the general ques-
tion as to the nature and extent of normal variation in the growth
of the body. It is known that this variation is greatly influenced
by external factors, such as the quantity and quality of food,
temperature and so forth. Slonaker (’12) has recently demon-
strated that the growth of the albino rat in body weight may
be greatly retarded by exercise, and that it is also much less with
vegetarian than with mixed diet.

These factors, however, were nearly alike in the case of Donald-
son’s rats and mine, though unavoidable differences in environ-
ment may account in part for their difference in growth. But
even when the environment is kept constant, there still remains
the variability due to intrinsic factors, which probably varies some-
what according to the ancestral strain from which the animals
were derived, as well as’ according to litter, sex and individual.
Pearson (’00, p. 473) states that ‘‘The individual contains in
itself, owing to a bathmie (i.e., intrinsic) law of growth, a varia-
bility which is quite sensible, being 80 to 90 per cent of the
variability of the race.’’ As before mentioned, it is therefore

12 Cc. M. JACKSON

evident that in general there is no such thing as a single ‘normal’
course of growth and variation for a given species. We may,
however, determine the process under a given set of conditions,
and also learn the effect produced by varying the individual fac-
tors. It is probable that some variation in the intrinsic growth
factors is largely responsible for the differences noted between
Donaldson’s data and mine. The possibility of unrecognized
pathological factors must also be kept in mind.

We may now consider the variability in body weight found
in the present series.2, The variability is best measured by the
coefficient of variation, which expresses the percentage of the
mean formed by the standard deviation. For the net body weight
(table 2), it is seen that the coefficient of variation, taking both
sexes together, increases from 12.3 at birth to 15.6 at seven days
and to a maximum of 28.4 at twenty days. Thereafter it de-
creases, being 21.3 at six weeks, 19.9 at ten weeks, and 19.1 at
five months. On account of the small number of observations
and probable abnormalities at one year, no calculations were
made for the total at this age; but the coefficient of variation in
body weight for the 20 females at this age was slightly greater
than for those at five months. Even when allowance is made
for accidental variations due to the small number of observations
(as indicated by the relatively large probable errors), the approx-
imate extent of variation, and the general trend according to age
are clearly evident. Hatai (08) in mature albino rats (over
150 days old) found the coefficient of variation for the gross
body weight in 53 males, 25.076+2.675 and in 51 females,
12.235 + 0.974.

The difference in variability between the sexes of the rats used
in the present study is indicated in table 2. It is seen that the
coefficient of variation appears greater in the males at every
age, except at twenty days. No great stress can be laid upon

2 The variability in body- length was not calculated. According to Donaldson
(09), however, the body length of the albino rat is less variable than the body
weight; but the two are closely correlated, the coefficient of correlation being
0.90. In the human species, the body length (height) is only about one-third as
variable as the body weight (measured by the coefficient of variation).

POSTNATAL GROWTH IN THE ALBINO RAT 13

this indication, however, as it must be remembered that the
number of observations is comparatively small, and the differ-
ences are usually within the limits of probable error. It is note-
worthy, however, that Hatai (’08) likewise found for the body
weight and for various skull measurements of the albino rat a
tendency to greater variability in the male.

At the ages of twenty days and six weeks, the standard devi-
ation and coefficient of variation were also calculated for the
corresponding gross body weights. At twenty days, the coeffi-
cient of variation was, for the males 28.7 (as compared with
28.4 for the net body), and for the female 18.4 (17.5 for the net) ;
at six weeks, for the male 17.7 (17.4 for the net), and for the
female 24.6 (24.7 for the net). It is therefore evident that there
is no material difference between the coefficients of variation for
the gross and for the corresponding net body weights.

For a larger series of 56 males and 36 females newborn, cor-
responding only in part to those given for net body weight, the
coefficient of variation for the gross body weight of the male was
14.0 (13.6 for the net), and of the female 9.2 (9.9 for the net).
A larger series was also calculated for the gross body weight at
twenty days and six weeks, including all those in table:1. In
this larger series (which was about twice as large as the smaller)
the coefficient of variation at twenty days for the male was
24.4 (28.7 in the smaller series) and for the female, 29.4 (18.4
in the smaller series). At six weeks, the coefficient of variation
in the larger series for the male was 20.8 (17.7 in the smaller
series) and for the female, 24.2 (24.6 in the smaller series). This
would seem to indicate that the great difference in the coefficient
of variation between the sexes of the smaller series at twenty
days and at six weeks is due to an abnormally small variation
among the females at twenty days and among the males at six
weeks. This should be remembered in considering the varia-
bility of the organs, which correspond to the smaller series and
are, as will be shown later, all more or less closely correlated with
the body weight. The necessity for caution in drawing con-
clusions as to variability from a comparatively small number of
observations is thus emphasized.

14 Cc. M. JACKSON

The foregoing data indicate clearly that the coefficient of vari-
ation in body weight is smaller in the younger rats (newborn
and seven days) and larger in the older (twenty days, six weeks,
ten weeks and five months). Less conclusive is the evidence that
the maximum variation, for the ages observed, occurs at twenty
days, and that the variation is in general greater in the male
than in the female. The changes in variation cannot be ascribed
to a selective death rate, as there were in the litters observed
very few deaths before the age of five months.

For the variation in the weight of the human body at different
ages, fairly complete data are available for comparison. For the
English newborn, Pearson (’99) finds the coefficient of variation
for the male to be about 15.7, and for the female 14.2. For
Cambridge University students, nineteen to twenty-five years of
age, he finds the coefficient for 1090 males to be about 10.8, and
for 160 females 11.2. For Oxford students (male) of eighteen to
twenty-three years, Schuster (11) similarly finds the average
coefficient of variation in body weight to be 10.8, varying from
10.2 to 11.1 for different years of age.

For the period from birth to the age of six years, no data are
available. But from six years onward we have the extensive
observations of Porter (’95) on St. Louis school children. Porter
uses the ‘probable deviation’ (which equals approximately 0.6745
times the standard deviation) as a measure of dispersion, but
for convenience of comparison this has been reduced to the
standard deviation for calculating the coefficient of variation in
table D.

Porter concludes that variation is correlated with rapidity of
growth, a conclusion preyiously reached by Thoma (’82). Boas
(97, 04) reaches similar conclusions. This relation will not hold
good for the human newborn, however; for although the growth
rate is then far more rapid than at any subsequent period, the
coefficient of variation is slightly less than at the age of puberty.
In the rat, as we have seen, the coefficient of variation is smaller
in the younger animals although the growth rate is far more
rapid then than later.

POSTNATAL GROWTH IN THE ALBINO RAT 15

We may therefore conclude that for the human (British and
American) the coefficient of variation for the body weight in the
newborn male is about 15.7. At six years, it has diminished to
a little below 11, and fluctuates in this neighborhood until the
twelfth year. During the acceleration of growth from thirteen
to sixteen years, the coefficient of variation increases to 16 or
17, thereafter diminishing rapidly to about 11 in the young adult.
For the female newborn the coefficient of variation (14.2) is
somewhat below that of the male. From six years onward, the
coefficient of variation appears to be correlated with the relative
rapidity of body growth, being somewhat greater in the female
up to the age of fourteen, after which the male exceeds it.

In comparison with the human species, the coefficient of vari-
ation in the body weight of the rat at birth appears somewhat
smaller. Although in all subsequent stages (except at the age of
puberty) the variation in the human body is smaller than at
birth, in the rat it appears constantly greater, and shows no
evident correlation with the rate of growth of the body.

TABLE D
Body weight of St. Louis school children (from Porter’s data)

NUMBER OF COEFFICIENT OF | RELATIVE-ANNU AL

AGE AT NEAREST BIRTHDAY CEBERV-ATIONS | VIMSTE MI ETOSS) | Tet wane sale yaa
Male Female | Male | Female | Male | Female
| | per cent per cent
6 ) meee "798° )- 10:7 11.3
7 (Sian 714 HG. avdse3 9.7 10.0
8 Dee 2047 12h2 12.6 9.7 9.9
9 2188 2055 | 11.9 13.2 9.6 9.6
10 206ml 1947 | 117 || 124 | 8.7 9.6
11 1743 1708 DRA AAR IE On 9.7
12 1644 1676 LONSM eel 4s Sys peau 11.6
13 1242 1343 1a 0) a Kes a pt ie 2) 14.3
14 946 1082 16.7 16:3> le AOr5 9.9
15 498 690 16.3 1219), shy FLAGS 10.4
16 203420) 1) 21758 TDA Wile te lal God) WS Te
17 7s 230 17 10.4 7.9 4.7
18 fee 155 10 +2
19 | 81 10.7
20 66 10.4

16 C.

M. JACKSON

3. Fraternal variability

It is a matter of common knowledge that yariation within
fraternities is always less than that of the general population.
In the various litters of rats used for the present study, it is
in most cases evident from mere inspection that variation in the
body weights within the litter is much less than the variation in

the total population of the same age.

To measure the variation

within the litter, three methods were used and the results are

summarized in table E.

TABLE E

Coefficient of variation in body weight for total population by ordinary method, and
on litter basis (fraternal variation) estimated by various methods

SEX

NEW- | 7 20

6 10 5

| BORN DAYS | DAYS | WEEKS | WEEKS MONTHS

Total population (ordinary
MeLHOG) Meek. fo he eee
Litter basis (average of lit-|
ters, calculated by ordinary

Litter basis (calculated from
enle/sstiormulla)) s.4.- secu
Litter basis (from eS s {|

formula).. Re) Saree |

male
| female

male
female

male

| female

male
female

13.6! | 16.91 | 24. 42

9.9: | 13.7} 29.42

Onan KYAT

20.8? | 18.81 | 18.5?
24.2? | 16.81 | 15.3!

! | For net body weight.

2 For gross body weight, larger series.

|

The first method of determining fraternal variation consists
simply in calculating the standard deviation and coefficient of
variation for each separate litter by the usual formula. The sexes
were calculated separately, only those litters with four or more
of either sex being used. The average coefficient for each sex at

each age is given in the preceding table.

On account of the small.

number of individuals in each litter, the results can of course be
considered only as grossly approximate.

The second method of calculating the fraternal variability is
based upon a formula by Yule (’11) who demonstrates (p. 142)
that ‘‘if a series ‘of (N) observations consists of 7 component
series with standard deviations a1, o.,
diverging from the general mean of the whole series by di,

o,, and means

POSTNATAL GROWTH IN THE ALBINO RAT 17

d,. . . . d;, the standard deviation o of the whole series
is given (using m to denote any subscript) by the equation:
N.o? = = (Np. Oe?) +E (Nm. dm?2).”’ If we let dm represent the
mean standard deviation of the component series (which may
be assumed to be fairly constant) then

> (N ea Om?)
becomes
N om?
and

eo No? — 2 (Nim dm’)
N

whence the value of c, 1s obtained. In applying this method
to the rat data N represents the total number of rats at a given
age; o, the standard deviation of the body weight of all the rats;
dm, the deviation of any litter mean from the total mean, Nm,
the number of individuals in the corresponding litter; and om, the
average standard deviation within a litter. The sexes are con-
sidered separately. The results, as shown by the preceding table,
do not differ materially from those by the first and third methods.

In the third method! of determining fratetnal variation, all the
litters at each age (sexes separately) are reduced to a common
litter basis. This is accomplished by multiplying the body weight
of each member of a given litter by the factor

ean Of ig a abopulaton (aizety en see)
Mean of given litter

3 Since the formula may be written in the form:

SHON E)
7) a Neo.
ad N

. nd . . oye . . . .
and since is necessarily a positive quantity, it is evident that accord-

p> (Nim Ge)
N

ing to this formula the standard deviation of the individual fraternity is
usually (and on the average necessarily) less than that of the total population.
The formula gives no indication as to the magnitude of the difference, however.

4 For this method, and for aid otherwise, I am indebted to my colleague Prof.
O. D. Kellogg, of the Department of Mathematics. He adds the caution that
under some circumstances the method might tend to give a spurious reduction
in the standard deviation and coefficient of variation, but this does not appear
to be the case with the present data.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 1

18 Cc. M. JACKSON

Since the coefficient of variation for each litter and also the mean
of the total population remains unchanged by this procedure, the
products may be considered as forming a single large litter, whose
coefficient of variation may be calculated in the usual way. The
results by this method are seen to correspond closely to those by
the preceding methods. In fact all three methods agree much
more closely than might have been anticipated, considering the
relatively small number of litters and of individuals.

Comparing the coefficients of fraternal variation, calculated on
the litter basis, with those of the total population calculated by
the ordinary method at corresponding ages, it is evident that
(excepting only the unusually large fraternal variation for the
female at ten weeks) the litter variation is always very much
smaller, being usually only from one-third to one-half as large as
the total variation. It also appears that the increased varia-
bility in the body weight of the total population from twenty
days onward is due to an increased variability between litters more
than to increased variability among the individuals within the
litter. Dunn (712) states (p. 137) that if conditions of growth
are favorable the rats of unlike initial weight within a litter
tend to approach each other in weight as growth goes on. If
this be true, the fraternal (intra-litter) variation should decrease
with age. |

Table F includes my observations upon the variation in indi-
vidual litters at successive ages. The sexes are here taken
together. It is evident that there is considerable variation, both
among different litters at the same age and in the same litter at
different ages. The data suggest that there is a tendency for
intra-litter variation to decrease up to the age of about twenty
days, followed by a tendency to increase. This is supported to
certain extent by table E, showing the coefficients of variation
on the litter basis.

It may be concluded from all the evidence available that in
general the variation in body weight within a given litter of
albino rats is probably less than half that of the general popula-
tion of the same age under similar environment. Nevertheless,
even within the litter, variations are sometimes so great that it

POSTNATAL GROWTH IN THE ALBINO RAT 19

is never safe to draw conclusions from a single litter, and there
is some risk even with several litters. These conclusions are of
practical importance in selecting animals for experimental work,
and in interpreting the results of such work. Although fraternal
variability was not directly calculated for the individual organs,
the same principle doubtless holds good with them; for, as will
be shown later, all the organs are more or less closely correlated
with the body weight.

In the human species, fraternal variation appears to be rela-
tively greater than in the rat. For example, Galton (94) found
the mid-stature for the human adult (male) population to be 68.2
inches. The quartile (or probable) deviation for the whole adult
population he found to be about 1.70 inches, and that for brothers
(average of four methods) about 1.06 inches. Thus for human
stature by this method the fraternal variability appears to be
only about 62 per cent of the racial variability. On the basis

TABLE F
Coefficient of variation in individual litters at different ages
NUMBER OF
LITTER NO. GUNG ERR | NEWBORN 7 Days 14pays | 20pays | 30pays | 6 WEEKS

Males |Females, | |
AOD ies: 3 5 5.81 3.82 |
NiO i> 5 3 | Waa 4.85 |
ING 3 a] 4.19 2.92 4.45 |
MR G88 eo 5 3 10.202 4.11 5.61
Vial OR ee 2 4 8.18 7.61 7.47 7.64 11.8
IVIEISE sees: 2 5 8.78 4.82 3.94 2.42 6.76 12.43
MSR eo onuee 0 7 12.00 6.67
AGRE ea 0 5 7.34 4.70 6.94
INTENT x ore: 2 4 3.95 18.00
1 Cae seers 1 4 21.30 30.60
1 BE aes 4 1 2.83 7.91
Dei 4 1 4.11 6.57
INR ZO ea 2 4 | 4.47 4.95
OSs F. ote: 4 2 | * 5.06 6.86

1 Two males killed in litter M9 at 7 days, and 2 females in A 33 at 20 days; one
(a ‘dwarf’) in litter M7 died between 20 and 30 days.

2 The large variation here was due to a ‘dwarf’ which caught up with the others
in body weight before the age of 14 days.

3 At 10 weeks the coefficient of variation in this litter was 17.0, the increase
being due chiefly to sexual differentiation in body weights.

20 Cc. M. JACKSON

of more recent biometric investigations in various lines by Pear-
son and others, it appears (cf. Encyclopaedia Britannica, 13th
edition, vol. 27, p. 911) that in general the fraternal 8. D. =
racial S. D. xX ble reckoning the fraternal correlation at 0.50.
According to this formula, the fraternal variability would amount
to about 87 per cent of the racial variability. Compared with
these figures the fraternal variability of the rat, which is less
than half of the racial, appears relatively small. This, however,
is intra-litter variability, which may be somewhat less than fra-
ternal variability in general. The latter could be determined for
the rat only by comparing numerous litters born from the same
pair of rats at different times.

GROWTH AND VARIABILITY OF THE INDIVIDUAL ORGANS
1. Head

In a previous paper by Jackson and Lowrey (712), the relative
growth of the head was considered. It was pointed out, as may
be observed in table 3, that the head (sexes together) of the albino
rat increases in relative size from an average of about 23 per
cent of the body at birth to a maximum of nearly 26 per cent
at seven days, after which it decreases gradually to about 10
per cent in the adult. The figures in the table for the percentage
at one year are probably somewhat high, as they include some
slightly emaciated individuals, in which the head is always rela-
tively large. In very large rats (above 300 grams in body weight)
the head continues to decrease in relative weight and may reach
8 per cent or less.

As is furthermore evident from table 3, the variability of the
head is less than that of the body as a whole, the coefficient
increasing from about 9 in the newborn to about 13 in the adult.
The variation also appears slightly greater in the male than in
the female (except at six weeks), as is also the case with the
whole body.

The coefficient of correlation between the head and the total
body weight varies at different ages from 0.76 to 0.95 (sexes
together). These figures are somewhat too high, however, the

POSTNATAL GROWTH IN THE ALBINO RAT 2h

true correlation being augmented by a ‘spurious correlation,’ as
will be explained later. There appear no constant differences
according to age and sex. If such exist, as is probably the case,
they are obscured by accidental variations in the comparatively
small number of observations.

Phe close correlation between the weights of the head and of
the whole body is also evident from the slight variability in the
percentage weight which the head forms of the whole body (table
3). If the head at each age formed a constant per cent of the
body, the variability of the percentage weight would be zero.
The coefficient of variation of the percentage weight of the head
is, however, usually not very much smaller than that of the
absolute weight, and in a few cases even appears slightly larger.
That is to say, at any given age the absolute weight of the head
can be predicted almost as accurately as the percentage weight.
This is not the case with most of the individual organs, as will
be seen later.

Whether reckoned upon the absolute or upon the percentage
basis, it is evident the head of the rat exhibits slight variability
when compared with most other organs as well as with the body
as a whole. This agrees with the observations of Hatai (’08)
upon the variability of the skull bones of the rat, as well as with
what has been found in the human body. Quetelet (71) and
other anthropometrists have observed that the head is the least
variable portion of the human body, a conclusion supported by
extensive and careful measurements on the skeleton of different
parts of the body by various observers.

2. Central nervous system

Since the growth of the brain and spinal cord has been care-
fully worked out by Donaldson (’08) from extensive data, it was
thought unnecessary to include these organs in the present inves-
tigation. Incidentally, however, some observations were made
upon the brain, and a few upon the spinal cord. From these,
together with those published by Donaldson (08) and some
unpublished data from The Wistar Institute, a few additional
conclusions may be drawn.

22 Cc. M. JACKSON

In regard to the brain, it may be noted that the maximum
postnatal relative size occurs, not at birth, but a short time
later. Using the data derived from Donaldson’s formula express-
ing the brain weight in terms of (gross) body weight (’08, table
1), calculations show that with a body weight of 5 grams (ap-
proximately that at birth) the brain weight forms 4.6 per cent
of the body weight. This increases to 6.7 per cent at a body
weight of 15 grams. From this (apparent) maximum, the relative
size of the brain decreases to 5.0 per cent at 25 grams, 2.7 per
per cent at 55 grams, 1.5 per cent at 115 grams, 1.2 per cent
at 155 grams, 0.9 per cent at 205 grams, and 0.62 per cent at
315 grams. In this case, the sexes are not separated, although
the male brain is relatively slightly heavier than that of the
female.

The foregoing figures for the relative size of the brain, as de-
rived from Donaldson’s formula, agree fairly well with the present
data grouped according to age. We find (combining Donaldson’s
data with my own) that in 92 newborn albino rats (56 males,
36 females) the brain averaged 4.8 per cent of the (gross) body
weight; in 22 (10 males, 12 females) at seven days, 6.4 per cent;
in 31 (28 males, 3 females) at three weeks, 5.6 per cent; in 30
(27 males, 3 females) at six weeks, 3.1 per cent. Only scattering
observations are available at the later stages. It therefore ap-
pears probable that the brain reaches its maximum relative size
about the second or third week, when the body weight is between
10 and 15 grams.

The combined data for the brain are sufficient for estimation
of variability and correlation with body weight only at birth,
three weeks, and six weeks. The coefficient of variation for the
brain was found to be at birth, male, 13.9; female, 9.1, total
(sexes together) 12.2. The coefficient of variation for the cor-
responding (gross) body weights was 12.8. At three weeks the
coefficient of variation of the brain, for males only, was 6.8,
which, excepting the eyeballs (table 4) was the lowest variability
found for any organ at any age. The corresponding coefficient
of variation for total body weight was 14.8. At six weeks, the
coefficient of variation for the brain, males only, was 12.2, the
coefficient for the corresponding total body weights being 36.2.

POSTNATAL GROWTH IN THE ALBINO RAT 23

For the same data, the coefficient of correlation between brain
weight and (gross) body weights was, at birth, male, 0.736;
female, 0.566, total, 0.690 +0.037; at three weeks, male only,
0.783 = 0.049; at six weeks, male only, 0.883+0.020. These high
figures are due in part to ‘spurious correlation’ (as will be explained
later). In general, Donaldson (’08) found (in 680 records at
various ages) the coefficient of correlation between brain weight
and body weight to be 0.7639= 0.0108.

Hatai (08) found for the cranial capacity of adult albino rats a
coefficient of variation of about 6.7 in the male, and 7.2 in the
female. The coefficient, of correlation between cranial capacity
and (gross) body weight was 0.516 = 0.074 in the male and 0.692 +
0.058 in the female.

For the spinal cord, the relative weight (according to Donald-
son’s formula) increases from about 0.66 per cent of the body
at 5 grams to a maximum of 0.77 per cent at 10 to 15 grams,
declining to 0.28 per cent at 205 grams and 0.22 per cent at 315
grams. From the combined data the coefficient of variation was
calculated for the newborn only (48 males, 29 females) and was
found to be as follows: male, 17.8 (for total body, 13.1); female
12.6 (total body, 9.4); total (sexes combined) 16.0 (for total body
12.5). The corresponding coefficient of correlation with the body
weight was 0.666 +0.048, which is somewhat lower than that
found by Donaldson (’08) for the spinal cord of the rat in general
(0.8564 = 0.0071).

In comparing these results with those for other organs, it must
be remembered that the rats from which the brain and spinal
cord were derived correspond only in part to those for all the
other organs.

3. Eyeballs

The relative or percentage weight of the eyeballs for various
body weights, when calculated from Hatai’s (13) formula, is
shown by the curve in figure 1 b. According to this curve, it
is seen that the eyeballs increase in relative weight from about
0.59 per cent of the body weight at 5 grams to 0.61 per cent at
10 grams, thereafter decreasing steadily, reaching 0.48 per cent

24 Cc. M. JACKSON

at 20 grams, 0.29 per cent at 50 grams, 0.18 per cent at 120 grams,
0.15 per cent at 170 grams, 14 per cent at 200 grams, and 0.11
per cent at 300 grams.

In my data grouped according to age periods (table 4, fig. 1 b)
the average weight of the eyeballs at birth forms about 0.52 per
cent of the (net) body weight in the male, and 0.54 per cent in
the female. The relative weight increases to a maximum at
seven days of about 0.60 per cent in the male, and 0.64 per cent
in the female.* Thereafter the eyeballs decrease steadily in rela-
tive weight, excepting a slight rise at one year (which, like that
for the head, is probably abnormal). It will be noted that the
percentage weight of the eyeballs is larger in the female at every
age except at six weeks. Whether this expresses a true difference
according to sex is uncertain, however, as the difference is small
and the data perhaps inadequate to determine this point.

In connection with this difference in relative weight accord-
ing to sex, it is noteworthy (table 4) that the absolute weight
of the eyeballs is nearly the same in both sexes at every age.
Since the average body weight of the female is smaller at each
of the ages noted (except six weeks), it follows that the relative
weight of the eyeballs must be correspondingly larger in the
females.

The similarity in the absolute weight of the eyeballs in both
cases at various ages suggests the possibility that the growth of
these organs may be somewhat independent of influences affect-
ing the growth of the body as a whole. This idea is to a certain
extent confirmed by the coefficient of variation in the absolute
weight of the eyeballs (table 4). Although at birth this coeffi-
cient (15.5) appears larger than that of the whole body (12.3),
after the age of seven days the variation in the absolute weight
of the eyeballs is exceedingly low (7.45 to 13.3), only the brain
approaching it in this respect.

5 In comparing the observed with the calculated values in this and the other
organs, it should be pointed out that even slight fluctuations in absolute weight
which may lie within the experimental error, may produce a much greater per-
centage deviation in the youngest animals, owing (1) to the small absolute size

of the organ, and (2) to the relatively greater weight of the organ in the earlier
periods.

POSTNATAL GROWTH IN THE ALBINO RAT 25

Furthermore, the idea of an independent growth of the eye-
balls is supported by the variation in the percentage weight rela-
tions (table 4). While in the newborn the coefficient of variation
in the percentage weight is less than that for the absolute weight
(the usual relation in other organs at all ages), it is a remarkable
fact that after seven days the variation in the eyeballs is decidedly
greater for the percentage weight. Finally, the coefficient of cor-
relation between body weight and weight of the eyeballs, as
might be expected from the foregoing, is comparatively low and
very irregular (0.21 to 0.67).

4. Thyroid gland

When the calculations are made according to Hatai’s formula,
the relative weight of the thyroid (fig. 3 a) is greatest in the
earliest stages, forming about 0.029 per cent of the body weight
at 5 grams. The relative weight decreases slowly to 0.028 per
cent of the body at 10 grams, 0.026 per cent at 20 grams, 0.022
per cent at 50 grams, 0.019 per cent at 100 grams, 0.016 per cent
at 200 grams, and 0.015 per cent at 300 grams.

On account of certain difficulties, the number of observations
upon the thyroid gland in my own series is somewhat limited.
The gland is small and difficult to separate accurately from the
adjacent muscles, especially in the younger stages. It was also
sometimes injured in the process of decapitation. The obser-
vations recorded, arranged according to age (table 5, fig. 3 a),
indicate that the thyroid is relatively largest in the newborn,
decreasing from about 0.04 per cent of the body weight to 0.018
per cent at one year. In comparison with Hatai’s theoretical
curve of growth, there is in my data a lagging behind during the
first week. The thyroid apparently increases but slightly during
this period while the body weight doubles. This causes a con-
siderable drop in the percentage weight of the thyroid at seven
days. No difference according to sex is apparent. On account
of the limited data, the coefficients of variation and correlation
were not calculated.

Watson (710) finds the thyroid (and parathyroid) glands usu-
ally enlarged in rats fed upon meat, and especially oatmeal, diet.

26 Cc. M. JACKSON

They were also found relatively much larger in wild rats, due
probably to difference in exercise and diet.

5. Thymus

On account of inadequate data, Dr. Hatai was unable to con-
struct a satisfactory formula for the growth in absolute weight
of the thymus in the albino rat.

In my own data grouped according to age periods (table 6,
fig. 2 b), it appears that the thymus increases, in the male, from
about 0.15 per cent of the body weight at birth to 0.24 per cent
at seven days, and to a maximum of 0.38 per cent at twenty
days. Thereafter it decreases, and at one year forms an average
of only 0.02 per cent of the body. The relative weight under-
goes a similar change in the female with no significant difference
according to sex.

A unique feature of the thymus is its decrease in absolute as
well as in relative weight. As shown in table 6, the average
absolute weight at five months is slightly smaller than at ten
weeks, and at one year it has undergone a very striking decrease.
This is of course in connection with the process of involution,
following the age of puberty, whereby the thymus is largely
transformed into a mass of adipose tissue.

As would necessarily follow from the decrease in the absolute
weight of the thymus during the process of involution, there is
actually a negative correlation at five months between the thymus
and body weights. During the earlier life, on the other hand,
there is a well marked positive correlation, the coefficient increas-
ing from 0.668 at birth to 0.904 at six weeks (the high io
being partly due to ‘spurious correlation’).

The coefficient of variation in absolute weight is high, increas-
ing from 30.9 at birth to 50.4 at six weeks. Later, however,
when a greater variation might naturally be expected during the
involution process, it is actually less, the coefficient being 25.3
and 22.2. For the percentage weight, the coefficient of variation,
though still high, is considerably smaller than that for the abso-
lute weight, excepting the periods at ten weeks and five months.

POSTNATAL GROWTH IN THE ALBINO RAT vat

6. Heart

According to the curve of relative (percentage) weight (fig. 1 a)
constructed from Hatai’s formula, the heart increases from 0.64
per cent of the body weight at 5 grams to a maximum of 0.70
per cent at 10 grams. Thereafter it gradually decreases to 0.67
per cent at 20 grams, 0.56 per cent at 50 grams, 0.45 per cent at
120 grams, 0.42 per cent at 170 grams, and 0.40 per cent at 200
grams, 0.37 per cent at 300 grams and 0.35 per cent at 400 grams.

When grouped according to age periods, my own data (table 7,
fig. 1a) show an increase from an average in the male of 0.65 per
cent at birth to 0.68 per cent at seven days and to a maximum
of nearly 0.73 per cent at twenty days; thereafter it decreases
to about 0.45 per cent at one year. In the female the relations
are similar, although the average relative weight is greater at
every age (except twenty days). This apparently greater rela-
tive weight of the heart in the female, however, is probably with-
out significance. In the first place, the relative weight of the
heart in the female at any given age beyond six weeks should
be slightly greater on account of the smaller body weight. More-

Fig.l a HEART Percentage of body weight.
Curve derived from Hatai’s formula (for males).
Means at seven age periods from Jackson's data; males, ©, females, ®.

Fig.1.5 EYEBALLS Percentage of body weight.
Curve derived from Hatai’s formula (for males).
Means at seven age periods from Jackson's data; males, 0, females, ®.

Percentage of body weight

150
Body weight in grams

28 Cc. M. JACKSON

=

Fig.2a LUNGS Percentage of body weight.
Curve derived from Hatai’s formula (for males).
Means at seven age periods from Jackson’s data; males, ©, females, ®.

Percentage of body weight

Fig.2b THYMUS Percentage of body weight.
Means at seven age periods from Jackson's data; males, O, females, ®.

150
Body weight in grams

over, the differences between the sexes in percentage weight
shown in the table are comparatively slight, usually well within
the limits of probable error. It is noteworthy that my data give
percentage weights which are slightly, but almost constantly,
higher than those of the curve derived from Hatai’s formula
Giga

The heart shows a considerable degree of variability in abso-
lute weight (table 7), the coefficient increasing from 18 at birth
to 33.7 at twenty days and 29.7 at six weeks. At ten weeks
and five months the coefficient decreases (18.4 and 21.3) to near
that at birth. For the relative weight, however, the relations
are quite different. At birth and seven days, the coefficient of
‘variation for the percentage weight of the heart is rather high
(15 to 18), though still somewhat less than that for the absolute
weight. From twenty days onward, however, there is remark-
ably little variation in the percentage weight of the heart, the
coefficient being usually less than 10. This corresponds to the
high coefficient of correlation between the weight of the heart
and the body weight after twenty days (0.840 to 0.968), which

POSTNATAL GROWTH IN THE ALBINO RAT 29

at birth and seven days is much lower (0.583 and 0.504). How-
ever, the high figures for the older rats are partly due to a
‘spurious correlation,’ as will be explained later.

7. Lungs

According to calculations made from Hatai’s (’13) formula, the
lungs, as shown by the curve in figure 2 a, decrease in relative
(percentage) weight from a maximum of 1.60 per cent of the
entire body weight at 5 grams to 1.32 per cent at 10 grams,
1.06 per cent at 20 grams, 0.80 per cent at 50 grams, 0.64 per
cent at 120 grams, 0.59 per cent at 200 grams, 0.55 per cent at
300 grams, and 0.54 per cent at 400 grams.

The relative weight of the lungs in the male according to my
data increases from about 1.59 per cent of the body weight at
birth to 1.93 per cent at seven days. From this maximum it
decreases to 0.68 per cent at six weeks, and 0.57 per cent at ten
weeks. The apparent increase at five months and one year is
probably due to the inclusion of unrecognized pathological cases.
Except at twenty days, the relative weight of the lungs appears
slightly higher in the female than in the male. This is chiefly
because the body weight is usually greater in the male. Any
sexual difference aside from this is very doubtful. It will be
observed that the curve derived from Hatai’s formula does not
show the preliminary increase in percentage weight which is
indicated by my data, and which is found so characteristic of
the viscera in general.

The variability of the lungs in absolute weight at birth (23.3)
is much greater than that of the whole body, but is later approxi-
mately equal to it in most cases (16.6 to 23.9). The variation
in percentage weight is usually much less than in absolute weight;
and the correlation with the body weight is correspondingly high
(73.6 to 94.3 except at ten weeks, 62.3). The high figures are
partly due to a ‘spurious correlation.’

The weights of the right and the left lungs were also taken
separately, although not recorded in the table. The right lung
is always much larger than the left, the ratio being approxi-

30 Cc. M. JACKSON

mately 2 : 1, which does not change appreciably according to age.
The variability in absolute weight of right and left lungs taken
separately, is about the same as for both together, as shown by
table G.

TABLE G

Coefficient of variation for the lungs (sexes together)

|
| » |
NEWBORN 4 DAYS 20 DAYS | 6 WEEKS

44m. 43 f. 30m. 27f. | 24mb25f. | 22m.176
, : | | | |
Bothvlungeys eres een | 23.3+1.3 | 16.6+1.1 23.9+1.7 | 19.4+1.5
Right lunes 5.) eee. cnet oe \) 22 path 0 24.9 19.7
2.9 | Lysis: Dp. 19.4

Aoektiwlum oe See ee A be 22.6

The coefficient of correlation between the body weight and
weight of lungs, including right and left lung separately, is given
in table H. While the coefficients are too high (due to ‘spurious
correlation’) those at any given age may be safely compared with
each other.

TABLE H

Coefficient of correlation between the lungs and the net body weight (sexes together)

NEWBORN | 7 DAYS 20 DAYS | 6 WEEKS

dom: 43f.. je 30am t | .ddmeee, |) 29 ne aiae
‘Both lungs...... | 0.736+0.033 | 0.799+0.032 0.868+0.024 | 0.943-£0.012
Right lung...... | 0.739+0.033 | 0.813+0.030 | 0.854+0.026 | 0.949+0.011
Left dune eee 0.693+0.038 | 0.687+0.047 | 0.876+0.022 | 0.902+0.020

From this table it is evident that the correlation between body
weight and right lung is practically the same as between body
weight and both lungs; and that the correlation between body
weight and left lung is usually but slightly less. These figures
show no evidence of the existence between the right and left
lungs of any compensatory regulation in size, for deviations
within the limits of normal variation. This question may be
complicated by other factors, however, so that it is unsafe to
draw any definite conclusion regarding the matter.

POSTNATAL GROWTH IN THE ALBINO RAT B31
r 8. Laver

The curve representing the relative (percentage) weight of the
liver, according to calculations from Hatai’s formula, is shown
in figure 6a. It is seen that the relative weight of the liver
increases from 4.87 per cent of the body weight at 10 grams to
a maximum of 7.80 per cent at 25 grams, after which it gradually
decreases to 7.17 per cent at 50 grams, 5.75 per cent at 120
grams, 5.02 per cent at 200 grams, 4.55 per cent at 300 grams,
and 4.27 per cent at 400 grams.

When grouped according to age periods, my own data (table
9, fig. 6 a) show that in the male the relative weight of the liver
forms an average of 4.74 per cent of the body weight at birth.
Unlike the organs previously considered, it apparently decreases
at seven days, to 3.39 per cent, increasing to 4.64 per cent at
twenty days and reaching a maximum of 6.78 per cent at six
weeks. Thereafter it decreases to an average of 4.42 per cent at
one year. The female exhibits a similar course of growth; but is
slightly larger relatively than the male at birth and seven days,
and smaller at all later periods.- My data therefore indicate a
sexual difference, although none was found by Dr. Hatai in his
data.

It will also be noted that the liver in my data is at all periods
relatively small, when compared with the formula derived from
Hatai’s data. It is possible that the discrepancy may be due to
a slight difference in the diet. Chalmers Watson (’10) finds a
marked decrease in the relative size of the liver in captured wild
rats, and ascribes the decrease to a diminution in the protein of
the bread and milk diet during captivity. If the lower figures
in my data were due to this cause, however, we should expect to
find a similar condition in the kidneys, which Watson finds also
to be larger with rich protein diet. As will be seen later, how-
ever, my data usually show for the kidneys a higher relative
weight than that according to the formula calculated from Hatai’s
data. The difference, therefore, can hardly be explained on this
basis.

ae Cc. M. JACKSON

The large but irregular variability of the liver in the present
data is shown in table 9. The coefficient of variation in abso-
lute weight appears at different periods from 18.6 to 40.8, but
with no definite change according to age or sex. The variation
in the percentage weight is seen to be, as a rule, very much less.
Accordingly, the coefficient of correlation between liver and body
weight is high, from 73.6 to 96.8 (high figures, however, partly
due to ‘spurious correlation’).

Fig.3 a THYROID GLAND Percentage of body weight.
Curve derived from Hatai’s formula (both sexes).
: Ty o Means at seven age periods from Jackson's data; males, 0, females, ®.
020

Percentage of body weight
xa

Ges ’
20H
Fig.3 b SPLEEN Percentage of body weight.
5 Curve derived from Hatai’s formula (males, enlarged spleens excluded).
J

Means at seven age periods from Jackson's data; males, 0, females, ®.

0 510 20 50 100 150 200 300
Body weight in grams

9. Spleen

The relative (percentage) weight of the spleen, calculated from
Hatai’s (13) formula, is represented by the curve in figure 3 b.
It is seen that the spleen increases in relative size from 0.16 per
cent of the body weight at 5 grams to 0.30 per cent at 10 grams,
and a maximum of 0.32 per cent at 20 grams, thereafter decreas-
ing slowly to 0.30 per cent at 50 grams, 0.28 per cent at 120
grams, 0.27 per cent at 200 grams, and 0.26 per cent at 300 to
500 grams.

POSTNATAL GROWTH IN THE ALBINO RAT 33

When grouped according to age periods (table 10, fig. 3 b)
my own data show an increase in the relative size of the (male)
spleen frdm 0.22 per cent of the body weight at birth to a maxi-
mum of 0.41 per cent at seven days. For succeeding ages, the
percentage weight agrees fairly with that derived from the for-
mula, excepting the unusually high figure (0.396 per cent) at
one year. ‘There is no distinct difference between the sexes, or
change in relative size according to age (after the first week).

It may be noted that my data for the spleen tend to run some-
what higher than the curve derived from Hatai’s formula. This
may be because Hatai’s formula is based upon data from which
the ‘enlarged’ spleens had been excluded. His figures for all
spleens average very much higher than mine.

The spleen is known to be an unusually variable organ and
this is certainly true for the rat. As shown in table 10, the
coefficient of variation in absolute weight is from 25 to 51 (ex-
cepting at five months, 18.9) The coefficient of variation in per-
centage weight is usually but slightly less than that in absolute
weight. The coefficient of correlation between spleen and body
weight is accordingly somewhat low, varying from 0.406 to 0.542
with an exceptionally high correlation (partly ‘spurious’) of 0.967
at twenty days.

10. Stomach and intestines

From the curve of relative (percentage) weight constructed
from Hatai’s (13) formula (fig. 5), it appears that the empty
alimentary canal increases from about 3.0 per cent of the body
at 10 grams, to 7.5 per cent at 20 grams, and a maximum of 8.0
per cent at about 35 grams, after which it decreases to 6.3 per
cent at 100 grams, 5.0 per cent at 200 grams, and 4.5 per cent
at 300 grams.

Observations were made upon the empty intestinal canal in
only a part of my own series. These observations, grouped
according to ages (table 11, fig. 5) indicate that the relative weight
of the empty tract increases from an average of about 2.4 per
cent of the body weight in the newborn to a maximum of about
8 per cent at six weeks, decreasing to 5 per cent at one year.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No 1

34 Cc. M. JACKSON

They therefore agree fairly well with the curve from Hatai’s
formula. The data are too few for calculation of the coefficients
of variation and correlation at the various ages.

A complete series of observations is available for the stomach
and intestines plus contents, however, a summary of which is
given in table 12 and figure 5. The increase in relative weight
runs somewhat parallel to that for the empty tract, increasing
from an average of about 6 per cent of the body weight in
the newborn toa maximum of about 16 per cent at six weeks,
decreasing thereafter to about 7 per cent at one year.

The coefficient of variation for the canal with contents (calcu-
lated only to six weeks) is somewhat high, 28.6 to 42.1, but not
so high as was anticipated. It is, as might be expected, highest
in the newborn (only a part of which had suckled) and at twenty
days (weaning period). The coefficient of correlation between
body weight and canal with contents is low in the newborn (0.29)
but comparatively high later (0.59 to 0.84), partly due to ‘spuri-
ous correlation.’ Although one naturally thinks of the alimentary
canal with contents as exceedingly variable in size, it is inter-
esting to note that it is actually less variable than some other
viscera. In general, after the newborn age, it is usually less
variable and more closely correlated with the body weight than
are the ovaries, spleen and suprarenals. It is on account of this
degree of correlation between body weight and alimentary canal
with contents that in the rat the gross weight serves almost as
well as the net body weight as a basis for estimating the relative
weight of the various organs.

11. Suprarenal glands

From the curves of relative (percentage) weight (fig. 4) cal-
culated according to Hatai’s formulas, it is seen that the relative
weight of the suprarenals in the male increases from 0.031 per
cent of the body at 5 grams to a maximum of about 0.040 per
cent at 10 to 15 grams, thereafter decreasing to 0.038 per cent at
20 grams, 0.030 per cent at 50 grams, 0.021 per cent at 120 grams,
0.018 per cent at 160 grams, and 0.014 per cent at 300 grams. In

POSTNATAL GROWTH IN THE ALBINO’ RAT 35

SUPRARENAL GLANDS Percentage of body weight.
Curve derived from Hatai’s formula, males, ___, females,
Means at seven age periods from Jackson's data; males, ©, females, ®.

Se mo ml
Mem ome ween ee meee = come senerens =--

Percentage of body weight

0 510 20 50 100 150 200 300
Body weight in grams

Percentage of body weight.

STOMACH-INTESTINE Percentage of body weight.

Curve, for empty canal, derived from Hatai’s formula (for males).
Means at seven age periods from Jackson's data.

For empty canal, males, ©, females, 8;

For canal with contents; males, e, females, +

0 15}10 »20 50 100 150 200 “300!
Body weight in grams

36 Cc. M. JACKSON

the female the relative weight increases from 0.037 per cent of
the body at 5 grams to a maximum of about 0.042 per cent at
10 grams, decreasing to 0.038 per cent at 20 grams, 0.032 per
cent at 50 grams, 0.029 per cent at 100 grams, 0.027 per cent at
160 grams, and 0.026 at 300 grams. Thus the suprarenal bodies
appear relatively much larger in the female, especially for a body
weight above 100 grams.

.Grouped according to age periods, my own data (table 13,
fig. 4) likewise show the relative growth of the suprarenals to be
quite different in the two sexes. In the male, the maximum rela-
tive size, 0.038 per cent of the body weight, apparently occurs
in the newborn, decreasing to 0.023 per cent at seven days, but
increasing again to 0.036 per cent at twenty days, which is nearly
as large as the maximum. Thereafter the relative weight de-
creases to 0.027 per cent at six weeks, 0.018 per cent at ten weeks,
and 0.016 per cent at five months and one year. The course of
relative growth is somewhat similar in the female, but the maxi-
mum (0.043 per cent) occurs at twenty days instead of at birth
(0.041 per cent). After six weeks, the relative size is much greater
in the female, being 0.026 per cent to 0.028 per cent of the body
weight. Even the absolute weight of the suprarenals (after six
weeks) is considerably greater in oe female, although the body
weight is much less.

When compared with the ee cal curve of growth, a marked
discrepancy may be noted in the present data (table 13) at seven
days. For some unaccountable reason, the suprarenals in my
series appear to lag behind during the first week, increasing in
weight only about 20 per cent, while the body weight is doubled.
There is also a corresponding irregularity in correlation with the
body weight at seven days, which is very low, especially in the
male.

The coefficient of variation in absolute weight is somewhat
high and irregular (20.4 to 38.7) with variations showing no
definite relation to age or sex. The coefficient of variation in
percentage weight is usually but little, if any, lower. The coeffi-
cient of correlation between suprarenals and body weight is cor-

POSTNATAL GROWTH IN THE ALBINO RAT ai

respondingly low, 0.347 to 0.583 (except at seven days) for the
sexes combined. In the sexes, separately, however, the correla-
tion is usually somewhat higher, as might be expected, since the
sexes show a characteristic difference in the relative weight of
the suprarenals.

12. Kidneys

The relative (percentage) weight of the kidneys, reckoned
according to Hatai’s formula, is shown by the curve in figure 6 b.
The kidneys appear in the newborn male to form 0.90 per cent
of the body weight at 5 grams, which increases to a maximum
of 1.53 per cent at 10 grams, thereafter decreasing to 1.38 per
cent at 20 grams, 1.10 per cent at 50 grams, 0.91 per cent at 130
grams, 0.85 per cent at 200 grams, 0.82 per cent at 300 grams,
and 0.80 per cent at 400 grams.

When grouped according to age periods, my own data (table
14, fig. 6 b) show the relative (percentage) weight of the kidneys
to increase in the male from 0.96 per cent of the body weight
at birth to 1.29 per cent at seven days and to a maximum of
1.44 per cent at twenty days. Thereafter it decreases to 1.27
per cent at six weeks, 1.03 per cent at ten weeks, 0.93 per cent
at five months, and 0.95 per cent at one year. In the females,
the average percentage weight of the kidneys is usually slightly
higher than in the male of the same age, but nearly the same as
that of a male, with the same body weight. The agreement of
my data with the curve derived from Hatai’s formula is remark-
ably close (fig. 6 b).

The coefficient of variation of the kidneys in absolute weight
(table 14) is greater than that of the body in the earlier periods,
reaching a maximum of 33.7 at twenty days. Later, however,
the kidneys become less variable than the body weight, the coef-
ficient being 15 to 19. The variation in the percentage weight
is low, especially from seven days onward. The coefficient of
correlation between kidneys and body weight is correspondingly
high, being 0.703.at birth, and 0.788 to 0.955 at later age periods,
(partly due to ‘spurious correlation’). No constant difference in
variation is noticeable according to age or sex.

38 Cc. M. JACKSON

RESO Lee

Fig.6 a LIVER Percentage of body weight.
Curve derived from Hatai’s formula (for males).
ee Means at seven age periods from Jackson's data; maies, O, fernales, ©.

Percentage of body weight

Fig.6 b KIDNEYS Percentage of body weight.
Curve derived from Hatai’s formula (for males).
Means at seven age periods from Jackson’s data; males, ©, females, ®.

0 510 20 50 100 150 200 300
Body weight in grams

1.50
%
1.20) —_|

30
Fig.7 a TESTES Percentage of body weight.
60 a) Curve derived from Hatai’s formula (epididymis not. included).
Means at seven age periods from Jackson’s data (epididymis included).
30
@
ie)
040/74
%

[o)
@®
(s)

Percentage of body weight

.020 32
bs Fig.7 b OVARIES Percentage of body weight.
. Curve derived from Hatai’s formula, *
010. eee . Means at seven age periods from Jackson's date.
0 510 20 50 100 _ 150 200 300

Body weight in grams

POSTNATAL GROWTH IN THE ALBINO RAT 39

13. Gonads

a. Testes. The relative (percentage) weight of the testes, cal-
culated from Hatai’s (13) formulas, is represented by the curve
in figure 7 a. This gives a relative weight of 0.40 per cent of
the body weight at 10 grams and about the same at 20 grams.
Unlike that of other organs, the relative weight of the testes con-
tinues to increase for a long time (up to age of puberty), being
0.80 per cent at 50 grams and reaching a maximum of 1:30 per
cent from 90 to 100 grams. Thereafter it decreases to 1.28 per
cent at 120 grams, 1.16 per cent at 170 grams, 1.09 per cent at
200 grams, 0.90 per cent at 300 grams, and 0.76 per cent at 400
grams.

Unfortunately, in my own data the epididymis is included with
the testes and the results are therefore not strictly comparable
with those of the formula above, which do not include the epi-
didymis. A few special measurements indicate that thé epididy-
mis forms one-fifth or one-sixth of the weight of the testes proper
in the younger rats, which however increases (with irregular vari-
ations) to one-third after the age of puberty, at about ten weeks.
In extreme cases, the weight of the epididymis is one-half that
of the testis proper. The weight of the testes proper, as well
as of accessory sexual apparatus (epididymis, seminal vesicles, et
cetera), probably undergoes considerable change during cycles of
sexual activity. This has been noted by Disselhorst (’98, ’08)
in various mammals and especially in birds, for the gonads in
both sexes.

My own data (table 15, fig. 7 a) indicate that the testes (in-
cluding the epididymis) form an average of about 0.13 per cent
of the body at birth, increasing gradually to a maximum of 1.50
per cent at ten weeks, and decreasing somewhat at later periods.
The variability in absolute weight is quite high, the coefficient
varying from 25.3 at birth to 40.7 at five months (excepting at
seven days, when it is only 17.7). The coefficient of variation in
percentage weight is much lower (10.3 to 32.8) and the correla-
tion with the body weight higher than might be expected (0.67
to 0.95, except at ten weeks, 0.48). These figures are somewhat
too high, however, due to ‘spurious correlation.’

40 C. M. JACKSON

b. Ovaries The growth of the ovaries is quite complex. In the
relative (percentage) growth curve (fig. 7 b) constructed from
data according to Hatai’sformulas, two distinct phases are notice-

able. In the first phase, beginning at birth, the ovaries increase
' in relative size from about 0.017 per cent to a maximum of about
0.030 per cent at a body weight of 10 to 15 grams. Thereafter
the ovaries decrease to about 0.015 per cent of the body weight
at 60 grams. Then begins the second period of acceleration,
corresponding to the advent of puberty, during which the ovaries
increase to a second maximum of about 0.037 per cent of the
body weight at 110 to 120 grams. Thereafter the ovaries lag
behind steadily in relative growth, and form only about 0.017
per cent of the body weight at 300 grams.

When grouped according to age periods, my data (table 15,

fig. 7 b), show considerable irregularity in the relative size of
the ovaries. From an average of about 0.017 per cent of the
body at birth they increase (after an apparent drop at seven
days) to a first maximum of about 0.022 per cent at twenty
days. Then they decrease to about 0.020 per cent at six weeks,
increasing to a second maximum of 0.034 per cent at ten weeks,
the age of puberty. Thereafter they decrease, averaging 0.025
per cent of the body weight at one year.
_ The extremes recorded in the size of the ovaries (table 15)
show a remarkable range. This is due partly to difficulty during
the earlier stages in dissecting out the ovaries accurately, and
partly to fluctuations in size in the later stages, probably on
account of cycles of ovulation. Coefficients of variation were
not calculated for the earliest stages, as the data were consid-
ered inadequate. For periods from twenty days onward, the high-
est coefficient of variation in absolute weight, 50.9, was found at
ten weeks (the age of puberty), and the lowest, 32.7, at five
months. The coefficients of variation in percentage weight are
somewhat lower (25 to 39); and the coefficient of correlation
between ovaries and body weight varies from 0.64 to 0.82 (some-
what too high, due to ‘spurious correlation’).

POSTNATAL GROWTH IN THE ALBINO RAT 41

14. General considerations

We may now review briefly certain phases of growth and varia-
bility of the viscera. First should be noted the growth of the
viscera, taking the group as a whole (including brain), in com-
parison with the remainder of the body. As found by Jackson
and Lowrey (12), the visceral group of the rat at birth forms
about 18 per cent of the body weight. It -increases in relative
weight to an average of about 19.2 per cent at seven days,
and to a maximum of about 21.3 per cent of the body weight
at three weeks. At six weeks, it has decreased slightly, to 20.4
per cent, but continues to decrease in relative weight to about
16 per cent at ten weeks, 14.8 per cent at five months, and 13.3
per cent at one year. ;

Data for comparison of the relative size in other animals are
cited by Jackson and Lowrey (712), who point out that in gen-
eral the smaller mammals have a relatively larger visceral appa-
ratus, probably correlated with a more intense metabolism. The
rat occupies a somewhat intermediate position, the relative weight
of the viscera being less than that of most of the smaller mammals,
but greater than that of the larger mammals.

Since the visceral group forms a comparatively small part of
the entire body, its relative size will evidently be influenced greatly
by the rate of growth of the other parts of the body.. Jackson
and Lowrey (’12) have shown that in the rat the relative increase
in the weight of the viscera during the first week is accompanied
_by a similar slight increase in the skeleton and a very marked
increase in the relative weight of the integument. The relative
increase in these three systems is apparently balanced by a small
decrease in the relative weight of the musculature, and by a
remarkable decrease in the ‘remainder,’ due chiefly to a disap-
pearance of excess body liquids. The slight further increase in
the relative weight of the viscera at three weeks is accompanied
by an increase in the musculature, and is balanced by decreases
in the skin, skeleton and remainder. Thereafter the slow gradual
decrease in the relative weight of the visceral group is accom-
panied by a similar decrease in the skin and skeleton. This is

42 Cc. M. JACKSON

balanced by a corresponding increase in the musculature, which
by its large size virtually dominates the further growth of the
body as a whole.

It is further evident that the growth of the body as a whole
is the resultant of unlike growths of its various component sys-
tems. This principle applies likewise to the growth of the vis-
ceral group, the various organs having each its own characteristic
mode of growth. As a matter of fact, only three individual
organs, thymus, heart, and kidneys (and perhaps also the supra-
renals) have their apparent maximum relative weight at the age
of three weeks, when the visceral group as a whole appears rela-
tively largest. The stomach and intestines and the liver appar-
ently reach their maximum relative weight at the latter period
of six weeks, and the gonads at ten weeks. Of those reaching
their maximum at an earlier period, the brain, spinal cord, eye-
balls, lungs, and spleen appear to be relatively heaviest in the
second week while the thyroid gland appears relatively heaviest
in the newborn.

Somewhat similar relations as to the relative growth of the
various organs were found by Kellicott (’08) in the dogfish. Of
the various organs observed, only the brain and rectal gland
appear relatively largest at birth. The heart, pancreas, spleen
and liver increase rapidly so as to reach their maximum relative
_ size a short time after birth, and thereafter decline steadily (with
a secondary increase in the liver, due to accumulation of fat).
The gonads reach their maximum relative size at sexual maturity
in the dog-fish.

Scattering data for the growth of the human viscera are re-
corded by various authors (for references, see paper by Jackson
09), but they are scarcely adequate to determine the question
as to the course of postnatal relative growth for the individual
organs. In general, so far as may be judged from the data avail-
able, the human visceral group appears relatively larger at birth
than at any subsequent age. The lungs, however, appear to
increase somewhat in relative weight so as to reach a maximum
after birth. This is perhaps also true of the heart, kidneys and
gonads, and it is quite possible that more extensive data would

POSTNATAL GROWTH IN THE ALBINO RAT 43

show this to be true also for other organs. Some difference
between man and rat might naturally be expected, however,
since the rat is born in a more immature condition.

The variability in the weight of the body must also depend
ultimately upon the variability of its component parts. As to
the variability of the component parts of the rat, we have data
for the head and viscera only. A brief summary of the coeffi-
cients of variation in round numbers (from tables 2 to 15; sexes
together, excepting gonads and brain) is given in table I.

The difference in variation between the sexes is usually not
very marked (as may be noted in tables 2 to 15); although there
appears to be a tendency to greater variability in the male, both
in body weight and in weight of the individual viscera. In the
table above it may be noted that the head and head organs
(brain, eyeballs) form a group of small variability (average coeffi-
cient 10 to 12) which is usually far below that for the body as a
whole (average 19). The other organs are more variable than
the body. The lungs, kidneys, heart, liver, and suprarenals form
a moderately variable group (average 21 to 26) while the gonads,
thymus, spleen and intestinal canal (with contents) are exceed-
ingly variable (average 29 to 43).

The average coefficient of variation for the viscera examined
is 25. It is noteworthy, however, that the average coefficient of
variation is lowest at birth and one week, and highest at three
weeks. This agrees with what is found also for the body as a
whole.

A brief summary of the coefficients of correlation between the
body weight and the weight of the individual viscera (from tables
3 to 15, sexes together, excepting brain and gonads) is given in
round numbers in table J.

In this table likewise the differences according to sex are dis-
regarded, but even when these are taken into account (cf. tables
3 to 15) the general relations are not materially changed. In
about two-thirds of the cases, however, the coefficient of correla-
tion is higher in the males than in the corresponding females;

6 The blood, the percentage weight of which in adult rats, according to Chisolm
(11), has a coefficient of variation of 10.7, possibly also belongs in this group.

44

Coefficients of variation in weight: albino rat

C. M. JACKSON

TABLE I

6 WEEKS

NEWBORN, 1 WEEK 3 WEEKS 10 WEEKS | 5 MONTHS | AVERAGE

isa Wy ale 7 12 , i 40
Eyeballs...... 16 15 13 8 11 Ores 12
ieadseneeenm: 10 iil 15 10 14 13 12
Total body... 12 16 28 21 20 19 19
unest aera 23 17 24 19 21 21
Kidneys...... 24 22 34 15 17, a 19 22
Eleantee eee 18 20 SA 30 1334 21 24
Miverss oe eeeen A | c.19 41 19 33 25 26
Suprarenals. . ph ea 20 33 22 21 39 26
iesteseee. 22-4 25 18 RO 27 35 4] 29
Thymus S| 32 43 50 25 224.1 | 34
Spleen...... BOW etl Bom 51 26 38 19° >|) « 84
Intestinal |

canal (plus © |

contents)... 38 29 42 30 | ) 018s
Ovaries....... 42 47 51 33 | 43

Average of | 5 |

viscera...| 23 | 22 Sia 24 26 24 25
1 From combined data as explained under Central nervous system.
TABLE J
Coefficients of correlation with the body weight: albino rat
NEWBORN 1 WEEK | 3 WEEKS 6 WEEKS | 10 WEEKS | 5 MONTHS | AVERAGE
| |

elec oc coed Osne 0.89 0.93 0.95 0.75 0.85 0.86
Kidneys...... 0.70 0.79 0.96 0.92 0.90 0.91 0.86
Teivier! Jace a OeiG 0.76 0.97 | 0.84 0.74 | 0.87 | 0.83
Lungs 0.74 0.80 Ors7 | 0.94 0.62 | 0.80
Brains: aoe 0.69 0.78 0.88 ae a 0.78
1siGaittve ae seus 0.58 0.50 0.91 0.97 0.86 | 0.84 0.78
Testes... 0.67 0.75 0.95 0) 75) 0.48 0.88 0.75
Mvanlesee. nee O73 0.64 0.82 0.81 0.75
Intestinal |

canal (plus | |

contents).. 0.29 0.59 0.84 0.76 0.62
Phymuses ts) 02 OF 0.74 0.89 0.90 0.51 | —0.09 0.60
Spleen face 0.54 0.44 0.97 0.50 0.41 0.46 0.55
Eyeballs...... 0.67 0.52 OnG729| ‘Oral 0.22 0.32 0.45
Suprarenals.. 0.51 0.13 0.58 0.41 0.41 0.35 0.40

Average....| 0.63 | 0.63 | 0.85 | 0.75 | 0.62 | 0.70 | 0.70

POSTNATAL GROWTH IN THE ALBINO RAT 45

so there appears to be a tendency to closer correlation, as well as
to greater variability, in the males. It will be observed, on com-
paring this table with the preceding, that (excepting the head)
the organs most closely correlated with the body weight are not
those of least variability in weight, but a group which is some-
what more variable than the body as a whole (kidneys, liver,
lungs). In these organs, the average coefficient of correlation
varies from 0.80 to 0.86.

Next come the brain and heart (0.78). The gonads are more
closely correlated (0.75) with the body weight than might be
anticipated from their variability in weight; while the eyeballs
(0.45) and suprarenals (0.40) are lowest in the scale.

The average coefficient of correlation for the organs studied
is 0.70. It will be noted that there seems to be a direct relation
between the variability of the viscera and their correlation with
the body weight. In both it is evident that the average coeff-
cient is lowest at birth and seven days, and highest at three
weeks.

As a matter of fact, however, a higher coefficient of correlation
is to be expected during growth at times when the body weight
and organ weight are most variable. This is due to a ‘spurious
correlation,’ which has been referred to repeatedly in connection
with the discussion of correlation of the individual organs with
the body weight at the definite age periods. When all the rats
at a given age are grouped together there is naturally, on account
of their unequal growth, a considerable scattering of the body
weights and corresponding organ weights. This causes an aug-
mentation of the real coefficient of correlation, in accordance with
the general principle of ‘“‘correlation due to heterogeneity of
material”’ (Yule ’11, p. 214). To determine the true correlation
between the body weight and organ weight at any given age, it
would be necessary to have a sufficient number of animals so
that they could be separated into groups of approximately the
same body weight or organ weight. The present data are unfor-
tunately inadequate for this purpose. It is evident, however,
that all the coefficients of correlation above given are somewhat
too high, and that no conclusions can be drawn from them as

46 Cc. M. JACKSON

to change in correlation with age. Coefficients of correlation in
different organs at the same age may be compared with each other,
however, since the body weights and the ‘spurious correlation’
factor would be the same for all.

The coefficient of variation of the relative or percentage weight
is usually much lower than that of the absolute weight of the
various organs.’ The difference is inconstant, however, and the
eyeballs form a marked exception to the rule. In most cases,
therefore, the percentage weight of an organ can be predicted
much more accurately than the absolute weight of the organ at
any given age. The data agree in general with the theory that
the growth of the individual organs is correlated with the growth
of the whole body more closely than with age, as Donaldson
(08) has found for the brain of the rat.

For comparison of the variability in the human viscera, a few
data are available. Pearson (’97) from data of Reid and Peacock
calculates the coefficients of variation for the human heart (abso-
lute weight), male, 19.8, female, 20.7; liver, male, 14.5, female,
22.2; kidneys, male, 20.5, female, 22.5. Greenwood (04) from
more extensive data finds the coefficients of variation for healthy
organs (males only) approximately as follows: heart, 17.7; liver,
14.8; kidneys, 16.8; spleen, 38. From the foregoing it would
therefore appear that the heart, liver and kidneys are less variable
in man than in the rat. The spleen is more nearly alike in the
two forms, and in both is by far the most variable of the organs
compared. Pearl (’05) classifies the human bodily characters
with reference to variability in three groups: (1) those with
coefficient of variation above 10, viscera in general, whose weight
depends largely upon the general metabolic condition of the body
and in which natural selection is concerned with functional ability
rather than with size: (2) those below 7, chiefly skeletal dimen-
sions, in which the conditions of (1) are reversed; and (3) those
with coefficient from 7 to 10, including brain and skull capacity,
in which intermediate conditions are found.

7 Chisolm (’11) finds the coefficient of variation for the percentage weights in

adult rabbits, for the spleen, 46.7; kidneys, 16.5; liver, 32.5 This would indicate
that these organs are more variable in the rabbit than in the albino rat.

POSTNATAL GROWTH IN THE ALBINO RAT 47

For various skull measurements, Hatai (’08) finds the varia-
bility in the adult albino rat slightly less than in man.

Finally, the question may be raised as to the significance of
the relative size of an organ. Why does it vary so greatly in
different species and even in individuals of the same species,
especially at different stages of development? While a satis-
factory answer to this question is impossible in the present state
of our knowledge, certain features of interest in this connection
may be noted. There are several conditions according to which a
priori differences in the relative size of organs might be expected;
even though, to a certain extent, a change in the relative size of
any part tends, through physiological correlation, to produce a
corresponding change in other parts of the body.

1. The relative size of an organ may vary according to the
size of the whole body. On account of mechanical principles,
with changes in the ratio of dimensions to surface and mass of
the body, and so forth, corresponding changes in the relative
size of skin, skeleton, musculature, et cetera, might be expected,
as has been pointed out by Welcker and Brandt (’03).

2. Unequal growth of any (especially of a large) part of the body
necessarily involves an inverse change in the relative size of the
remaining parts of the body. For example, the relative increase
in the musculature during the later periods of growth involves
a corresponding decrease in the relative size of other organs.
Fluctuations in the relative amounts of intestinal contents, body
fat, hair, and so forth, may likewise produce changes in the rela-
tive size of the various organs, as has been emphasized by E.
Voit (’05).

3. Differentiation in histological structure may result in more
efficient physiological activity, so that a relatively smaller organ
may suffice to perform a given function. For example, it is
doubtless partly on this account that the embryonic heart and
other organs are relatively larger than later.

4. Changes in functional activity, as is well known, occasion
an atrophy or hypertrophy of the corresponding organs, with
resultant changes in their relative size. Thus an increase of
protein in the diet throws more work upon the liver and kidneys,

48 Cc. M. JACKSON

causing them to become relatively larger. This principle of ‘use
and disuse’ is widespread in its application as an explanation of
changes in the relative size of organs. Joseph (’08) suggests that
the relatively large heart in the smaller mammals may be due
to a ‘physiological hypertrophy,’ correlated with the more rapid
rate of pulsation.

5. Within certain limits, however, an organ is normally some-
what larger than is necessary to perform the functional activity
usually demanded. Fluctuations in the amount of this excess of
structure (which Melzer has termed the ‘factor of safety’) may
account for a part of the individual variations in relative size
found to a greater or less extent in every organ.

6. Various pathological conditions may be associated, either
directly or indirectly, with changes in the relative size of indi-
vidual organs or parts of the body.

The foregoing illustrate conditions associated with changes in
the relative size of organs, and representing for the most part
teleological explanations, rather than actual causes of varying
growth rates in the different organs and parts. For any given
organ the immediate cause of its characteristic rate of growth is,
like that for the whole body, a complex of factors. These may
be divided into (a) intrinsic structure and chemical composition
of the organ and (b) its environment, especially the conditions
affecting the vascular supply, which transmits the respiratory,
nutritive and excretory materials, as well as specific hormones
or substances which may stimulate the organ to growth and
activity.

SUMMARY

Although the extensive data presented cannot satisfactorily be
summarized, some of the more important general conclusions con-
cerning growth and variability of the albino rat are included in
the following:

1. At birth the males invariably exceed the females of the
same litter in average body weight. Although growth is in gen-
eral more vigorous in the females during the first six weeks, they
fail to overtake the males in the majority of litters (at observed
ages). At six weeks, however, the total average body weight of

POSTNATAL GROWTH IN THE ALBINO RAT 49

the females approaches or slightly exceeds that of the males.
After six weeks, growth is more vigorous in the males and the
females lag behind.

2. Variability in body weight is lowest at birth (the coefficient
being about 12) and.is not much higher at seven days (16). It
appears highest at three weeks (28), and at later periods varies
from 19 to 21. The average coefficient, taking all ages together,
is 19. There is in the rat no evident correlation between varia-
bility and rapidity of growth. The coefficient of variation is
practically the same for the gross as for the net body weight.
The coefficient usually appears higher in the male, but the differ-
ence is slight and of doubtful significance.

3. Fraternal variability (within the litter) in body weight is
very low, usually less than half that of the total population of
the same age.

4, As the growth rate of the whole body is a resultant of the
varying growth rates of the component systems (musculature,
skeleton, viscera, and so forth) so the growth rate of the vis-
ceral group is a resultant of the different growth rates of the
individual organs. While the visceral group as a whole reaches
its maximum relative (percentage) weight at about the age of
three weeks, the thyroid gland apparently is relatively largest at
birth, the brain, spinal cord, eyeballs, lungs and spleen about the
second week, the thymus, heart, suprarenals (?) and kidneys at
three weeks, intestinal canal and liver at six weeks, and the
gonads at ten weeks. Differences according to sex, aside from
the gonads, are most marked in the eyeballs (?) and suprarenal
glands.

5. Similarly the variability in the weight of the body as a whole
depends upon the variability of its component parts. In this
respect the individual organs may be classified in three groups:
(1) the head and head organs (brain, eyeballs) form a group of
comparatively slight variability (average coefficient 10 to 12); (2)
the heart, lungs, liver, suprarenals and kidneys form a group of
moderate variability (average coefficient 21 to 26); (8) the thy-
mus, spleen, gonads and intestinal canal (with contents) form
an extremely variable group (average coefficient above ,29).

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 1

50 Cc. M. JACKSON

The average coefficient of variation for the viscera, taking all
ages together, is about 25. The average however is lowest at
birth and one week (22 or 23) and highest at three weeks (31),
resembling in this respect the variability in the weight of the
body as a whole.

6. The coefficient of variation for the relative or percentage
weight is usually much lower than that for the absolute weight
of the various organs, as the growth of the organs is correlated
with that of the whole body more closely than with age. The
eyeballs, however, form a conspicuous exception to this rule.

7. The highest degree of correlation with the body weight
(average coefficient 0.80 to 0.86) is found, not among the organs
of least variability (excepting the head), but among those of
moderate variability (kidneys, liver, lungs). Next come the
brain, heart and gonads (0.75 to 0.78). The remaining organs
are much less closely correlated with the body weight, the lowest
being the eyeballs and suprarenals (0.40 to 0.45). The average
coefficient of correlation, all ages together, is about 0.70. The
figures for coefficient of correlation are somewhat too high, being
augmented by a ‘spurious correlation’ due to the heterogeneity
of the weights when grouped at the age periods. Like the varia-
bility, the correlation appears lowest at birth and one week (0.63)
and highest at three weeks (0.85). Similarly the coefficient of
correlation appears usually higher in the male, as is also the
case with the coefficient of variation.

BIBLIOGRAPHY

Boas, F. 1897 The growth of Toronto school children, Report of U. 5S. Com-
missioner of Education, II.

Boas, O..0., aNd WissLER, O. O. 1904 Statistics of growth. Report U.S. .
Commissioner of Education, I.

Cutsotm, R. A. 1911 On the size and growth of the blood in tame rats. Quar-
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Davenport, C. B. 1904 Statistical methods with especial reference to biologi-
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DisseLHorst, R. 1898 Uber Asymetrien und Gewichtsunterschiede der Ge-
sehlechtsorgane, Archiv f. wiss. u. prakt. Thierheilk. Bd. 24.

~

POSTNATAL GROWTH IN THE ALBINO RAT ol

DissevHorst, R. 1908 Gewichts- und Volumszunahme der minnlichen Keim-
driisen bei Végeln und Saéugern in der Paarungszeit; Unabhingigkeit
des Wachstums. Anat. Anz., Bd. 32, 8. 113-117.

Donaupson, H. H. 1906 A comparison of the white rat with man in respect to
the growth of the entire body. Boas Memorial Volume, New York.
1908 A comparison of the albino rat with man in respect to the growth
of the brain and of the spinal cord. Jour. Comp. Neur., vol. 18.

1909 On the relation of the body length to the body weight and to the
weight of the brain and of the spinal cord in the albino rat. Jour.
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Dunn, EvizasetH H. 1912 The influence of age, sex, weight and relationship
upon the number of medullated nerve fibers and on the size of the
largest fibers in the ventral root of the second cervical nerve of the
albino rat. Jour. Comp. Neur., vol. 22.

GALTON, Francis 1894 Natural inheritance. New York.

GREENWOOD, M. 1904 A first study of the weight, variability and correlation
of the human viscera, with special reference to the healthy and diseased
heart. Biometrika, vol. 3.

Hartar, S. 1908 Studies on the variation and correlation of skull measurements
in both sexes of mature albino rats. Amer. Jour. Anat., vol. 7.

1913 On the weight of the abdominal and thoracic viscera, and sex
glands, ductless glands, and the eyeballs of the albino rat according
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Jackson, C. M. 1909 °On the prenatal growth of the human body and the rela-
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Jackson, C. M., anp Lowrey, L. G. 1912 On the relative growth of the com-
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JosppH, D. R. 1908 The ratio between the heart weight and body weight in
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52 Cc. M. JACKSON

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TABLE 1

Litters of albino rats used _

g | MALES FEMALES

@ | AGE OF lm —=

8 | LITTER Average gross Average net | | Average gross Average net
aa No. weight weight |No. weight weight

4 | (and range) (and range) | (and range) | (and range)

| grams grams | grams grams

20 | newborn | 2 | 4.12 (8.67-4.57) 4.04 (3.59-4.48) | 2 | 3.71 (3.56-3.86) | 3.65 (3.50-3.79)
22 “newborn | 5 | 4.78 (3.48-5.56) 4.69 (3.43-5.17) 3 | 4.43 (4.03-4.70) | 4.24 (4.01-4.62)
23 |newborn| 1 | 4.44 4.30 0 |

24 | newborn 5 | 5.00 (4.68-5.25) | 4.90 (4.56-5.17) 4 | 4.74 (4.62-4.91) 4.63 (4.50-4.84)
25 | newborn| 1 | 5.19 | 4.98 | 1 | 4.28 4.07

47 |newborn| 3 | 6.50 (6.40-6.62) | 6.03 (5.88-6.25) | 0 |

48 | newborn| 3 | 5.73 (5.53-5.97) 5.42 (5.24-5.59) 3 | 5.47 (5,29-5.59) 5.05 (4.90-5.20)
52 |newborn| 4 | 3.96 (3.41-4.75) | 3.82 (3.30-4.60) 1 | 3.88 | 3.72

53a| newborn) 1 | 5.27 5.09 5 | 4.93 (4.67-5.14) | 4.77 (4.52-4.98)
58 |newborn| 4 | 5.48 (5.30-5.62) 5.32 (5.16-5.45) 3 | 4.95 (4.23-5.41) | 4.82 (4.12-5.26)
61 |newborn| 4 | 5.49 (5.23-5.66) 5.35 (5.10-5.52) | 5 | 5.27 (5.00-5.53) | 5.13 (4.91-5.44)
64 |newborn| 8 | 4.77 (4.35-5.09) | 4.46 (4.06-4.74) 3 | 4.37 (4.24-4.55) | 4.07 (3.96-4.22)
M9 |newborn| 5 | 5.42 (5.1-5.8) : 3 | 5.13 (4.9-5.4)
M10 newborn} 2 | 5.85 (5.7-6.0) 4 | 5.58 (4.8-6.3)

A28|newborn| 4 | 4.71 2 | 4.37

A29)newborn| 2 | 4.70 4) 4.63

A32 newborn) 3 5.11 (4.95-5.38) 5 | 4.73 (4.50-4.91)
M8 | . 1 day 3 | 6.08 (5.75-6.35) 5 | 5.43 (4.70-6.05) |

62 | 1 day 3 | 5.41 (5.30-5.56) 5.13 (4.97-5.33) 8 | 4.96 (4.45-5.19) 4.74 (4.27-4.98)
63 | 1 day 0 5 | 4.90 (4.68-5.07) | 4.53 (4.15-4.78)
18 | 7 days 5 | 9.92 (8.54-10.46) | 9.56 (8.29-10.02)| 3 | 9.18 (8.88-9.57) 8.78 (8.58-9.11)
19 | 7 days 6 | 9.42 (9.11-9.58) 9.15 (8.85-9.33) 3 | 8.04 (7.56-8.72) 7.71 (7.19-8.32)
42 | 7 days 2 11.62 (10.80-12.43) 10.59 (9.84-11.33)| 4 12.07 (11.32-12.70) [11.02 (10.04-11.77)
46 | 7 days 1 15.56 14.95 | 1 |14.13 /13.23

49 | 7 days 7 12.36 (10.58-13.06) [11.77 (9.94-12.41) | 3 (11.60 (10.80-12.17) |11.07 (10.33 11-68)
51 | 7 days 2 |11.95 (11.89-12.00) (11.16 (11.04-11.27)) 3 |11.67 (11.50-11.77) 10.93 (10.71-11.07)
56a| 7 days 0 3 10.46 (9.87-10.96) |10.01 (9.48-10.43)
59 | 7 days 4 | 9.39 (8.07-10.13) 8.78 (7.40-9.55) ; 3 |10.12 (9.78-10.42) 9.44 (9.08-9.85)
60 | 7 days 3 | 8.48 (6.46-10.46) | 8.24 (6.32-10.15)| 4 | 9.40 (9.05-9.78) 9.10 (8.70-9.63)
M6 | 7 days 5 12.01 (9. 10-12. 95) 3 /11.55 (11. 15-12. 15)
M7 | 7 days 2 10.75 (10.20-11.30) 4 10.96 (10.55-11.40)
M8 | 7 days 3 12.22 (12.05-12.55) 5 |11.52 (10.55-12.25)
M9 | 7 days 5 | 9.26 (8.8-9.7) 3 | 8.7 (8.4-9.1)
M10! 7 days 2 {11.3 (11.1-11.5) 4 10.75 (9.2-11.8)

A287 days 4 | 7.52 2 | 7.56 e

A29| 7 days 2 12.42 4 |10.42

A32| 7 days 3 | 9.83 (9.44-10.14) 5 | 9.50 (9.04-10.08)

A383) 7 days O° 7 | 9.44 (6.70-10.12)

|

M6 | 14days | 5 21.0 (20.4-21.9) 3 /19.5 (19.1-19.9)
M8 | 14days | 3 /18.8 18.3-19.2) 5 /17.8 (16.9-18.7)
M9 | 14days | 3 18.7 (18.5-19.0) 3 /17.9 (17.5-18.6)
M10} 14 days | 2 17.7 (17.0-18.4) 4 |17.9 (15.3-19.3)

26 | 20 days | 5 16.66 (13.92-18.52) |16.12 (13.82-17.74) | 3 |16.98 (15.39-17.82) |16.20 (14.99-17.03)
43 | 20days | 3 22.68 (21.90-23.15) |21.67 (20.98-22.04) | 3 |22.58 (22.45-22.65) |21 59 (21.52-21.71)
45 | 20 days | 3 /14.02 (13.49-14.56) /13.55 (13.00-14.09)| 8 |13.31 (12.71-13.76) [12.85 (12.34-13.23)
53b| 20 days | 5 29.42 (27.25-30.94) 28.31 (25.80-29.79) | 0

56b| 20 days | 2 33.84 (29.70-37.97) |32.05 (28.20-35.90)| 0 |

57 | 20 days | 4 (22.11 (21.16-22.43) 20.50 (19.64-21.90) | 3 19.46 (18.30-20.22) 18.05 (17.34-18.60)
M1 | 20 days | 4 23.7 (22.9-24.1) | 1 25.0
M2 | 20 days | 4 26.9 (25.8-27.9) 1 |24.8
M6 | 20 days | 5 27.2 (23.7-28.8) 3 26.3 (25.8-26.8)

M7 20 days | 2 21.0 (15.7-26.4) 4 |24.4 (19.7-27.0)

53

TABLE 1 (Continued)

S MALES FEMALES
c AGE OF 2 aes = ro
B | LITTER | Average gross Average net | Average gross Average net
e | |No. weight weight No weight weight
Bi | | (and range) (and range) | (and range) (and range)
grams grams grams grams
M8 | 20 days | 3 | 26.2 (25.8-26.5) 5 | 25.1 (24.7-25.5)
M9 | 20 days | 3 | 26.9 (25.6-27.9) | 3 | 25.8 (24.3-26.6)
M10} 20 days | 2 | 24.7 (22.6-26.8) | 4 | 24.7 (21.8-26.5)
A28| 20 days | 4 | 25.4 (23.5-27.0) | 2 | 25.2 (24.3-26.2)
A29| 20 days | 2 | 24.1 (23.4-24.8) | 4 | 23.2 (21.5-24.1)
A33| 20 days | 0 | 7 | 24.6 (21.0-26.1)
15 | 2l1days | 2 | 18.22 (17.71-18.72) | 17.77 (17.45-18.08)| 8 | 17.72 (15.27-19.19) | 16.95 (14.77-17.89)
M7 | 30 days st | 45.2 (1 died) 4 | 40.8 (24.1-48.7)
M8 | 30days | 3 | 47.9 (44.8-50.2) 5 | 44.0 (41.1-48.1)
M9 | 30 days | (killed) ees | 49.9 (47.2-51.4)
29 | 41 days | 2 | 54.55 (52.9-56.2) | 49.65 (48.9-50.4) | 6 | 49.65 (42.8-53.7) | 46.0 (40.5-48.6)
30 | 41 days | 4 | 56.58 (55.1-58.4) | 50.78 (49.6-52.2) | 3 | 52.67 (40.8-58.7) | 48.43 (39.4-53.9)
M2 | 41 days | 4 | 73.5 (70.2-80.9) | 1 | 66.7 |
M9 | 42 days (killed) 3 | 82.5 (75.2-86.6) |
M10| 42 days | 2 | 89.0 (83.0-95.0) 4 | 80.0 (66.0-92.0) |
A28 | 42 days | 4 | 60.8 (57.3-63.5) 2| 65.3 (59.8-70.7) |
A29| 42 days | 2 | 66.7 (66.1-67.4) 4 61.1 (59.6-63.6) |
A33 | 42 days | 0 | 5 | 62.1 (58.4-69.8) | (2 killed)
31 | 42 days | 10 | 48.02 (39.4-53.4) | 44.63 (38.1-49.6)| 1 | 42.4 39.5
82 | 42 days | 5 | 51.78 (48.1-57.4) | 49.24 (41.3-54.9)| 4 | 46.27 (42.9-48.2) | 44.05 (39.6-46.3)
50a | 42 days | 1 | 89.0 | 82.5 | 8 | 84.57 (83.5-85.6) | 78.07 (76.8-78.8)
M6 | 42 days | 3 | 62.7 (55.0-69.0) 1 | 66.0 |
33 | 48 days | 0 3 | 49.83 (42.9-60.8) | 45.4 (40.1-54.5)
M1 | 43days | 4 | 73.1 (64.4-81.6) 1 | 70.0 |
M7 | 44 days | 1/| 57.1 | 4 | 56.8 (23.5-71.8) |
M8 | 48 days | 3 |105.9 (93.5-114.2) 5 | 86.5 (81.6-95.0)
jessy fe ~ | = ———
4 | 69days | 3 |117.1 (107.4-125.8) |112.5 (103.6-120.8) 0 |
5 | 70 days | 0 | 3.|100.7 (86.1-127.3) | 97.2 (83.2-122.2)
12 | 70 days | 3 | 89.9 (77.5-97.9) | 86.2 (74.1-93.7) | 2 |100.2 (98.2-102.1) | 89.2 (87.2-91.1)
40 | 70 days | 1 \151.1 1145.3 | 1 |101.9 97.6
41 | 70days | 4 117.8 (110.1-124.8) 112.7 (105.4-119.1)| 4 | 91.5 (82.2-106.9) | 88.1 (78.2-102.3)
50b| 70 days | 1 |182.2 (175.2 | 2 129.6 (123.9-135.4) 125.3 (120.3-130.2)
1 |; 7i days | 2 j149.9 (140.8-159.) 144.1 (184.1-154.) | 7 110.7 (85.4-126.9) 107.1 (82. 7-123.5)
44 | 71 days | #6 119.0 (111.8-134.1) (114.0 (108.4-128.6) 4 | 93.0 (75.5-100.) | 88.2 (71.1-94.8)
M8 | 70 days | 3 {187.3 (177.-193.) | | 5 /135.0 (124.-145.) |
10 149days| 0 | | 2 137.8 (126.-149.6) 134.4 (122.5-146.3)
6 150 days| 5 1158 1 (142.5-167.8) 154.7 (139.4-164.6) | 4 |145.3 (120.6-159.9) (142.1 (115.9-156.7)
11 | 150 days| 4 |157.2 (150.7-166.2) [151.7 (145.9-157.1) | 3 160.0 (150.-164.0) (151.9 (140. 1-158. 1)
36 150 days | 3 172.6 (163.3-189.8) 168.0 (156.3-186.5) | 3 127.9 (111.8-136.3) 125.2 (110.3-133.)
38 | 150 days} 6 152.0 (120.6-176.8) 149.7 (118.8-176.) | 4 114.9 (97.3-130.3) 111.9 (94. 6-126. 2)
28 | 151 days | 2 |247.2 (241.8-252.5) 243.1 (239.4-246.8) | 5 |160.8 (183.5-174.1) |158.2 (131.8-171.5)
2 | lyear 0) 2 1184.3 (183.5-185.5) (181.9 (181.-182.7)
3 | lyear 0 | | 3 192.0 (156.2-209.7) {189.1 (154.-207.3)
4 | lyear | 0 | | 2 195.8 (188.7-202.8) |190.6 (182.6-198.5)
7 | Lyear | 1 (290.5 287.3 | 2120.9 (107.3-134.5) |119.5 (105.8-133.1)
8 | lyear | 1 |142.8 139.6 1 188.3 186.0
13 | 1 year 0) 2 |174.8 (142.-207.5) 172.5 (141.-204.)
34 1 year 0 | | 1 /115.8 114.2
37 | lyear | 1 [281.1 276.4 0
39 | 1 year 2 175.3 (118.4-232.1) 172.4 (115.6-229.2) | 3 113.8 (86.1-165.0) 110.8 (83.4-161.1)
35 | 13mo. | 0 4 |175.0 (167.2-181.3) |170.7 (161.9-178.6)

54

55

POSTNATAL GROWTH IN THE ALBINO RAT

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57

POSTNATAL GROWTH IN THE ALBINO RAT

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THE ALBINO RAT

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MUSCLE DEGENERATION AND ITS RELATION TO THE
ORIGIN OF EOSINOPHILE LEUCOCYTES IN
AMPHIBIA (SALAMANDRA ATRA)

J. A. BADERTSCHER
Department of Histology and Embryology, Cornell University, Ithaca

SEVEN FIGURES

The investigation presented in this paper has a twofold object:
Primarily, to offer an explanation for the source of large numbers
of free eosin-staining granules and eosinophile leucocytes found
in connection with degenerating muscle and with the absorption
of the gills taking place during the period of transformation of
Salamandra atra; and, secondarily, to present briefly the manner
of muscle degeneration as it occurs in this form. In Salamandra
atra an intimate relation exists between the origin of the eosino-
phile leucocytes and degenerating muscle tissue so that the one
cannot be studied without considering the other.'

An investigation into the origin of the eosinophile iedoouwien
seemed proper for the reason that the source and nature of the
granules in the granular leucocytes is still a much debated ques-
tion. Also, as far as I was able to determine, no work has yet

1 The material used was prepared by Dr. B. F. Kingsbury to whom it was kindly
furnished by Professor Mollier of the Histological-Embryological Institute, Mu-
nich. I wish to express my thanks to Professor Kingsbury for the use of the mate-
rial and for the valuable suggestions given me on this work. Twenty-seven series
were examined, of which six were of animals still in the larval condition, ranging
in length from 12.5 to 38 mm.; thirteen in the period of transformation, ranging in
length from 44 to 48 mm. and seven in the adult condition. Different approved
fixers (Helly’s, Zenker’s, Tri- chlor-acetic) were used in fixing the material. Only
the head and anterior part of the body, including the heart, were sectioned. The
sections were cut from 8 to 15 micrain thickness. The greater number of the series
were stained with iron or copper (Weigert’s) hematoxylin, and eosin or orange-G
was used as a counter stain.

69

70 J. A. BADERTSCHER

been done on amphibians with a view of tracing out the origin of
eosinophile leucocytes.

Brown (’98) is of the opinion that the products of degenerated
muscle tissue are a source of eosinophile granules. In cases
of trichinosis in man, he observed that the muscle fibers invaded
by Trichina spiralis undergo a granulation in the immediate
neighborhood of the parasite. All through the affected parts of
a muscle are found large numbers of leucocytes, many of which
are of the eosinophile type. The latter are very numerous in areas
of marked degeneration. Though there were no evidences that
- the leucocytes ingested bodily fragments of muscle tissue, he
thinks that possibly the degenerated substance is taken up in a
soluble form and transformed into eosinophile granules.

Weidenreich (’08, ’11), who worked with mammals, is the chief
exponent of the theory that the eosinophile granules are hemoglo-
bin, containing products of degenerated erythrocytes. Accord-
ing to him, leucocytes may ingest entire erythrocytes which then
undergo disintegration, resulting in the formation of eosinophile
granules, or the disintegration of the red cells may take place
extracellularly and the eosinophile granules thus formed may be
taken up, in their fully formed condition, by the leucocytes. In
referring to the work of Brown he says (’11, p. 635): “‘.
auch die Moéglichkeit ist nicht a accachloe em dass das Heme
globin der Muskelfasern, . . . . eine Rolle bei der Bildung
der Granula spielt.”’

The amphibians furnish most suitable material to test tie
correctness of this theory, for during the period of metamorphosis
marked disintegration of both erythrocytes and muscle tissue takes
place. Also the degeneration of muscle tissue in this class of
animals, in contrast to the pathological degeneration in cases of —
trichinosis, is a normal process that regularly occurs while they
are adapting themselves to a different mode of life. While ~
Brown (’98) pointed out a possible relation of the origin of eosino-
phile cells to pathological degenerating muscle, no work appar-
ently has yet been done on any form to show a relation of the
origin of those cells to normally degenerating muscle.

MUSCLE DEGENERATION IN AMPHIBIA 7!

Among some of the muscles that undergo degeneration in this
salamander are the gill muscles, some of the muscles of the lower
jaw (fig. 1) and some of the dorsal muscles along the spinal column
(fig. 4). In the specimens studied, muscle degeneration was most
pronounced in those far along in their period of transformation or
in the young adult, thus indicating that even after the external
signs of metamorphosis have been obliterated, there are still mor-
phological changes taking place internallv. In the degenerating

Fig. 1 Part of a transverse section through the head of a 51 mm. young adult,
showing the position in the lower jaw of a muscle well advanced in degeneration.
M.c., Meckel’s cartilage; d.m., degenerating muscle; ¢., tongue. Photo. X 30.

muscles and gills there is also a degeneration of blood capillaries.
As the capillaries are broken down there is a marked destruction
of erythrocytes which can be satisfactorily demonstrated espe-
cially in the degenerating gills. If the eosinophile granules are
derived from degenerating red cells and degenerating muscle tis-
sue, both granules and eosinophile leucocytes should be found in
connection with those tissues. On examining the different series
from the larval to the adult condition the evidence is conclusive
that the free eosinophile granules are products of the degenerating

* P 4 .
lr. Ae 3

Fig. 2. Section of a part of the degenerating muscle represented in Fig. 1, show-
ing a large accumulation of eosinophile leucocytes in connection with the degener-
ating muscle fibers. The section being rather thick the granules in the eosino-
phile cells could not be photographed sharply. Photo. X 370.

Fig. 3 Degenerating muscle taken a few sections posterior to that represented
in figure 1. Free eosinophile granules, represented by black dots about the size of
an ordinary pin head; some a little smaller, can be seen scattered promiscuously
along two degenerating muscle fibers and in the spaces not occupied by cells, most
of which are eosinophile leucocytes. On account of the section being rather thick
not all parts could be focused sharply. Photo. X 700.

(2

MUSCLE DEGENERATION IN AMPHIBIA 73

tissues above named and are taken up by the leucocytes, trans-
forming them into the eosinophile type. These observations
extend the scope of Weidenreich’s views to the amphibia.

During the larval period only an occasional eosinophile leuco-
cyte can be found in the blood stream. A few cells containing
eosinophile granules can also be demonstrated in the connective
tissue. The number of eosinophile cells at this stage of develop-
ment is so small that only after considerable searching could one
be found. During the period of transformation, eosinophile cells
can invariably be demonstrated in connection with degenerating
muscle, the number of those cells corresponding approximately
with the advancement of degeneration. Where degeneration is at
its greatest height a large accumulation of eosinophile cellsis
present (fig. 2). Also more eosinophile cells can be demonstrated
in the blood stream and especially in the connective tissue inthe
neighborhood of the degenerating parts during the period of trans-
formation than before or after this period. No differential counts
were made of the leucocytes in the blood stream. The greater
number of eosinophile cells during the perod of metamorphosis
was evident by the great frequency in their occurrence when
searching for them in the sections.

In the section from which figure 3 was photographed can be
seen free eosinophile granules lying among the debris of degenerat-
ing muscle. That these granules lie free in the regions mentioned
is beyond doubt for when the sections are followed along in serial
order they are not found to lie in the bodies of leucocytes but lie
loose in the intercellular spaces. These granules, apparently
formed from the degenerating muscle and degerating erythrocytes,
vary somewhat in size, but the majority closely approximate in
size and hue those found in the eosinophile leucocytes. They
have various positions in respect to the degenerating muscles fibers.
They may be found among lymphocytes or eosinophile leucocytes
some distance away from a muscle fiber, scattered promiscuously
among them, strung along in a row between two adjacent fibers,
or often in small recesses of a muscle fiber. Some muscle fibers
can be seen, the central portions of which are completely degen-
erated, the gap between the two remaining ends being filled in

Fig 4 Transverse section through the neck of a 47 mm. adult, showing the posi-
tion of a muscle in which degeneration has just begun; sp.c., spinal cord; d.m.,
degenerating muscle. Photo. 30.

Fig. 5 Portion of the degenerating muscle represented in figure 4, showing the
accumulation of lymphocytes among the muscle fibers in the first stage of muscle

degeneration. Photo. * 450.
74

MUSCLE DEGENERATION IN AMPHIBIA 75

with numerous eosinophile granules and while blood cells, thus
suggesting the origin of the granules from the degenerating muscle
fiber. Disintegrated erythrocytes can also be seen in connection
with degenerating muscle tissue. However, from the material
studied the majority of eosinophile granules found in degenerating
muscle are products of muscle degeneration while the smaller por-
tion is contributed by degenerating red cells. Eosinophile leuco-
cytes can often be seen in connective tissue in the neighborhood of
degenerating muscle. These through their amoeboid movements
have perhaps wandered away from their source of origin.

In the degeneration of the gills numerous blood vessels are
necessarily broken down, producing in some cases stasis in the
small vessels resulting in the disintegration of the erythrocytes
in those parts. Degenerating red cells can be demonstrated espe-
cially where the degeneration of the gills is well advanced. They
can also be found isolated or in smaller and larger groups outside
of the walls of blood vessels. The degeneration of some is evident,
as can be seen from the appearance of their nuclei which stain
intensely, lose their oval shape and become irregular in outline
and a few were seen apparently about to be extruded from the cell
body. The cell envelope also becomes very irregular in outline
and in some cases even ruptured. In some the cytoplasmic mate-
rial is coarsely granular, losing its seemingly homogeneous nature.
In others globular masses of varying sizes are apparently about to
be detached from the cell. Many of these large masses or coarse
granules together with smaller granules can often be seen lying
free in close approximation to degenerating erythrocytes. These
larger masses and granules stain with eosin, the smaller granules
staining more deeply than the coarse granules. A difference readily
observed between the eosinophile granules formed from the degen-
erating erythrocytes and degenerating muscle tissue is that the
granules formed from the former vary much in size as stated above
while those formed from the latter, while varying somewhat in
size, approximate more nearly the dimensions of those found in
the eosinophile cells. That these eosinophile granules are taken
up by cells of the character of leucocytes is indicated by the pres-
ence of such cells, sometimes in large numbers, in the immediate

76 J. A. BADERTSCHER

neighborhood or in contact with them (fig. 6). I have been
unable to find leucocytes ingesting entire or large fragments of
erythrocytes as was observed by Lewis (04) who worked with the
ungulates and by Weidenreich (’08) who worked with guinea-
pigs. A possible reason for amphibian leucocytes not ingesting
entire erythrocytes is found in the large size of the red blood cells
in this class of animals.

Fig. 6 Lymphocytes among free eosinophile granules found in degenerating
muscle. Camera lucida drawing.

Fig. 7 Series of diagrams, a, b, c, to show the formation of eosinophile leuco-
cytes from lymphocytes. The limit of the cytoplasm is marked by a dotted line.
Camera lucida drawings.

The first evidences of muscle degeneration are marked by a
collection of leucocytes among the muscle fibers. The number of
leucocytes that accumulate between the fibers is so great that
the width of the intercellular spaces is increased to several times —
their normal extent (fig. 5). In this accumulation of white blood
cells the great majority are the smallest of the leucocytes. Their
nuclei are large, taking up almost the entire cell body. The out-
line of the nuclei which vary, may be round, slightly elongated,
gently dented on one side or irregularly crescent-shaped, indicat-

MUSCLE DEGENERATION IN AMPHIBIA yh

ing amoeboid movement. The rim of cytoplasm around the nu-
cleus is very narrow, in‘some cells so scanty in its amount that it is
difficult to see. The minority of cells in this accumulation are
larger than the ones described. They have large nuclei which
-may vary somewhat in outline as those of the smallest white cells.
They have a wide rim of cytoplasm around the nuclei to which the
large size of the cell is due. According to Friedsohn’s work (’10)
on the amphibian blood, the smaller cells appear to be the small
lymphocytes and the larger ones the large lymphocytes. An
occasional eosinophile leucocyte can also be seen, while partially
and completely degenerated erythrocytes are thinly scattered
throughout the accumulation of leucocytes. No perceptible
change in the muscle fiber itself has yet taken place in this stage of
degeneration.

After the degeneration of the muscle tissue has advanced to the
stage in which the degeneration of the fibers is readily perceptible
the number of white blood cells is as numerous as at the time when
the degeneration was first evident, but the lymphocytes have
greatly decreased, while the eosinophile leucocytes have increased
in number. In this stage of degeneration numerous eosinophile
granules can be seen lying free in the intercellular spaces and
often in contact with lymphocytes and eosinophile leucocytes.
The number of eosinophile granules varies greatly in the eosino-
phile cells, from only a few granules to the gorged condition of
some of the cells (fig. 7), thus suggesting again that the lympho-
cytes take up eosinophile granules. The presence of the large
number of lymphocytes during the first stages of degeneration and
a subsequent reduction of their number and replacement by large
numbers of eosinophile leucocytes as degeneration advances,
clearly indicates that the eosinophile granules are taken up by the
lymphocytes. The point in.case is that the lymphocytes are the
main cells that take up the granules and are thus converted into
eosinophile cells.

The majority of the eosinophile leucocytes found in connection
with degenerating muscle and erythrocytes have round or nearly
round nuclei. In some the nuclei are of the transitional type

78 J. A. BADERTSCHER

while in others they are of the polymorphic type. In studying
the eosinophile leucocytes with the various types of nuclei, one
can find sufficient evidence—as can be indicated by a series of grad-
ual transitions from one type of nucleus to another—to con-
firm the conclusion that all are derived from the round nucleated -
type of eosinophile cells. This transformation takes place by a
gradual change in the shape of their nuclei. They are thus geneti-
cally related, the round nucleated type being the forerunner of the
transitional and polymorphic types.

No attempt was made to determine the chemical nature of the
eosinophile granules. The material was not prepared for that
purpose. However, the hemaglobin nature of the eosinophile
granules as held by Weidenreich and others, is indicated by circum-
stantial evidence in that the free eosinophile granules and group-
ings of eosinophile leucocytes are found only in connection with
tissue containing hemoglobin, namely, muscle tissue and erythro-
cytes. The hemoglobin-containing material of degenerating
muscle and erythrocytes apparently does not break up directly
into the eosinophile granules, that is, this compound is altered in
some way before it is taken up by the lymphocytes. This is indi-
cated by the fact stated above that the very coarse granules or
masses are stained more faintly than the smaller granules, which
are often stained as deeply as the eosinophile granules in the cells.

Although the evidences are strongly in favor of a hemoglobin-
nature of the granules, yet when their complex chemical source
(erythrocytes and muscle tissue) is considered, it would not
be unreasonable to assume that other elements besides hemoglo-
bin also enter into their composition. In considering the chemical
nature of the granules Weidenreich (’11, p. 622) says: ‘‘Der von
Sherrington angeblich festgestellte Phosphorgehalt der Granula- .
tion braucht nun keineswegs, wie dieser Autor annimmt, fiir eine
Nucleinnatur zu sprechen; denn wir wissen heute, dass die roten
Blutkérperchen auch aus dem phosphorhaltigen Lecithin aufge-
baut werden, so dass also unter der Voraussetzung, die Granula
sind Zerfallsprodukte der Erythrocyten, auch Phosphor in ihnen
enthalten sein kénnte.” If the granules do contain both phos-
phorous and hemoglobin then the term ‘phosphorous-nature’ would

MUSCLE DEGENERATION IN AMPHIBIA 79

represent the chemical nature of the granules as well as ‘hemo-
globin-nature,’ for both muscle tissue and erythocytes contain
phosphorous-containing compounds. The term ‘hemoglobin-
nature’ does not seem to be comprehensive enought to express their
chemical nature. The simple phrase ‘‘Zerfallsprodukte der
Erythrocyten” to which I add ‘products of degenerating muscle
tissue,’ would express a greater probability of the granules con-
taining other elements beside hemoglobin, than does the term
‘Himoglobinnatur.’

To give a detailed account of the different theories relative to
the degeneration of muscle tissue in different classes of animals is
beyond the scope of this paper. Observations by different inves-
tigators of various animal groups indicate that the processes
involved in the degeneration of muscle tissue vary considerably.
Briefly stated, the interpretations of different workers of the man-
ner by which a muscle degenerates and the part played by the leu-
cocytes in this phenomenon has resulted in the establishment of
three general views: (1) a purely aphagocytic process by which
the muscle fibers undergo liquefaction, either through the activity
of internal conditions of the fiber or through the action of the
fluids surrounding them; (2) a purely phagocytic process by which
the phagocytes break up the muscle fibers into fragments which
are taken up and removed by them; (3) by a combination of the
aphagocytic and phagocytic processes. The views enumerated
above are stated only in a general way and the theories as worked
out by different investigators have detailed modifications.

Barfurth (’87), in his work on the frog larvae, came to the con-
clusion that a muscle degenerates independently of the action of
phagocytes. The myofibrillae break up into comparatively long
fragments, bundles of which make up the sarcolytes. The cross-
striations are present in the sarcolytes when they are first formed
but gradually disappear as the muscle fragments undergo disso-
lution. The liquefaction of the fragments takes place within
the sarcolemma, which is comparatively resistent to the action of
the liquefacient, and disappears only after the sarcolytes have
been reduced to granules—the débris of degeneration. The ini-
tiatory causes of degeneration are unknown to him but he thinks

SO J. A. BADERTSCHER

that they are extrinsic in their nature and is of the opinion that a
lack of nutrition or atrophy through disuse after the appearance
of the forelegs, are factors to be considered.

According to Katz (00), who worked with transforming toads,
the initiatory causes of degeneration appear to be intrinsic in the
muscle itself. Sarcolytes are formed only in some fibers. In the
course of degeneration a homogeneous material is produced, which —
disappears by liquefaction in situ.

Looss (’92) arrived at practically the same conclusions as those
of Barfurth, with the exception that a small portion (less than 10
per cent) of the sarcolytes are taken up and destroyed by the pha-
gocytes. The remaining large proportion undergo dissolution in
place through the activity of the surrounding fluids.

Metchnikoff (’84), in his work on larval frogs, held that muscle
degeneration was effected entirely through the action of phago-
cytes which collected in large numbers among the degenerating
fibers. The source of the numerous phagocytes he then ascribed
to the blood. Further investigations (’92) led him to modify the
phagocytosis theory of muscle degeneration. In his later work he
holds that the phagocytes, which play the important part in muscle
degeneration, are formed in the muscle fiber. The first percep-
tible changes in degeneration are a proliferation of muscle nuclei
and an increase in the amount of sarcoplasm. ‘The muscle nuclei
and protoplasm then differentiate into cells, the muscle phago-
cytes, which find their way among the fibrillae, breaking them up
into fragments. These fragments (sarcolytes) are ingested and
removed by them. The usual type of phagocytes takes no part
in muscle degneration, that work being entirely limited to the
muscle phagocytes.

Muscle degeneration in Salamandra atra is, to all appearances,
a purely aphagocytic process. However, on comparing the degen-
erating jaw muscles in this class of animals with those in the
tails of larval toads, a marked point of difference is recognized in
that the formation of sarcolytes does not occur in the former.
On examining a series of toads from the larval to the adult condi-
tion, sarcolytes in the degenerating tail muscles were found as
described by the investigators mentioned above. In the Sala-

MUSCLE DEGENERATION IN AMPHIBIA 81

mander the myofibrillae, instead of forming sarcolytes, break up
into fragments at short intervals in their course, thus obliterating
the cross-striations and giving the affected part of a muscle fiber
a granular appearance. ‘These fragments or granules fade away
as if by liquefaction. Their dissolution does not take place uni-
formly along the course of a fiber, so that in the extent of a well
advanced degenerating fiber indefinitely outlined groups of dis-
membered fibrillae can be seen. Coincidently with the lique-
faction of the fibrillae appear numerous spherical granules that
stain intensely with eosin, the eosinophile granules, which are
taken up by the leucocytes in the manner described above. Ac-
cording to Barfurth, in the frog larvae degeneration begins at
one end and gradually advances to the opposite end of the fiber.
While this is the case in some of the degenerating fibers in Sala-
mandra atra, it is not so with all, for in some fibers can be seen a
large gap, produced by degeneration, and containing numerous
eosinophile granules and leucocytes, while the ends of the fibers
appear quite normal. The sarcolemma degenerates simultane-
ously with the fibrillae. There are no evidences indicating that it
spans the gap of a degenerated portion of a fiber, which would be
the case if it were more resistant to the agencies bringing about
the liquefaction of the fibrillae. The muscle nuclei and sarco-
plasm are not transformed into phagocytes as is the case, accord-
ing to Metchnikoff, in larval frogs. No perceptible increase in
the amount of protoplasm or in the number of nuclei was noticeable.
They are, however, very resistant to the agencies that cause the
liquefaction of the fibrillae. During the last stages of degenera-
tion they can be seen lying free among the numerous leucocytes
and débris of degenerated muscle. On account of their large size
they cannot be mistaken for lymphocytes or for connective tissue
cells. Their form varies. Some retain their natural rod shape,
some are crescent in outline, some are bent double so that the two
ends are in close approximation, while others are bent and twisted.
A considerable number show marked signs of degeneration by the
massing of their chromatin. Their fate is unknown to me but
from the appearance of some I am of the opinion that they undergo
degeneration.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 1

82 J. A. BADERTSCHER

The origin of the lymphocytes found in a muscle during its first
stages of degeneration can be attributed to two sources, namely,
connective tissue cells and the blood. The connective tissue cells
in the affected part of a muscle lose their stellate form, become
rounded, their nuclei grow denser, thus acquiring all the appear-
ances of nongranular leucocytes. This process is much like the
transformation into lymphocytes of the mesenchymal cells which
surround the epithelial anlage of the thymus in Axolotol as de-
scribed by Maximow (712). Metchnikoff (’84) also holds that in
Triton larvae transformed connective tissue cells assume phago-
eytic functions. It is difficult to determine to just what extent
the formation of lymphocytes takes place in muscle. However,
I am confident that the connective tissue cells do not transform
rapidly enough to account for the large numbers of lymphocytes
present. No mitotic figures were seen and only an occasional
dumbbell-shaped nucleus suggested amitosis. It is, therefore,
necessary to assume that the blood also is a source of a part of the
lymphocytes. According to an hypothesis of Metchnikoff (’84)
the endothelial walls of the blood capillaries are acted upon chemi-
cally, rendering them more permeable to leucocytes and permit-
ting of a ‘passive diapedesis’ of the red blood cells. Although
this is only an hypothesis, it seems to be a very reasonable way to
account for the presence of such large numbers of leucocytes and
also of red blood cells which are scattered in small numbers
among them, as was stated above. Since muscle degeneration in
Salamandra atra is an aphagocytic process it seems probable that
the cells of the delicate endothelial walls of capillaries may be
among the first elements to be affected. Another view, however,
may be taken. Dantschakoff (’08), in her work on the develop-
ment of the blood in birds, gives evidence that the cells of the vas- |
cular endothelium have the power of transforming into lympho-
cytes. This genetic relationship of the leucocytes to the vascular
endothelium has also been held by other investigators. If this
view be correct, is it not probable that the vascular endothelial
cells. are transformed into leucocytes? This process also would
permit leucocytes and erythrocytes to escape into the intercellular
spaces.

MUSCLE DEGENERATION IN AMPHIBIA 83

The only apparent réle of the leucocytes in the degenerating
muscles of Salamandra atra is the taking up and removal of the
products of degeneration. They take no perceptible active part
in breaking down the muscle tissue. According to a theory of
Anglas (’00), the leucocytes produce an enzyme which acts upon
the tissues, causing their dissolution. The only indication of the
correctness of this view is the large accumulation of leucocytes in
the first stages of degeneration, even before any changes of the
fibers are apparent under the microscope.

CONCLUSIONS

The conclusions of this work are summarized as follows:

1. The eosinophile granules are exogenous in their nature, that
is, they are derived from material outside of the cell containing
them and not directly a product of the activity of the leucocyte
itself. The presence of free eosinophile granules found to be so
plentiful among degenerating erythrocytes and muscle tissue and
later taken up by lymphocytes, support this conclusion.

2. The eosinophile granules are products of degenerated muscle
tissue and degenerated erythrocytes. This is supported by the
fact that they are found only in connection with the above named
degenerating tissues. The fact also that more eosinophile leu-
cocytes are found in the blood during the period of metamor-
phosis than in the larval or in the adult condition, supports this
conclusion.

3. As they are found only in connection with tissue containing
a relatively large amount of hemoglobin, it is believed that hemo-
globin is a part of their chemical composition. This conclusion
by no means excludes the probability that the eosinophile granules
contain chemical compounds besides hemoglobin.

4. The eosinophile granules are taken up by the lymphocytes
(large and small) which are thus converted into eosinophile cells.
The eosinophile leucocytes are, therefore, the white blood cells
ingested with the products of degenerated erythrocytes and degen-
erated muscle tissue.

5. The eosinophile cells with different types of nuclei are gene-
tically related to one another.

84 J. A. BADERTSCHER

6. Muscle degeneration in Salamandra atra is a process of
liquefaction (a purely aphagocytic process), and is apparently
brought about through the activity of the surrounding fluids.

7. The only apparent function of the leucocytes in degenerating
muscle is the removal of the products of degenerated muscle tissue
and erythrocytes. They apparently play no part in the processes
bringing about the degeneration of those tissues.

BIBLIOGRAPHY

AumgvisT, J. 1902 Uber die Emigrations-Fahigkeit der Lymphocyten. Virch.
Arch., Bd. 169, S. 17.

AnGLAS, J. 1900a Note préliminaire sur la métamorphose internes de la guépe
et del’abeille. Lelyocytose. C.R. Soc. Biol., T. 52, p. 94.

1900 b Sur la significance des termes ‘phagocytose’ C. R. Soc. Biol.,
T. 52, p. 219.

BarFurtTH, D. 1887 Die Riickbildung des Froschlarvenschwanzes und die so-
genannten Sarcoplasten. Arch. f. mikr. Anat., Bd. 29, p. 35.

Brown, T.R. 1898 Studies on trichinosis, with especial reference to the increase
of the eosinophilic cells in the blood and muscle, the origin of these cells
and their diagnostic importance. Jour. Exp. Med., vol. 3, p. 314.

Bryce, T. H. 1904a The history of the blood of the larva of Lepidosiren para-
doxa. Part I. Structure of the resting and dividing corpuscle&8. Trans.
R. Soc. Edinburgh, vol. 41, P. II, p. 291.

1904 b Part Il. Haematogenesis. Trans. R. Soc. Edinburgh, vol.
ALE: Aly p. As5.

DantscHakorr, W. 1908 Untersuchungen iiber die Entwickelung von Blut und
Bindegewebe bei Végeln. Das locktre Bindegewebe des Hiihnchens im
fetalen Leben. Arch. f. mikr. Anat., Bd. 73, S. 117.

FRrIEpsoHN, A. 1910 Zur Morphologie des Amphibienblutes. Zugleich ein Bei-
trag zu der Lehre von der Differenzierung der Leucocyten. Arch. f.
mikr. Anat., Bd. 75, S. 435.

GuuLanp, G. L. 1906 Classification, origin and probable réle of leucocytes,
mast cells and plasma cells. Fol. haemat., Bd. 3.

Gitie, K. 1907 Ein Beitrag zur Morphologie des Schweineblutes. Arch. f.
mikr. Anat., Bd. 70, S. 629.

Katz, L. 1900 Histolysis of muscle in the transforming toad. (Bufo lentigi-
nosus) Science,’ vol. 49.

Lewis, T. 1904 Further observations on the functions of the spleen and other
haemolymph glands. Jour. Anat. and Physiol., vol. 38, p. 144.

P2)
Or

MUSCLE DEGENERATION IN AMPHIBIA

Lirtin, M. 1880 Ueber embolische Muskelveriinderung und die Resorption
todter Muskelfasern. Arch. f. Pathol. Anat. Bd. 80.

LOEWENTHAL, N. 1909 Contribution 4 l'étude des globules blanes du sang éosin-
ophiles chez les animaux vertébres. Journal d. l’Anatomie et de la
Physiologie, Ann. 45.

Looss, A. 1892 Phagocyten und Phagocytose. Centralblatt fiir Bakteriologie,
Bas2:

Maximo, A. 1909 Untersuchungen iiber Blut und Bindegewebe (1) Die friih-
esten Entwickelungstadien der Blut- und Binde-gewebszellen beim
Saugetierembryo, bis zum anfang der Blutbildung in der Leber. Arch.
f. mikr. Anatomie, Bd. 73, S. 444.

1912 Untersuchungen iiber Blut und Bindegewebe IV. Uber die Histo-
genese der Thymus bei Amphibien. Arch. f. mikr. Anat., Bd. 79, p.
560.

Mercuntkorr, E. 1884 Untersuchungen iiber die mesodermalen Phagocyten
einiger Wirbeltiere. Biologische Centralblatt, Bd. 3.

1892 Atrophie des muscles pendant la transformation des batraciens.
Annals de Vinstitut Pasteur, vol. 6.

Murr, R. 1904 Discussion on the réle of the leucocyte. Brit. Med. Journ.,
D.5S., p. 585.

Opin, E.L. 1904 a The occurrence of cells with eosinophile granulation and their
relation to nutrition. Amer. Journ. Med. Sc., vol. 127, p. 217.

1904 b An experimental study of the relation of cells with eosinophile
* granulation to infection with an animal parasite (Trichina spiralis),
Amer. Journ. Med. Se., vol. 127, p. 477.

PappENHEIM, A. 1905 Zur Frage der Entstehung eosinophiler Leukocyten.
Folia. haemot., Bd. 2, p. 166.

1909 Uber die Deutung und Bedeutung einkerniger Leucocyten Formen
in entziindlichen Zellanhaufungen mit besonderer Riicksicht auf die
lokale Eosinophilie. Fol. haemat., Bd. 8, S. 1.

SHERRINGTON, C.S. 1904 Note on some changes in the blood of the general cir-
culation consequent upon certain inflammations of acute and local
character. Proceed. Roy. Soc., London, vol. 55, p. 161.

Werenreicu, F. 1901 Uber Blutlymphdriisen. Die Bedeutung der eosinophilen
Leucocyten, itiber Phagocytose und die Entstehung von Riesenzellen.
Anat. Anz., Bd. 20, 8S. 188.

1903 Das Schicksal der roten Blutkérperchen im normalen Organismus.
Anat. Anz., Bd. 24, S. 186.

1908 a Morphologische und experimentelle Untersuchungen iiber
Entstehung und Bedeutung der eosinophilen Leucocyten. Anat. Anz.,
Bd. 32, p. 81, Erganzungsheft.

86 J. A. BADERTSCHER

WeIpENREICH, F. 1908b Beitrage zur Kenntniss der granulierten Leucocyten.
Arch. f. mikr. Anat. Bd. 72, S. 209.

1909 Zur Morphologie und morphologischen Stellung der ungranulier-
ten Leucocyten-Lymphocyten des Blutes und der Lymphe. Arch. f.
mikr. Anat., Bd. 73.

1911 Die Leucocyten und verwandte Zellformen. Ergebn. d. Anat. u.
Entwickelungsge.

ON THE WEIGHTS OF THE ABDOMINAL AND THE
THORACIC VISCERA, THE SEX GLANDS, DUCTLESS
GLANDS AND THE EYEBALLS OF THE ALBINO RAT
(MUS NORVEGICUS ALBINUS) ACCORDING TO
BODY WEIGHT

SHINKISHI HATATI
The Wistar Institute of Anatomy and Biology

TWELVE CHARTS

Complete quantitative data on the various anatomical com-
ponents of the body are important not only for the study of
growth, but also for cross reference. A lack of such reference
data interferes in many cases with a clear recognition of altera-
tions which are taking place in the animal body under various
experimental conditions. Fortunately the following data on
the growth of the albino rat are already available:

Growth of body in weight in respect to age (Donaldson ’06)
and body length (Donaldson ’09); weight of the brain and spinal
cord (Donaldson ’08) ; weight of total amount of blood and hemo-
globin content (Chisolm 711), the growth of the head, trunk,
extremities, skin, skeleton, musculature and viscera in weight
according to age (Jackson and Lowrey 712); and finally the growth
of the dry substance in the albino rat (Lowrey 713). Besides
these we have several other series of data on the albino rat,
though not so comprehensive as those just cited.

The addition of the present data on the weight of individual
organs to the records above cited will make available observa-
tions on nearly all the important anatomical components of the
albino rat in a form useful either for the study of growth or for
cross reference. Indeed in no other mammal are such adequate
data available at the present moment.

Since the object of the present paper is to report the objective
findings, mainly for reference purposes, many interesting points

87

88 SHINKISHI HATAI

relating to growth will not be discussed at length, but merely
mentioned as they come, in connection with the presentation
of the formulas and charts.

The data used were secured mainly from two colonies of al-
bino rats: (1) a colony kept at The Wistar Institute (during
1911 and 1912), and (2) a colony kept at the University of Mis-
souri (during 1910 and 1911). For the latter, the present writer
is under great obligations to Professor Jackson, who not only
supplied numerous rats for the purpose of control examination,
but has also granted the free use of his entire collection of data
on the weight of the viscera.

I take this opportunity therefore to acknowledge my indebted-
ness to Professor Jackson for his courtesy.

In addition, I have secured some rats from both New Haven
and Chicago for the purpose of determining the range of vari-
ability under different climatic and nutritional conditions. For
these rats I am indebted to Professor Mendel of New Haven
and Professor Carr of Chicago, and I desire to thank both of
these gentlemen for their generous assistance.

For the interpretation of the results which follow, it is of the
greatest importance to bear in mind that the animals examined
were nearly all less than one year old—a very few having at-
tained this age. If we take the normal span of life of the albino
rat as three years, then it is plain that we are dealing with rats
in the first third of their life and that the end of the records gives
the conditions at the close of the growing period only, leaving
untouched the changes which may be expected to occur during
the next two years. For the discussion of the span of life in
the albino rat, the reader is referred to Donaldson (’08, p.
368), and to Slonaker (712). i

TECHNIQUE

Although most of the organs are clearly marked off from the
surrounding tissues, and thus may be readily removed in an
exact manner, nevertheless I shall describe my method of dis-
section in detail so that others may be fully informed concern-
ing it.

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 89

Method of dissection

The animal was removed from the colony in the morning be-
fore feeding and killed by chloroform. The gross body weight
(to one-tenth of a gram) and the linear measurements were next
taken. For the latter determinations, the animal was placed
with the abdominal side up and the following measurements
made with the calipers: (1) Total length, the distance between
the tip of the nose and the tip of the tail; (2) body length, dis-
tance between the tip of the nose and anal opening; (3) tail
length, obtained by the difference between measurements (1)
and (2). The linear measurements were made to the nearest
millimeter.

The animal was then at once opened with the scissors along
the abdominal line from the anus to the level of the diaphragm.
Thus the thorax was left closed to prevent evaporation from the
thoracic viscera while the abdominal organs were being removed.

All the dissections were made beneath a glass hood designed to
protect the operator against draughts and so prevent a loss of
moisture from the organs during their removal.

The details of the procedure for each organ will be given in
the order of removal. The following entries give the names of
the organs in this order, unless otherwise stated. Each organ was
prepared rapidly and then put alone or with others into a closed
weighing bottle. Only the special treatment (if any) of an organ
is noted in each case:

1. Liver: The blood which fills the main vessels was gently ©
squeezed out before the liver was placed in the weighing bottle.

2. Spleen: The blood vessels were cut close to the hilum.

3. Testes: Accessory organs such as the epididymis, are not
included.

4. The alimentary tract was removed from the level of the
diaphragm to the anus, together with the pancreas and mesentery,
and any other adherent structures, such as fat, were left intact.
The oesophagus was therefore not included. The stomach was
cut open and the contents removed. The contents of the small
intestine were removed by gentle pressure along its entire course

90 SHINKISHI HATAI

from above downward. In the case of the rectum and large
intestine, it was usually necessary to cut them open in order to
remove the contents. The entire group of the structures above
named but minus contents, is here called ‘alimentary tract.’
The removal of contents can be accomplished easily and uniformly
after short practice. The body weight was not corrected for
the contents of the alimentary tract.

5. The suprarenal glands were carefully separated from the
surrounding structures. These glands are usually imbedded with-
in some fatty tissue but a little familiarity will enable one to
dissect them out without difficulty.

6. The ovaries were carefully dissected out of the capsule.
The fallopian tube was not included. On account of its minute-
ness, the ovary is often difficult to remove when the animal is
young. A dissecting microscope is sometimes necessary.

7. The kidneys: All blood vessels were cut close to the hilum
and any masses of fat carefully removed.

All these organs having been removed and placed in weighing
bottles, the thoracic cavity was next opened along the median
ventral line by a cut extending as far as the upper end of the
neck. Care was taken not to injure the thyroid gland, which
lies close to the trachea.

8. Thymus gland: Large lymphatic glands together with fat
lie close to this organ but were not included. Because the
weight of this gland is so closely correlated with age, the data
already obtained will not be presented at this time but reserved
until the study of an extended series of animals of known age
has been completed.

9. The heart was removed by cutting all the vessels close to
their proximal ends. The heart was next cut open by longitudi-
nal slits and any blood clots carefully removed.

10. The lungs were severed from the trachea. The esophagus
which lies near to the lungs must be removed. The infected
lungs of rats suffering from so-called ‘pneumonia’ are more or
less filled with pus. Such lungs were weighed as removed but
these data were not used in computing the formula for lung weight.

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 91

Associated with the infected lung a pathological condition of
the alimentary tract is usually found. The tract is diminished in
weight.

11. Thyroid gland: The removal of the thyroid is rather diffi-
cult on account of the minute muscles, similar in coloration to
the gland, which adhere very closely to it. <A little experience
enables one to avoid this difficulty.

This completes the list of viscera removed by the ventral
incisions. |

The animal was now turned with the dorsal side up, an inci-
sion made along median dorsal line, from the root of the tail to
the tip of the nose, and the skin laid back on each side.

12. The eyeballs were removed free from all the muscles.

13. The vertebral canal was then opened and the spinal cord
removed, free from the spinal nerves, which were severed at the
point of their emergence from the cord. The cord was separated
from the brain at the tip of calamus scriptorius, equivalent to
the level of the first spinal nerve.

14. The brain: Care was taken not to injure the olfactory
bulbs or the flocculus; the latter is embedded in a bony capsule.

15. The hypophysis was removed last. Removal of this organ
is very simple in the rat as it can be lifted from the floor of the
cranium with a fine forceps without further dissection. Both
the glandular and infundibular portions were included.

For weighing, these fifteen organs were placed as removed
in four closed bottles. Bottle 1 contained liver, spleen, kidneys,
testes, heart and lungs. Bottle 2 contained alimentary tract,
ovaries, suprarenals, thymus, thyroid, eyeballs and hypophysis.
Bottle 3 contained brain. Bottle 4 contained spinal cord.

In each instance the bottle with all the organs in it was first
weighed, then the organs were successively removed, one at a
time, and the bottle reweighed after each removal. The emptied
bottle with such small quantities of fluid as drained from the
organs was finally weighed, and from these data the weight of
each viscus was computed. It is seen from this that each organ
was weighed with its contained blood. The weighings were all
made on delicate balances to one-tenth of a milligram.

92 SHINKISHI HATAI

In making these weighings I had the help of Miss Wolfe and
Miss Conrow and desire to acknowledge here my indebtedness
to both of them for their accurate work.

The formulas and their application

By means of a mathematical formula the relations between the
weight of the body and that of the organ have been expressed in
each case. Such formulas have an evident use for the purpose of
interpolation, while for the study of various growth phenomena,
such as relative rate of growth, form of graph during various
stages of life, and so forth, they give a proper basis for discussion.

In expressing the weight relations mentioned above, I have em-
ployedatypeof formula (Hatai’11) which may bewritten as follows:

Y =aX +b logX +c
or in some cases a simpler form was used
Y=blogX +c

where Y = weight of organ; X = body weight; a, b, c = con-
stants; to be determined from the observed data.

In the case of the sex glands it was necessary to use the two
formulas in order to express the several phases of growth, that is

Y =a+b0X + cX? for one phase and
Y = b log X +c for the other phase.

In determining the constants of the formulas, I have used as
far as possible the data obtained from the rats reared in The
Wistar Institute. This procedure was important in order to
avoid the mixture of data.

In some instances however, I have made use of the data ob-
tained by Dr. Jackson, when my own observations were too few for
an adequate treatment. Dr. Jackson’s data were used, as follows:
- 1. For studying the eyeballs and ovaries I have used only the
data obtained by Dr. pedis None of my own observations
were employed.

2. For studying the suprarenals, thyroid and alimentary tract,
I have used the data obtained by Dr. Jackson to fill the interval

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 93

between 5 and 50 grams in body weight. I have however, tested
with my own limited data the weights of the organs belonging
to the rats of these body weights (5 to 50 grams) and found that
the two sets of data agree perfectly.

In fact, in this period all the organs determined by both of
us agree closely, though I have not used the data obtained by
Dr. Jackson, except in the cases named. Thus in the cases of
the three organs mentioned above, the observed data are those
furnished by Dr. Jackson and none of my own have been added,
while after 50 grams in body weight, all the data are from my
own observations made on the rats from the colony at The
Wistar Institute. In the case of all of the other organs, the
formulas are based on my own data alone.

The total number of The Wistar Institute rats examined by
me for the present purpose was 271 (220 males and 51 females)
while Dr. Jackson examined 344 (165 males and 179 females)
at the University of Missouri. The actual number of rats used
in the case of each organ will be stated when the formula is
presented.

It should be added that when a rat with infected lungs was
found—the rest of the organs except the alimentary tract being
apparently normal—the records for the lungs and alimentary
tract were discarded, while the datg on the remaining organs
were employed. Furthermore, in order to simplify the matter,
the determinations of the constants were based on the male data
alone, as in the case of most of the viscera, there is no clear evi-
dence of a sex difference.

When however the sex differences are evident, the constants
were determined separately for each sex. As will be seen later,
there are no distinct differences between the two sexes, except
in the case of some of the ductless glands. The formula deduced
for the males can therefore also be used for the females unless
the contrary is explicitly stated.

It should be noted also that in determining the form of the
graphs, especially at the upper end where both my data and
those of Dr. Jackson were scanty, I have been much helped by

94 SHINKISHI HATAI

making cross references to the data on a series of inbred albino
rats which have been grown here by Dr. King for several years
past. There are already on hand data for over 800 inbred rats
of various body weights—mostly very large. In addition to
these, the records for several hundred Norway and hybrid rats
were also employed for cross reference. Therefore, although
our data on the stock albino rats are not very extensive, never-
theless, by the aid of the various cross references, it has been
possible to produce graphs which are believed by the present
writer to be nearly correct in their essential features.

In table 1 the calculated weights of the organs for various values
of the body weight are given. This table has been used in making
the charts and for the purpose of graphic interpolation. Com-
plete tables giving the calculated weights of all the individual
organs for every millimeter of the body length will be published
later for the purpose of laboratory reference. Table 2 gives the
mean values of the observed weights for all the organs examined.

We now pass to a brief description of the formulas, the graphs
based on them and the relations between the observed and com-
puted values.

In charts 1 to 12 the growth of organs in weight for increasing
body weight is illustrated. Since I have not taken the age into
consideration, any given body weight may be represented by

‘individuals of various ages. In general, the weight of the organs
follows closely the body weight, and therefore in rats growing
normally, the weight of the organs may be considered as a func-
tion of the body weight. We notice in all cases that the greater
fluctuations occur among the larger individuals. This is due,
in part at least, to the fact that the relation between body weight
and body length varies much more in the larger than in the smaller
animals, owing either to abnormal fat deposition or emaciation.
Such fluctuations may be reduced to a minimum by computing
the normal body weight from the observed body length (Donald-
son ’09) and substituting the computed value of the body weight.
However, I have not adopted this method except in the case of
a few observations where the emaciation was very considerable.

95

WEIGHTS

ALBINO RAT VISCERA, GLANDS, EYEBALLS

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96 SHINKISHI HATAI

TABLE 2

Showing the mean values of the observed weights for all the organs examined. The
weights are given in grams

(eae _ | KID- | : malts 1 : . | il
POEL PAP | io.| BORE | somes | ol ARE | AOE mo ROPE, LOR de eos ze Are
: = == =
5.3| 0.030| 2/ 5.8/0.044-| 2] 5.3] O.271| 2] 5.3| 0.081 | 2] 5.3 | 0.01251)
14.3) 0.103} 4] 14.3/0.199 | 4) 143] O.711| 4/ 14.3/0.156 | 4] 14.3 | 0.036 | 4
26.7] 0.140) || 26:7)0.370 | 1) 26u%9) e782) 1) 26.7 |0r243, | CaN 926.7 | 0708it) ea
37.6 | 0.241| 6] 37.6| 0.431 | 6] 37.6| 2.600] 6] 37.6|0.329 | 6] 37.0)0.134 | 4
44.2| 0.252| 5| 44.2/0.472 | 5] 44.2] 3.066| 5] 44.2/0.386 | 5] 46.9|0.139-| 2
54.8 0.286), 7] 54.8|0.558 | 7] 54.8] 3.385| 7] 54.8/0.489 | 7| 54.5/|0.208 | 5
65.1 0.339| 6| 65.1| 0.673 | 6| 65.1/ 4.739) 6] 65.1|0.478 | 6] 66.0| 0.186 | 3
76.8 | 0.387| 7] 76.3|0.775 | 7] 76.3] 5.111) 7 76.3| 0.563 | 7 77.6| 0.200 | 4
83.0 | 0.432| 4| 83.0/0.955 | 4! 83.0] 5.999| 4] 83.0|0.620 | 4! 80.6|0.218 | 2
93.4| 0.487| 2) 93.4|1.005 | 2] 93.4] 5.980| 2] 94.9|0.584 | 1] 94.9|0.229 | 1
107.7 | 0.493] 4] 107.7/1.090 | 4) 107.7] 6.325] 4] 107.1/0.614 | 3] 107.1| 0.294 | 2
115.9} 0.571] 2] 115.9] 1.119 | 2] 115.9] 7.784] 2] 113.2 | 0.680 | 1] 115.9 | 0.368 | 2
124.4 | 0.575 | 11) 124.4/ 1.357 | 11) 124.4] 7.611] “11) 124.4 0.805 | 7] 124.0 0.350 | 7
138.1 | 0.587 | 3] 138.1] 1.232 | 3] 138.1 | 7.165) 3] 138.4/ 0.983 | 2] 138.4] 0.432 | 2
145.2} 0.616 | 2) 145.2| 1.202 | 2) 145.2] 8.005) 2| 145.2/1.141 | 2! 141.9 | 0.288 | 1
156.8 | 0.669| 3] 156.8| 1.496 | 3] 156.8 | 10.206| 3] 155.2 | 0.944 | 2] 157.1 | 0.487 | 2
164.9 0.706 | 6| 164.9| 1.496 | 6| 164.9] 9.666| 6/ 164.9/0.982 | 6| 165.4 0.484 | 2
173.6 | 0.705} 8| 173.6/1.425 | 8] 173.6] 9.025] 8| 175.4|1.040 | 2l 173.9/ 0.508 | 7
185.1 | 0.757] 4] 185.1] 1.599 | 4] 185.1 | 10.385| 4] 180.5|1.198 | 1] 185.1 | 0.499 | 4
195.0| 0.786 | 4] 195.0] 1.624 | *4) 195.0/ 9.955] 4] 199.5] 1.137 | 1} 197.3| 0.496 | 1
204.9 | 0.853] 4] 204.9]1.547 | 4]. 204.9/ 8.967] 4) 209.4|1.202 | 1} 205.1/ 0.501 | 3
215.7 | 0.834] 1] 215.7] 1.647 | 1] 215.7| 11.174] 1] 226.4|1.200 | 4] 224.0 | 0.438 | 2
226.0 0.840| 5) 226.0/1.649 | 5] 226.0| 10.916) 5| 237.6|1.356 | 2] 236.4 / 0.571 | 5
236.4| 0.884 | 5] 236.4| 2.035 | 5| 236.4| 9.717| 5) 240.6/1.175 | 1] 254.7/ 0.804 | 3
240.6 0.929| 1) 240.6) 1.982 | 1] 240.6 /13.699| 1/ 260.5|1.507 | 3) 260.5/0.508 | 2
254.2| 0.988 | 5] -254.2| 2.166 | 5] 254.2 | 11.698) 5] 279.7| 1.760 | 1) 273.2 | 0.429 | 1
263.5 | 0.988) 3] 263.5) 2.264 | 3) 263.5 | 12.246] 3) 296.8/1.104 | 1] 294.6) 0.758 | 3
274.4} 1.016| 3] 274:4|2.098 | 3] 274.4| 12.871) 3] 314.7] 1.685 | 2) 315.4| 0.933 | 4
204.6 1.011] 3) 294.6 / 2.200 | 3) 204.6] 12.159) 3) 324.4/1.713 | 2) 323.8 0.612 | 3
314.6| 1.171| 5] 314.6|2.550 | 5] 314.6|/12.580| 5) 331.4/1.820 | 1| 358.7| 0.729 | 1
323.8 | 1.150|- 3 323.8/2.855 | 3i 323.8| 12.794) 3] 307.0) 1.697 | 1) 373.4|1.042 | 1
332.0} 1.182 | 2] 332.0} 2.722 | 2] 332.0| 15.276 | 2) 454.0/2.376 | 1 454.0| 1.286 | 1
343.7 | 1.299] 1|| 343.7|2.867 | 1] 343.7| 13.628 | 1] ~90| 87
358.7 1.194] || 358.7| 2.805 | 1) 358.7|16.175| 1 |
| 373.4] 3.007 | 1) 373.4 | 16.152| 11
397.0 1.489] 1\| 397.0| 3.179 | 1] 307.0| 16.964] 1
454.0 1.504 1) 454.0 3.954 | 1) 454.0 | 21.786 | 1) |
135, 136 136, |
\ | | |
| ALI- | | ‘ Nhat
- MEN- | | y - OVA- |} YE- = THY- 5
geomet eae NO. Bestia TESTES NO. WEIGHT pure | NO. Hea ie pas NO.| your oe NO.
iz | |
5.0) 0.120| 37 5.3] 0.004 | 2) 5.2| 0.0008 13) 5.4/ 0.026 34, 5.2 0.0019 | 12
10.4 | 0.310] 25] 14.3] 0.095 | 4! 9.8] 0.0009) 3] 7.2|0.04 | 7 9.8] 0.0021} 3
15.9| 0.734 | 12) 26.7/0.096 | 1] 14.2] 0.0025) 30) 9.5 |0.057 | 8] 13.2 | 0.0033 | 12
23.5 | 1.640| 13] 37.6]0.272 | 6] 22.0| 0.0054| 4) 11.4|0:061 | 17] 22.2] 0.0063] 5
36.1| 2.261, 3] 44.2]0.311 | 5] 45.2] 0.0093, 11) 15.9] 0.087 | 12] 45.2] 0.0090] 4
46.7| 3.800) 8) 55.3/0.451 | 6] 55.0] 0.0088 5) 25.2| 0.116 | 12) 66.1/ 0.0162) 2
54.3 | 4.253 | 14|- 65.1] 0.691 | 6] 60.8 | 0.0080, 1] 36.1/0.124 | 3] 95.5 | 0.0236) 3

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 97

TABLE 2—Continued

| | | \|

ALI- ]
MEN- | ova- | __ || _ | BYE- | | THY-
Sse apd NO: SiGe TESTES [NO larnrca ay ee Bec Ai re |\0-leranrarrr| eo ee
\ a | |
i]
64.0) 4.779) 5] 76.3 | 0.886 7| 75.5 | 0.0140 | 45.8 | 0.153 6| 120.4 | 0.0196 | 3
75.8 | 5.146/ 7|| 82.1 | 0.822 3) 85.7; 0.0194, 10) 54.3 0.159 | 14) 146.1 | 0.0216 | 2
91.0] 6.485 | 5] 98.4 | 1.323 2 95.0 | 0.0216) 6] 77.1 | 0.179 5] 155.2 | 0.0268 | 2
105.0 5.740 2) 107.7 1.460 4) 104.3 | 0.0318 6] 99.8 | 0.209 3) 164.5 | 0.0268 | 2
115.0 | 7.609 | 11] 115.9/ 1.452 | 2} 118.9] 0.0482; 4|/ 114.9 | 0.194 8] 177.8 | 0.0288 | 4
124.7 | 7.647 | 11) 124.4) 1.455 | 11) 124.2| 0.0442) 8] 124.6 | 0.207 5|| 185.2 | 0.0305 | 2
142.1} 8.062) 5] 138.1 | 1.512 3], 135.1 | 0.0431) 5] 142.1 | 0.252 5|/ 193.9 | 0.0319 | 5 ~
154.2; 8.921 | 11) 145.2| 1.873 | 2) 144.2/| 0.0464 5] 153.6 | 0.240 5. | 63
164.5 | 9.796 |. 7) 156.8 | 2.207 3] 155.7 | 0.0457) 5]| 164.0 | 0.255 6
186.3 | 8.889 | 6) 164.9 | 2.030 6/- 165.1 0.0519] 7] 181.0 | 0.241 4 |
245.5 | 10.853 | 2) 173.6 | 2.072 8] 171.8 | 0.0423] 3] 259.6 | 0.318 5
259.5 | 11.683 | 6) 185.1 | 2.138 4 184.7, 0.0508} 5) | | 149! |
293.2 | 13.878 | 2] 195.0| 2.069 | 4|| 207.5 | 0.0455) 4 |
347.8 | 14.721 | -2)| 204.8 | 2.288 | 3 “136.
| 94) 215.7 | 1.854 1 | |
226.0 | 2.291 5 }
236.4 2.144 5 | |
240.6 | 2.495 | 1 | |
| 255.1 | 2.355 4
263.5 | 2.508 3 |
274.4 | 2.687 3 |
294.6 | 2.545 3 | |
314.6 | 2.872 5
323.8 | 2.616 | 3 |
| 332.0 | 2.714 2 I
| 358.7/2.901 | 1 | |
| 373.4 | 2.852 | 1} |
| 397.0 | 2.583 1 |
454.0 | 3.429 1
| | | 121
| ! |
at |
: — = ee
= | SUPRA SUPRA | | FeO= 4 HYPO- |
eel OID NO. IWwarern=NA1* NO. [pemronn RENAL NO. aera ean | NO. mien ‘eo NO.
i ery ae | | iets | | | me
i | | |
5.5 | 0.0018} 6) 5.4| 0.0020, 24) 5.2] 0.0021) 20/ 5.4 | 0.0005 | 5] 9.2 | 0.0009 | 2
7.0| 0.0022} 4! 9.4 | 0.0022 | 32/ 8.7] 0.0030, 12] 13.7|0.0012| 2] 16.2|0.0010| 2
11.8 | 0.0025} 10] 15.9 | 0.0053 12) 13.9 | 0.0049) 36 15.0] 0.0009; 2 21.2; 0.0018) 3
14.1} 0.0041) 5) 23.5 | 0.0085 | 13) 22.2) 0.0080 5 19.0 0.0013 5| 64.3 0.0032 4
23.5 | 0.0071) 13) 36.1 | 0.0120) 3) 45.2) 0.0115 11) 47.6 | 0.0030) 2 90.0 0.0057 3
34.5 | 0.0103} 2 46.7|0.0122| 8] 66. 4 0.0222 2 64.0 0.0035 5) 104.3 0.0062 8
47.6) 0.0103 2) 64.0) 0.0176) 5) 95.5 0.0243) 3) 83.3 0.0033 6) 116.0 | 0.0074 9
64.0} 0.0116} 5|| 74.5 | 0.0255 | 2] 120.4 | | 0.0344, 3) 95.2 | 0.0040 3!) 127.4 0.0073 4
74.5 0.0126 2) 86.6 | 0.0218) 2) 146.1| 0.0405) 2) 105.0 0.0047 2! 136.6 0.0006 4
86.6 | 0.0163, 2) 105.0 | 0.0212| 2 156.3/ 0.0494 3) 115.7 | 0.0053) 6|| 147.2 0.0088 | 6
105.0 | 0.0207, 2) 115.2 0.0191 | 3) 164.5 | 0.0403 4) 123.9 0.0050 8) 155.5 | 0.0114 | 7
115.2 | 0.0193} 3), 124.5 | 0.0266) 5|| 177.8 | 0.0406 3|| 137.7 | 0.0057 | 3|| 163.7 | 0.0106 | 7
124.5 | 0.0251) 5) 136.1 0.0317} 2|| 185.2) 0.0507) 4]) 154.5 | 0.0062 8] 174.2 | 0.0112} 8
136.1] 0.0274 2] 154.3 | 0.0296 | 5) 193.9) 0.0493 5| 178.8| 0.0065 4) 184. 5 | 0.0127 | 6
154.3 | 0.0263 : 186.4 | 0.0323 | 3) “113| 202.5 | 0.0082 2) 202.5 | 0. 0162 | 5
| | |

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 1

98 SHINKISHI HATAI

TABLE 2—Continued

3 ; | (eee | ao
SUPRA | SUPRA HYPO HYPO
Bee dr, HOI NO. Seer RENALS NO. ace nants NO" ween PHYSIS | NO. eel PHYSIS | NO.
| | |
186.4 | 0.0313) 3]) 202.5 | 0.0370) 2 | 214.6] 0.0077] 2\) 249.0] 0.0153 | 2
202.5} 0.0373) 2|) 211.4 | 0.0400} 2}, | 235.3 | 0.0083 | 4 “80
211.4 | 0.0306 2) 224.4 | 0.0339) 2 | | 244.9 | 0.0104] 2) |
224.4 | 0.0374 2\| 240.8 | 0.0404 3 | 284.0 0.0109] 2) |
240.8 | 0.0376, 3] 252.6 | 0.0444 | 2 | | 310.4 | 0.0104] 2 |
252.6 |- 0.0331, 2 269.5 / 0.0490} 2 356.8 | 0.0125 | 3 |
269.5 | 0.0390) 3|| 284.2 | 0.0353 | 3 | 78 | |
284.2 | 0.0449} 2) 293.9| 0.0445] 4
293.9 0.0466 4) 348.6 0.0450 | 4 |
348.6 0.0477 4 145 | |
| 91]

In all charts the observed mean values are indicated by either
black dots (male) or clear circles (female) while those calculated
from the formulas are represented either by continuous (male)
or discontinuous (female) lines. Since the calculated values rep-
resent the observed data very closely the formulas may be used
not only for the purpose of interpolation, but as the basis for «
the comparison of the growth of the various organs.

In connection with the amount of fluctuation, it should be
recalled that the number of individuals was not large, particu-
larly in the case of the heavier rats, in which a greater fluctuation
is to be expected, so that with a larger number of cases, the fluctu-
ations would probably be much diminished.

To those using the formulas which are given below, the fol-
lowing practical suggestions are offered:

1. When its value differs markedly from the normal, the body
weight should be corrected to the observed body length, as the
body length is least modified in animals exhibiting emaciation

‘or abnormal fat deposition. The proper correction can be made
according to the following formula (Donaldson ’09).

Body length = 1438 log (Bd. wt. + 15) — 134

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 99

Or expressing the body weight in terms of body length, we have

Body length + 134

Body weight = 10 143 — 15

2. Under some conditions the growth of rats is considerably
accelerated in both body weight and body length for given ages.
Such rats show generally a marked disproportion in the weight
relation between body and organs; that is, the viscera are, as it
were, overgrown by the remainder of the body. In such cases
the body weight normal for the viscera may be estimated from
the weight of any one of the organs, such as the weight of the
heart. Thus one determines the body weight from the observed
heart weight, and then compares the weights of the other organs
as observed with those computed for the body weight thus
obtained.

3. In any experiment where the controls are compared with
experimented animals, modifications in the experimented group
can be determined in the following manner: First determine the
percentage deviation of the controls from the calculated values
as given by the formulas and then the corresponding percentage
deviation of the experimented animals. The algebraical differ-
ence of the two values thus obtained will be the difference
between the experimented and controls. This method is im-
portant when the body weights of the two groups of rats (experi-
mented and controls) differ widely since the weight relations
between body and organs are not constant but vary with the
change in body weight.

We now turn to a brief description of the formulas! for the
weights of the viscera and the graphs which represent them.

1 Tn order to bring together all the formulas which so far have been worked
out in connection with the albino rat the following additional formulas are in-
serted here. This addition will, I believe, be very convenient for reference.

Weight of brain = 0.569 log (Bd. wt. — 8.7) + 0.554 [Bd. wt. > 10]

Weight of spinal cord = 0.585 log (Bd. wt. + 21) — 0.795

Tail length, male = 0.853 Bd. wt. + 38.8 log Bd. wt. — 90.5

Tail length, female = 0.874 Bd. wt. + 48.2 log Bd. wt. — 98.1

100 SHINKISHI HATAI

Ea SEEM Sees NT TT)
ie GSS USS CO ECE Ree Seo aoe
. Ameell eee a Sage ge oa oce Coo eee coco
1.5; WEIGHT GRAMS EOD SRRBE SSE er aC CoReeeRBSs aCe eae
ve EEE EEEEEEEE EEE EEE EEE EEE SeE EEE
a BECCEEEE EEC Be ae aioe ahaa
; BEBRREOS CeCe eee Breet 2 ieletetel slat aa
i2EEEEEEE EEE EEE eee EEE
9 V4
3 Bee esters oi ol atletattelescL-P1°eoeaie o
£4 eal ef foment a en wef rt
1.0 PERERA 10
CCF =455 ia
ee SEEEEEEEEEEE EEE Eee ssee5 58
AA anne 2 oes eeee Zoos =SEGREESEE =< [alse
epee km ef ae a oY |
7 i Fe i ff a a Bie
@ EERE ECC HoH
0.6 ea ea fe P= EEEecnas baa
GEE Res eSe See See eae aialatalela [isa a ae
p soane nue aausuuus danse es sseeeseeeeeeeeSeeeees
SEEPS EEE EEE Eee CEEEEEEH
03a facia engender a
oa CI] FE PEC feu E a
qgunres Soom na EERE Bee ==
0.1] REE EEEECECEEEECEECH| BODY WEIGHT GRAMS 7
L*] ! aes BORE ESE en ! ! ' (65
0 50 100 150 200 250 300 350 400 450

Chart 1 Showing the heart weight of the male albino rat according to body
weight. The observed weights are represented by 134 male rats.
@ Observed weight. —Calculated weight.

1. Heart.2. The growth of the heart in weight is represented
by the following formula: Weight of heart =

0.0026 (body weight + 14) + 0.249 log (Bd. wt. + 14) — 0.336

The constants of the formula were determined from 134 male
rats and the relation between observed and calculated values is
shown in chart 1. The greater fluctuation which occurs in the
rats of larger size has already been noted and explained. As has
been mentioned earlier, variations in body growth alter more or
less the relation between heart weight and body weight. The ~
heart weight is usually high when the rat is small for a given age

? The weight relation between heart and body has been examined by numerous
investigators at various times. A list of important papers, as well as a table
which illustrates this relation found in the various mammals is given by Joseph
(08) including his own extensive observations on dogs of various sizes.

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 101

—

4 t 1a ls T T r
[ i a i ea fi | a al
35; KIDNEYS 2. LILTT = iE Het aeenn78 aes
| WEIGHT GRAMS. | WH serge |
Bist ito ose ‘a am
so FEET EEE saaeuaneee
- |
Fe FA
+4195
Hf |
aoeeee
[= a soa
a 4 4
| Eee
| =I
a [ SSS
[ [|
Lope ale
HH va BREE
a aan SaSaea
BESS [| Bees =a Biel
sor aaeeeeeeeerl al aan
oe HEALTH PERCE es
han] erica
eH Hae PEEEEEEEEE ae
(iam [| Gl BODY WEIGHT GRAMS |
{ome i fe fe Ue fe [ea] fin [Ds] Pe Ss a) youn
aa 200 250 . 300 350 400 450

Chart 2 Showing the weight of kidneys of the male albino rat according to
body weight. The observed weights are represented by 136 male rats.
@ Observed weight. Calculated weight.

and it is usually low when the rat is large. A change in the rela-
tive weight of the heart is usually accompanied by a Corresponding
change for all the other organs.

2. Kidneys. The growth of the kidneys in weight is represented
by the following formula: Weight of kidneys =

0.00718 (Bd. wt. — 3) + 0.132 log (Bd. wt. — 3) — 0.009

The constants of the formula were determined from 136 male
rats and the relation between observed and calculated values is
shown in chart 2. We notice in this case a much greater fluctua-
tion than in the case of the heart, but at the moment no ade-
quate explanation for this can be given. Possibly it is due to
the neglected factor of age.

102 SHINKISHI HATAI

im ; o | 20.

cH

isle)
LIVER BEC AEOSRSooSTREeE
19.
WEIGHT GRAMS SegeebSe0ea000

18.
SEE ESE eta tateleietenaet

ol

a

CI
TT
loa
ale
Be
EE
on
Biel
aa
oe.
rr

a
al

\
‘I

a
|
i
r |
ia
I |
|
i
a
a
a
i
CI
B
| |
|
B
a
:
Oa
Be
al
cH
|
ae
A
B
B
Ne
Ni
yy
al
B
|

eo
Hh
EHH
i
Perio
HH a

ag
laa
Scr.
HHA
Sela
Be
BEIEIC
AE
asa
aaa
mela
Eels

Bee

HH
NH
NEC
Ne

Ny

Fy

4

=

A

Fy

=

FJ

a
||
a
|_|
a
|_|
|
|_|
|
a
7
aaleae
|_|
a
a
IN
|_|
a
a

BEE CEE EE EEE EEE
lea eae] 4 GRO

Hl
a
:
|]
a
a
a
|
“4
|
fal
|
o
|_|
a
FH
re
Oo
i
|
|

TBE ae ES VO er Se eh = ee)
:
i
[exc]
J H
|
| |
[es
| |
||
[|
|
ie
es
B
i
| |

EBeSaee BODY WEIGHT GRAMS
c (Et [ {| canes rae
10) 50 100 150 200 250 300 350 400 450

Chart 3 Showing the weight of liver of the male albino rat according to body
weight. The observed weights are represented by 136 male rats.
@ Observed weight. * —_ Calculated weight.

3. Liver. The growth of the liver in weight is represented by
the following formula: Weight of liver =

0.0303 (Bd. wt. +5) + 3.340 log (Bd. wt. +5) — 3.896
[Bd. wt. > 10]

The constants of the formula were determined from 136 male
rats and the relation between the observed and calculated values .
is shown in chart 3. We notice here a much greater fluctuation
of the observed values than in the two previous cases. Since the
liver is the seat of food storage, as well as performing several other
important and complex functions, one might naturally expect
a high variability as the consequence of its varied physiological
activities. It is possible also that the weight of the liver may

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 103

2.5

+

EEE

See\reeee
aH

oH
ECHR

(mpc | oa] oa Oe | aac a i fi eae

PEP

Je a ns a | a | BODY, WEIGHTAGRAMS
& Ee (f HEEL iia ea ea

350 400 450

Chart 4 Showing the weight of lungs of the male albino rat according to body
weight. The observed weights are represented by 90 male rats.
@ Observed weight. — Calculated weight.

alter according to the nature of the diet as well as according to
other conditions not yet studied. It is interesting to note that
a heavy liver is usually associated with a heavy spleen.

4. Lungs. The growth of the lungs in weight is represented
by the following formula: Weight of lungs =

0.00471 (Bd. wt. + 2) +0.122 log. (Bd. wt. + 2) — 0.056.

The constants of the formula were determined from 90 male
rats and the relation between the observed and calculated values
is shown in chart 4. All evidently pathological cases were elim-
inated from the records used. Nevertheless on account of dif-
ficulty in determining the infection at an early period, some
diseased cases may have been retained. This pulmonary trouble,
commonly called ‘pneumonia,’ seldom appears in rats less than
100 grams in body weight, but after this period almost 90 per
cent of the ordinary rat population is affected. This is true not
only for the albino rat kept in captivity but for the Norway rat
when freshly trapped. Although an elimination of the patholog-
ical records was difficult on account of the great frequency of

104 SHINKISHI HATAI

o SPLEEN _ all maa?

oe
0.8 DEbZza
Ee
0.7 paral alloy
Ccoum EcCoSeee
0.6 SOS0005000000000058
as HEH
0.4 Coo
Be co
oS ea a rata ne | pct
ae Hy Bee ge ee eeae
co SESS 0000505000085
Olam fen} Poa _l_] BODY WEIGHT GRAMS
Co | 1 ! |g ae eee ee
i¢) 50 100 150 200 250 300 350 400 450

Chart 5 Showing the weight of spleen of the male albino rat according to
body weight. The observed weights are represented by 87 male rats.
@ Observed weight. Calculated weight.

infection, nevertheless when this was made as far as possible,
the resulting data were quite uniform as will be seen from the
chart.

5. Spleen. The growth of the spleen in weight is represented
by the following formula: Weight of spleen =

0.00245 Bd. wt. + 0.0301 log Bd. wt. — 0.025

The constants of the formula were determined from 87 male
rats and the relation between observed and calculated values is
shown in chart 5. The weight of the spleen is highly variable
owing to a- great frequency of cases of ‘enlarged’ spleen. In
treating the records I have excluded all such plainly altered —
spleens. Such enlarged spleens are usually darker in color, soft
to the touch, and exhibit all over the surface dark or grayish
patches. A slight acquaintance with this organ will remove any
difficulty in distinguishing the ‘enlarged’ from the normal spleen.

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 105

8 ALIMENTARY tract titi iit
7 WEIGHT GRAMS = CCT TTT
Bales

|

|_|

a
| :

HL
sar
18: Sei Ber an eb
=| suucussvetessetessiesezaeeeec
14. at elejonaoialelisieleleizfollatal
SUE OeeSsEEZ ala an
13., SESE 0USES002 c00nSama0 5505
Seen eeeee JoSeoeeee
aanEEZas
dawn

+
|
[|
2 :
FEE
eH
a

BHEGeeeeneeoo

-
FEE

:
| 4

i ECE HEHE
hy

if EERE %

| A a a
FE sasecaed
BEES

EEEGae

Bea

lomiamifsee =f] TTT at a veep
ye © OL mee) 150 200 250 300 350 400 450

Chart 6 Showing the weight of alimentary tract of the male albino rat ac-
cording to body weight. The observed weights are represented by 112 (Jackson)
rats, below 50 grams in body weight, and 82 (Wistar) rats, above 50 grams in body
weight.

@ Observed weight. — Calculated weight.

The cause of this enlargement, as found in the rat, has not been
studied.

6. Alimentary tract. The growth of the alimentary tract in
weight is represented by the following formula: Weight of ali-
mentary tract =

0.0245 Bd. wt. + 4.720 log (Bd. wt. + 7) — 5.753

The constants of the formula were determined from 194 male
rats and the relation between observed and calculated values is
shown in chart 6. Despite the fact of some difficulty in removing
this organ, the weight of the alimentary tract is highly uniform
in the rats up to 150 grams in body weight, but beyond this pe-
riod it becomes decidedly variable owing to the greater frequency
of intestinal disturbance. This intestinal disturbance seems to

106 SHINKISHI HATAI

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| WEIGHT GRAMS saan Toccooee
Sama Tsial

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BODY WEIGHT GRAMS
i arta

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200 250 300 350 400 450

Chart 7 Showing the weight of testes of the male albino rat according to
body weight. The observed weights are represented by 121 male rats.
@ Observed weight. — Calculated weight.

be associated mainly with the. infected lungs. It is quite safe
to conclude that the alimentary tract is more or less altered in
cases where pulmonary infection is well advanced. This means
that nearly 90 per cent of adult rats are unfitted for this deter-
mination. The alteration in question causes a loss of weight.
The present formula was determined from the rats in apparently
good health and thus practically free from this infection. Con-
sequently the upper end of the graph will naturally be higher
than when based on the values obtained from a random sampling
of the general population without regard to the condition of the
alimentary tract.

7. Testes. Owing to dissimilar rates of growth during the
earlier days of life, the graph illustrating the growth of the testes
has three distinct phases. The first phase however occupies
such a brief interval (5 to 10 grams in body weight) that it was
thought, from a practical standpoint, not worth while to work

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 107

out a special formula for it. Consequently the following for-
mulas represent only the two remaining phases. The weight of
the testes on body weight between 5 and 10 grams may be cal-
culated with a sufficient degree of accuracy from the values given
by a straight line which joins the observed weight at 5 grams with
the calculated weight at 10 grams in body weight.

Testes = 0.043 — 0.000966 Bd. wt. + 0.000163 Bd. wt.?
[10 < Bd. wt. < 80]
Testes = 2.910 log Bd. wt. — 4.520 [Bd. wt. > 80]

The constants of the formula were determined from 121 male
rats, and the relation between observed and calculated values
is shown in chart 7.

Although I have not considered such factors as the season
and age, yet from the functions of these organs, it seems probable
that data controlled for both those factors would be less vari-
able than that here presented.

.06

OVARIES 2
WEIGHT GRAMS

03

02

02

| CECeo
BODY WEIGHT GRAMS

it

J ts sd
250 300 350

+ | cl
50 - 150 200

Chart 8 Showing the weight of ovaries of the female albino rat according to
body weight. The observed weights are represented by 136 (Jackson) rats.
© Observed weight. ---- Calculated weight.

8. Ovaries. The growth of ovaries in weight is even more
complicated than that of the testes. We notice in the ovaries

108 SHINKISHI HATAI

three distinct phases of growth. The first and last phases are
represented by the logarithmic curves, while the second is rep-
resented by a parabolic curve. Weight of ovaries:

(Phase 1) = 0.010 log (Bd. wt. + 3) — 0.0082
[Bd. wt. < 50]
(Phase 2) = 0.0425 — 0.00121 Bd. wt. + 0.0000108 Bd. wt.?
[50 < Bd. wt. < 80]
(Phase 3) = 0.007 log (Bd. wt. — 105) + 0.0352
[Bd. wt. > 110]

The constants of the formulas were determined from 136
female rats and the relation between observed and calculated
values is shown in chart 8. The data used here were obtained
by Dr. Jackson from the rats kept at the University of Missouri.
These rats were all unmated excepting a few of the oldest (at age
of one year). It is therefore possible that the ovaries belonging
to the mated females may show some deviation from the values
given by the present formulas.

9. Suprarenal glands. In this gland a sexual difference is clearly
shown. Consequently the growth of the suprarenals in weight
is treated separately according to sex and is represented by the
following formulas respectively.

Weight of suprarenals:

Male = 0.0000855 (Bd. wt. + 3) +0.0113 log (Bd. wt. +3) — 0.0093
Female = 0.00023 Bd. wt. + 0.00388 log Bd. wt. — 0.0020
[Bd. wt. > 30]

The constants of the formulas were determined from 145 males
and 113 females respectively. The relation between observed and
calculated values is shown in chart 9. As is shown in the chart,
the sex difference becomes clearly marked in the rats of about
30 grams in body weight. This difference becomes greater as
the rats increase in weight. The calculated value of the female
glands is represented by the discontinuous line, and the ob-
served values, by circles. The male values are given by the
continuous line record and the black dots. The physiological
significance of the greater weight of the suprarenals of the female
has still to be investigated.

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 109

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“TLE} BODY WEIGHT GRAMS
(0) 50 100 150 200 250 3

Chart 9 Showing the weight of suprarenals of the albino rat according to
body weight. The observed weights are represented by 92 (Jackson) male rats,
below 50 grams in body weight, and 53 (Wistar) male rats, above 50 grams in body
weight; and 84 (Jackson) female rats, below 50 grams in body weight, and 29
(Wistar) female rats, above 50 grams in body weight.

@ Observed weight, male. oO Observed weight, female.

— Calculated weight, male. ---- Calculated weight, female.

10. Hypophysis. Like the suprarenal glands, the hypophy-
sis also shows a distinct sex difference in weight. This appears
in rats weighing morethan 50 grams. Consequently the growth
of this gland in weight is represented after 50 grams in body
weight by the two formulas, one for each sex. Weight of
hypophysis:

Male =0.0000257 (Bd. wt. +3) +0.00140 log. (Bd. wt. +3) —0.00097
Female = 0.00205 + 0.000081 Bd. wt. — 0.00196 log Bd. wt.
[Bd. wt. > 50]

110 SHINKISHI HATAI

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Chart 10 Showing the weight of hypophysis of the albino rat according to
body weight. The observed weights are represented by 78 male and 80 female
rats.

@ Observed weight, male. oO Observed weight, female.
— Calculated weight, male. ---- Calculated weight, female.

The constants of the formulas were determined from 78 males

and 80 females. The relation between observed and calculated
values is shown in chart 10.
. The sex difference is clearly noticeable in the rats at and after’
50 grams in body weight, and it becomes greater as the rats
increase in weight. Before 50 grams in body weight the sex
difference is not evident, consequently the weight of the female
hypophysis prior to 50 grams should be calculated from the male
formula. This sex difference in the weight of the hypophysis
has not been shown previously in any mammal.

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 111

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Chart 11 Showing the weight of thyroid gland of the albino rat according to
body weight. The observed weights are represented by 42 (Jackson) female rats,
below 50 grams in body weight, and 49 (Wistar) male rats, above 50 grams in body
weight; and 36 (Jackson) female rats, below 50 grams in body weight, and 27 (Wis-
tar) female rats, above 50 grams in body weight.

@ Observed weight, male. Calculated weight for both sexes.

© Observed weight, female.

11. Thyroid gland. Unlike the two ductless glands already
mentioned, the thyroid gland does not exhibit any weight differ-
ence between the two sexes. The growth of the thyroid gland
in weight is represented by the following formula: Weight of
thyroid =

0.0000973 (Bd. wt. +27) + 0.0139 log (Bd. wt. + 27) — 0.0226.

The constants of the formula were determined from 91 males
and 63 females. The relation between calculated and observed
values is shown in chart 11. We notice in the chart that the
two sexes do not differ fom each other, as the observed mean
values for the two sexes are not segregated and those for both
sexes cluster round the theoretical line.

The thyroid gland is the most variable among all the ductless
glands treated in the present paper. The variation is due to cases

1 SHINKISHI HATAI

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(pet ES ACCS 2 oni aia aaa aaa alia ala cA Sasso
| WEIGHT GRAMS BEER EEE EEE EEE CEE
ase estate a leah: Iasi

04 ECEE EEE eee BECEEEEEEEEEEEEEEEEe
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Chart 12 Showing the weight of eyeballs of the male albino rat according to
body weight. The observed weights are represented by 149 male rats (Jackson)
@ Observed weight. Calculated weight.

of enlargement. In some instances this enlargement amounts
to almost ten times the average value. From the data here
used, all hypertrophied cases were excluded. The enlargement
of the gland can be easily recognized not only by the size, but
also by the color. The color of the normal gland is pink while
that of the abnormal gland is milky owing perhaps to an exces-
sive accumulation of the colloidal material. A slight acquaintance
removes any difficulty in distinguishing the abnormal from the
normal gland.

' 12. Eyeballs. The growth of the eyeballs in weight is rep-
resented by the following formula: Weight of eyeballs =

0.000428 Bd. wt. + 0.098 log Bd. wt. — 0.041

The constants of the formula were determined from 149 male
rats. The relation between observed and calculated values is
shown in chart 12. The entire data used were furnished by Dr.
Jackson and obtained by him from the rats belonging to the col-
ony kept at the University of Missouri.

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 113

For the weight of the Brain and of the Spinal Cord, formulas
have already been published (Donaldson ’08) (Hatai ’09).

As already stated, in the case of the Thymus no treatment of
the data has yet been attempted.

Types of graphs

A comparison of the graphs reveals several interesting points
touching the form of growth characteristic for each organ. A
little study of the graphs makes it evident that we can class them
under three general types.

Type 1. The graph represented by the growth of eyeballs.
This is characterized by very rapid growth in weight at an early
period and after this period the rate of growth is much reduced.
This type is characteristic for the growth of the brain and the
spinal cord in weight (Donaldson ’08).

Type 2. The graphs representing the growth of all the other
viscera except the sex glands. These graphs are characterized
by a relatively rapid rise at an earlier period followed by an
almost straight line which makes an angle of varying degree with
the base line; an angle always much greater than that of Type 1.

Type 8. The graphs representing the growth of the sex glands.
‘These graphs are characterized by fluctuating rates of growth be-
tween birth and sexual maturity. For example, in the case of
the ovaries, the first phase (from birth to 50 grams) is repre-
sented by a logarithmic curve. This is followed by a phase of
rapid growth represented by a parabolic curve (50 to 110 grams).
This second phase is followed in turn by a phase of slower growth
represented again by a logarithmic curve. Similarly, the growth
of the testes has three phases, though the time relations of the
corresponding phases are not the same as in the case of the
ovaries.

The difference between Type 1 and Type 2 is very clear when
the graphs are plotted to an equal scale on one sheet. When
this is done the graph for Type 1 appears almost parallel to the
base line soon after the very rapid early rise is completed, while
the graph of Type 2 continues to rise at a more rapid rate. When

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 1

114 SHINKISHI HATAI

however, animals lower in the zoological scale are taken, Type
1 tends to resemble Type 2.

This can be seen from the graphs for the growth of the brain
weight in the fish (Kellicott ’08) and in the frog (Donaldson
703). Type 3, however, seems to be quite characteristically
differentiated from the rest even in the fish, as shown by the
graph for the gonads. The relative growth of the organs rep-
resented by these three types of graphs is represented in table 3.

The data giving the percentage growth of the brain and spinal
cord (Donaldson ’08) are included for comparison. In the case of
each organ in table 3 the percentages were calculated by taking
the final weight of the organ, at a body weight of 450 grams for
the male, as 100 and then expressing the preceding weights as
percentages. In the case of the female, the final organ weight
(100) was taken from the rats of 300 grams in body weight. This
is about the average maximum body weight the female can
attain.

Examination of table 3 reveals many interesting points. First
of all the three types of growth are clearly shown; that is the
eyeballs and the nervous system are characterized by a precocious
growth in an earlier stage. Fifty per cent of the final weight is
attained by the brain in rats when still below 20 grams in body
weight—in the case of the spinal cord, below 100 grams and in
the case of the eyeballs, below 130 grams. In the viscera and
ductless glands on the other hand, 50 per cent is attained at a
body weight of about 200 grams or more. In the case of the
sex glands, half of the final weight is attained at about the same
stage as the eyeballs 130 grams. Nevertheless the characteris-
tic double or triple phases of growth serve to distinguish clearly
the type of the gonads from that of the eyeballs or nervous
system.

It is highly interesting to notice the similarity of growth rate
of all the abdominal and thoracic viscera and ductless glands,
especially after the 50 per cent increment has been attained.

This similarity of the growth rate of the organs just named
throughout this first third of the span of life suggests a close
quantitative interrelation between these various organs and their

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 115

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116 SHINKISHI HATAI

relative independence of both the gonads and the nervous sys-
tem. <A direct comparison between the two sexes with respect
to the relative growth rate, as here given, cannot be made because
the final body weight taken in the case of the female is different
from that taken for the male. Nevertheless a study of both
table 3 and the charts shows that both the ductless glands and
the ovary grow in the same manner as the corresponding organs
in the male.

GENERAL CONSIDERATIONS

The usual view that the viscera are highly variable, in man
for example, seems to have arisen largely (1) from the use of in-
dividuals past the prime of life, that is, more than thirty years
of age; (2) from confusing normal and pathological material,
and (3) from treating the data without proper regard for the
controlling factors of race, age, body weight, stature and sex.
Indeed from a standpoint of curve fitting the viscera are prob-
ably no more variable than other parts of the body.

This optimistic view is based on the data presented in this
paper. It will be noticed that the total number of rats used is
certainly not large, when the range of body weight is considered.
Nevertheless, the resulting data show a considerable uniformity
and the observed values do not usually deviate to any great extent
from those calculated.

It should be possible, however, to get even a better agreement
between the observed and computed values for the albino rat
were it possible to control certain important modifying conditions.
What these conditions are may be briefly stated.

Factors modifying the relative weights of the organs

1. Food, care and physical condition of rats. It is generally
assumed that the rat can eat anything and can grow and multiply
under almost any conditions. It is true that one can raise rats
with an ordinary bread and milk diet, or even with some arti-
ficial diet, for one or,two generations if the original strain was
thoroughly sound.

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 117

Our experience with rats for many years, however, shows that
they can not be kept in normal health and reared for any length
of time without great care and a highly complex diet. For a
time rats may grow and multiply under almost any condition,
but if one takes the trouble to record the fertility, death rate,
growth rate, and so forth, the importance of the proper food and
surroundings will at once be recognized. The relations between
the body and organs in weight are clearly modified according to
whether the growth of the individual was retarded, accelerated,
or normal. Although I have not arranged the data to demon-
strate such modifications, the records for such a demonstration
are at hand. Recent work by Watson (’10) shows more or less
histological alteration of various organs following the different
kinds of food given to the rats.

2. Different strains. Another cause of fluctuations in the rec-
ords may be due to the use of different strains of rats. How
many strains are there, and how these strains originated or fur-
ther how stable such strains will be under varying conditions we
do not yet know.

Nevertheless, it has come to our notice that certain strains,
represented by rats coming from a new source, tend to show
constantly some degree of deviation always in the same sense.
For example, we had a strain called ‘“‘the Ridley Park strain.”’
The rats belonging to this strain had for a given body weight a
relatively shorter tail and body and possessed a markedly heav-
ier brain. We had again a strain called ‘‘the summer strain’’—
animals which had been born and reared under unfavorable sum-
mer conditions. The rats belonging to this strain had a small
body weight for their age and an unusually small nervous system.
Such strains may occur under various climatic, nutritional or
other environmental conditions. It is important therefore to
consider the conditions of the particular colony of rats which
may be used for any series of observations. If the fluctuations
found are not merely statistical, but deviate constantly in one
direction, this fact must be given its proper weight when com-
paring these animals with those represented in the tables here
presented.

118 SHINKISHI HATAT

We found by comparing our data with those obtained by Dr.
Jackson from his rats that our rats had a constantly, though
slightly, smaller heart and kidneys, but a heavier liver. Just
what the cause of these differences may have been was not
determined.

3. Influence of age and season. A third cause of disturbance
may be due to the age and season. As has been already stated,
I have not taken either age or season into account, but have
purposely taken the rats at random in order that my data may
meet the usual conditions. However, there can be little ques-
tion that age and season influence the weight of some of the
organs at least. For instance, the weight of the heart is slightly
larger in older than in younger rats of the same body weight.
The weight of the sexual glands also may vary slightly according
to the season. Recent work by Seidell and Fenger (’13) demon-
strates a seasonal variation of the iodin content of the thyroid
eland.

4. Sex differences. The sex differences are so marked in the
case of some of the organs that different formulas were needed
to represent the two sexes. However, in the majority of in-
stances, we failed to distinguish any difference according to sex,
so far as weight relations are concerned, and consequently only
one formula for each organ, namely, that based on the male
data, was worked out. It may however so happen that certain
organs react differently according to sex under similar condi-
tions. For instance, in the albino rat removal of the sex glands
from the male produces an enlargement of the hypophysis, while
in the spayed female, the hypophysis is not affected (Hatai).

Doubtless a more precise analysis of the environmental con-
ditions would enable us to extend the list of factors modifying .
the growth of the organs, but those which have just been enu-
merated are evidently worth consideration and the control of
them will doubtless go far to reduce the variability of the data.

ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 119

LITERATURE CITED

Cutsoim, R. A. 1911 On the size and growth of the blood in tame rats. Quat.
Jour. Exp. Pliysiol., vol. 4, no. 3. pp. 207-229.

Donatpson, H. H. 1903 Ona formula for determining the weight of the central
nervous system of the frog from the weight and length of its entire
body. University of Chicago, Decennial Publications, Series 1, vol.
10.

1906 A comparison of the white rat with man inrespect to the growth
of the entire body. Boas Anniversary Volume, New York.

1908 A comparison of the albino rat with man in respect to the growth
of the brain and of the spinal cord. Jour. Comp. Neur., vol. 18, no.
4, pp. 345-392.

1909 On the relation of the kody length to the body weight and to
the weight of the brain and of the spinal cord in the albino rat (Mus
norvegicus var. albus). Jour. Comp. Neur., vol. 20, no. 2, pp. 119-144.

Harar, S. 1909 Note on the formulas used for calculating the weight of the
brain in the albinorats. Jour. Comp. Neur., vol. 19, no. 2, pp. 169-173.
1911 An interpretation of growth curves from a dynamical stand-
point. Anat. Rec., vol. 5, no. 8, pp. 373-382.

Jackson, C. M., anp Lowrey, L. G. 1912 On the relative growth of the com-
ponent parts (head, trunk, and extremities) and systems (skin, skeleton,
musculature and viscera) of the albino rat. Anat. Rec., vol. 6, no. 12,
pp. 449-474.

Kewuicort, W. E. 1908 The growth of the brain and viscera in the smooth dog
fish (Mustelus canis, Mitchill). Amer. Jour. Anat., vol. 8, no. 4, pp.
319-353.

JosepH, D. R. 1908 The ratio between*the heart weight and body weight in
various animals. Jour. Exp. Med., vol. 10, pp. 521-528. -

Watson, C. 1910 The influence of diet on the structure of the tissues. Lon-
don. Appendix to ‘‘Food and feeding in health and disease,’’ pp. 555-
615.

SLoNAKER, J. R. 1912 The normal activity of the albino rat from birth to
natural death, its rate of growth and the duration of life. Jour. Anim.
Behavior, vol. 2, no. 1, pp. 20-42.

SEIDELL, A., AND FenceER, F. 1913 Seasonal variation in the iodine content of
the thyroid gland. Jour. Biol. Chem., vol. 13, no. 4, pp. 517-526.

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THE NUCLEUS CARDIACUS NERVI VAGI AND THE
THREE DISTINCT TYPES OF NERVE CELLS WHICH
INNERVATE THE THREE DIFFERENT TYPES OF
MUSCLE

EDWARD F. MALONE
The Anatomical Laboratory of the University of Cincinnati

THREE FIGURES

When one has carefully and critically studied in series of Nissl
preparations the brains of various mammals, there is revealed
the presence of constant cell groups whose cells invariably pos-
sess certain definite characteristics as to size, form and _ struc-
ture. A separation of two groups of cells based merely upon
differences in their histological characters is justified in the
present state of our knowledge, only when such differences are
constant and striking. When these conditions are fulfilled we
may conclude that such constant and striking differences in cell
character correspond to a difference in cell activity, just as in
other portions of the body. I have pointed out elsewhere that
very real differences in cell character have been neglected by
experimental workers, and that their results have been rendered
thereby of less value. Since the dorsal motor (sympathetic)
nucleus of the vagus is known to contain centers for the con-
trol of both heart muscle and smooth muscle, one would sup-
pose that any real difference in the cell character of various
portions of this nucleus would at once claim the attention of
the experimental worker and that he would attempt to inform
us as to the relation of these different types of cells to the
various functions of the vagus nerve. But such is not the case;
we are informed casually that some cells are large and others
small, and thereafter the cells are considered as if they were all
of the same type.

121

123 EDWARD F. MALONE

The discovery of two different types of cells in the sympa-
thetic vagus nucleus was not accidental; I was led to look for
this difference on the following grounds. In the first place, I
had recently shown that all cells concerned in transmitting effer-
ent impulses to striated muscle possess a fundamental similarity
of structure, whether the axone of the cell be in direct relation
to the muscle or whether the cell act on the muscle through the
mediation of one or more efferent neurones. This observation
naturally strengthened my belief in the significance of the rela-
tion of cell character to cell function. In the second place, I
had observed the striking difference between the cells supplying
striated and those supplying smooth muscle. Since in a recent
paper Molhant had shown that all fibers of the vagus supplying
heart and smooth muscle arise from the sympathetic vagus
nucleus, I concluded that in cells having such diverse functions
there must exist a fundamental difference in histological charac-
ter. As was anticipated, two different types of cells were found;
the evidence in favor of ascribing to the cells of one type the
innervation of heart muscle, and on the other hand, to cells of
the other type the innervation of smooth muscle, will be con-
sidered later.

The material available consisted of two complete series. The
first was a series of the brain of a lemur, while the second was of
the brain of macacus rhesus. Both brains were fixed in 95 per
cent alcohol, and after the usual treatment with absolute and
chloroform, were imbedded in paraffin. Serial sections were
stained in a 1 per cent aqueous solution of toluidin blue (Griibler),
differentiated in 95 per cent alcohol, dehydrated in absolute,
cleared in xylol, and mounted in Canada balsam. Series of brains
of other forms will have to be prepared and studied before I |
feel justified in committing myself upon many points, and the
present article has therefore been limited, especially as to the
exact location and distribution of the different types of cells.

The efferent fibers of the vagus nerve arise from two distinct
columns of cells. From the nucleus ambiguous arise the fibers
which supply striated muscle, while from the so-called dorsal
motor nucleus arise fibers which innervate heart muscle and

NUCLEUS CARDIACUS NERVI VAGI 123

smooth muscle. This fundamental difference as to function,
which has been proved beyond doubt by the recent investigation
of Molhant, had not been clearly recognized; this obscurity was
probably favored by the fact that the nucleus ambiguous, to-
gether with the motor nuclei of the eleventh, seventh and fifth
cranial nerves have often been regarded as visceral, regardless of
the fact that their cells cannot be distinguished either histologi-
cally or functionally from the cells of the other motor nerves
supplying striated muscle. Thus this classification giving undue
emphasis to a condition which in mammals no longer exists, has
contributed to the general lack of appreciation that the dorsal
motor nucleus of the vagus is composed of cells which differ
radically both histologically and functionally from those of the
nuclei supplying striated muscle, regardless of whether the stri-
ated muscle be of somatic or of visceral origin. The name
‘“‘dorsal motor nucleus” does not indicate the true function of
this cell group, and I shall use the name ‘‘sympathetic or vis-
ceral nucleus of the vagus.”

The location and extent of the sympathetic nucleus of the
vagus is well known and will not be considered in this paper,
except to call attention to the fact that it extends as a long
column of cells dorso-lateral to the hypoglossus nucleus from the
lowest portion of the medulla to almost the level of the oral
pole of the inferior olive. An excellent description of the loca-
tion of this nucleus is given in Jacobsohn’s monograph. The
oral portion of the nucleus is composed of small cells of the type
shown in figure 2; this is true both in the case of the lemur and
the monkey. As one follows the nucleus caudally a second type
of cell begins to appear (fig. 1). The portion of the nucleus in
‘which both types of cells occur is at the level of the oral portion
of the hypoglossus nucleus, and here the sympathetic nucleus
attains its greatest diameter. The cells of each type are partly
separated from each other, although no sharp line of separation
is evident. In the lemur the large cells (fig. 1) form a fairly
compact group dorsal from the small cells, whereas in the mon-
key their relative position is reversed. Proceeding further in a
caudal direction, the small cells become rapidly less numerous

124 EDWARD F. MALONE

and finally disappear. After the disappearance of the small cells
the sympathetic nucleus, consisting now entirely of the large
cells (fig. 1) proceeds caudally as a well developed and definite
group. In the most caudal portion of the medulla the sym-
pathetic nucleus is much reduced; only a few cells are seen in
each section, and these cells become smaller and have the appear-
ance of the smallest cell in figure 1; in this portion of the nucleus
(the caudal end) are probably also cells of the type shown in
figure 2, that is, similar to those in the oral portion of the nucleus,
but at present I cannot be absolutely sure of this, as the sur-
rounding cell groups have not been sufficiently studied. The
smallest cell shown in figure 1, is probably a transition type
between the other cells of figure 1, and those of figure 2. To
sum up, the sympathetic vagus nucleus consists of three por-
tions: (a) an oral portion whose cells are of the type in figure 2;
(b) a middle portion whose cells are shown in figure 1; and (ce)
a caudal portion composed of cells shown in figure 2 (same as
oral portion) and also of cells such as the smallest cell in figure 1
(probably a transition type).

It is not my intention to present in this paper a detailed de-
scription of the types of cells in the vagus sympathetic nucleus,
but rather to point out the fact that there are very definite
differences in histological character between the cells of the vari-
ous groups; a study of the illustrations will make this evident.
Since these differences in cell character exist, and since such
differences must necessarily be an indication of corresponding
differences of cell activity, we may now consider whether these
different cell groups of different character may be brought into
relation with definite functions. In the first place, it has been
shown by Molhant, in his excellent and extensive work on the
vagus nerve,’ that the sympathetic nucleus of the vagus gives
origin to all the fibers of the vagus which supply smooth
and heart muscle, and that all its cells give origin to such
fibers. Further, he has shown that the oral portion sup-
plies smooth muscle (stomach, lungs), the function of the
extreme caudal portion is doubtful (possibly connected with the
trachea and bronchi), while the intermediate portion supplies

NUCLEUS CARDIACUS NERVI VAGI 125

heart muscle, but he has failed to connect these different func-
tions with different types of cells. Concerning the function of
the caudal portion of the nucleus, which is composed of cells
of the type shown in figure 2, together with cells resembling
the smallest cell of figure 1, we can draw no definite conclusion.
The oral portion consists exclusively of the type of cells shown
in figure 2, and we may conclude that this type of cell supplies
smooth muscle; of course this does not justify us in concluding
that this type of cell (fig. 2) is the only type of cell which may
supply smooth muscle, or that this type may not in other regions
have a different function. Overlapping thecells supplying smooth
muscle (fig. 2) and extending caudally unaccompanied by other
types of cells is the type of cell shown in figure 1, and this por-
tion of the sympathetic nucleus has been shown (Molhant) to
supply heart muscle.

It is evident therefore that the cells of figure 1 supply heart
muscle, while those of figure 2 supply smooth muscle (stomach
and lungs). In addition there is purely histological evidence to
support the functional relations of these two types of cells (figs.
1 and 2), since the cells supplying heart muscle (fig. 1) are a type
intermediate in histological structure between those supplying smooth
muscle (fig. 2) and those supplying striated muscle (cells of hypo-
glossus nucleus, fig. 3). The relative size of the Nissl bodies in
the three types of motor cells illustrated in figs. 1 to 3 is espe-
cially worthy of notice. The fact that nerve cells supplying
heart muscle are of a type intermediate between those supplying
striated and smooth muscle constitutes one of the strongest
arguments in support of the importance of the relation of cell
character to cell function, since heart muscle is histologically
intermediate between the two other types of muscle.

The cell group which supplies heart muscle, composed of the
characteristic cells shown in figure 1, I shall name provisionally
“nucleus cardiacus nervi vagi.” I do not feel justified in assign-
ing any name to the other portions of the vagus sympathetic
nucleus, but shall be content. with pointing out that the cells of
the oral portion which supply smooth muscle are of a definite
type (fig. 2). A further division is at present not advisable

126 EDWARD F. MALONE

because the functional relations of the caudal group are not
understood, and because the pigmented cells described by Jacob-
sohn in man, have not been identified and studied (of course,
in lower animals pigment is wanting, although homologous
non-pigmented cells may exist). Further subdivision of the
sympathetic nucleus, together with a detailed description of the
location and extent of the various cell types, the consider-
ation of transition types, and of the relations of the nucleus to
the cells of surrounding nuclei, must await a thorough study of
numerous series of various animals (including man).

CONCLUSIONS

1. The -histological character of a nerve cell is an indication
of its function. Differences in connections with portions of the
organism which differ merely in spatial relations do not involve
a difference in the character of the nerve cells, but are associated
merely with the location of the nerve cell; for instance, arm and
leg muscles, flexors and extensors are all innervated by the same
type of cell, although such differences in peripheral connections
correspond to differences in the position of the corresponding
nerve cells.

2. The three types of muscle are innervated by three distinct
types of nerve cell, which, however, are related to one another
in such a manner that the cell innervating heart muscle is of a
type intermediate between the other two types of cells. Heart
muscle, smooth muscle, and striated muscle are innervated by
cells such as are illustrated in figures 1, 2 and 3 respectively, the
cells of figure 1 constituting a type intermediate between the
other two.

3. The nucleus cardiacus nervi vagi is situated in the middle
portion of the sympathetic nucleus of the vagus and is composed
of cells shown in figure 1.

4. The time has passed when experimental workers can afford
to neglect to inform themselves of the existence of definite types
of cells situated in the region under investigation, and to attempt
to bring cell character into relation with cell function.

NUCLEUS CARDIACUS NERVI VAGI LWP /

BIBLIOGRAPHY
Jacopsoun, L. 1909 Uber die Kerne des menschlichen Hirnstamms. Aus dem
Anhang zu den Abhandlungen der kénigl. preuss. Akad. d. Wiss.

Matonr, E. 1910 Uber die Kerne des menschlichen Diencephalon. Aus dem
Anhang zu den Abhandlungen der kénigl. preuss. Akad. d. Wiss.

1913 Recognition of members of the somatic motor chain of nerve
cells, etc. Anat. Rec., vol. 7, no. 3.

Moruant, M. 1910 Le nerf vague (primiére partie). Le névraxe, tom. 11.

Mo.uant, M., et VAN GenUCHTEN, A. 1912 Contribution 4 1’étude anatomique
du nerf pneumo-gastrique chez Vhomme. Le névraxe, tom. 13.

«

PLATE 1

EXPLANATION OF FIGURES

1to3 The cells illustrated in the three figures were all drawn from the same
section with the aid of the.camera lucida, and for all cells the magnification is
580 diameters. I have attempted to reproduce as nearly as possible the actual
appearance of the cells, combining to a certain extent different levels of focus.
These three figures clearly show that the cells supplying heart muscle (fig. 1)
are histologically intermediate between the cells supplying smooth muscle (fig.
2) and those supplying striated muscle (fig. 3).

1 Cells from nucleus cardiacus nervi vagi of lemur. The smallest cell repre-
sents probably a transition type to the cell type of figure 2, and this type occurs
more frequently in the caudal portion of the vagus sympathetic nucleus where
it is found together with the cells of the type shown in figure 2. 580 diameters.

2 Cells of vagus sympathetic nucleus of lemur which innervate smooth mus-
cle. In the oral portion of the sympathetic vagus nucleus these cells occur alone;
more caudally they occur together with the cells of the nucleus cardiacus (fig. 1)
in the most oral portion of this nucleus. In the caudal portion of the sympa-
thetic nucleus such cells probably reappear and are accompanied by the small
cell type shown in figure 1 (smallest cell). 580 diameters.

3 Cells from hypoglossus nucleus of lemur, innervating striated muscle. 580
diameters.

PLATE I

NUCLEUS CARDIACUS NERVI VAGI

EDWARD F. MALONE

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No.1

129

‘

ve

herd

HISTOGENESIS AND MORPHOGENESIS OF THE THO-
RACIC DUCT IN THE CHICK; DEVELOPMENT OF
BLOOD CELLS AND THEIR PASSAGE TO THE BLOOD
STREAM VIA THE THORACIC DUCT

ADAM M. MILLER

«
The Anatomical Laboratory of Columbia University

TWENTY-EIGHT FIGURES (SEVENTEEN PLATES)
I. INTRODUCTION

The study of the development of the jugular lymph sac in the
chick, the result of which was published in this Journal,! (1) led to
the investigation also of the developing thoracic duct and the
means whereby its communication with the lymph sac is estab-
lished. This investigation has been carried on with the advice
and under supervision of Dr. Huntington and in the light of his
recent work on reptiles (2) and the eat (3).

Within the past few years different investigators have shown
that in the frog (4), (5), the chick (1), (8), the rabbit (6), the cat (9),
and in man (7) each jugular lymph sac develops directly from a
venous capillary network adjacent to the junction of the early
precardinal with the postcardinal vein to form the duct of Cuvier.
It has also been pointed out by Huntington (14) that the jugular
lymph sacs, regarded as of venous origin, constitute the connecting
links between the hemal vascular system and the general system
of lymphatic vessels.

The origin of the systemic lymphatic vessels is a problem on
which investigators are sharply divided. A summary of the
different views, including the bibliography, can be found on pages
10-13 of Huntington’s monograph in the Memoirs of The Wistar
Institute (3).

' References, by number, will be found on page 162.
" 131

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 2
SEPTEMBER, 1913

132 ADAM M. MILLER

As regards the particular case of the thoracic duct, Sala (10)
in 1900 published the results of his work on the chick in which
he holds that it develops by canalization of solid mesenchymal
cords. In 1902 Sabin (11), after working with the injection
method, published her account of the development of the lym-
phatic system in pig embryos. In this she reinforces and extends
the view of Langer (12) and Ranvier (13), that lymphatics arise
from veins by a process of sprouting and centrifugal growth,
maintaining that the system as a whole is developed by blind
ducts that ‘bud off’ from the veins of the cervical and inguinal
regions, widen out to form sacs from which lymphatics grow to
the skin, stating also that “at the same time a growth of ducts
occurs along the dorsal line following the aorta to make a tho-
racic duct from which lymphatics grow to the various organs.”’
The two views expressed by Sala and Sabin are thus diametrically
opposed, the one being that the lymphatics arise in the mesen-
chyme independently of the veins, the other that the lymphatics
are outgrowths from the veins.

In 1905 Lewis (6) expressed the view that in the rabbit the
lymphatic system is derived directly from the embryonal veins,
multiple detached portions of these becoming confluent to form
the permanent systemic lymphatics, stating that the thoracic
duct ‘‘arises from a plexus of lymphatics surrounding the aorta’’
(p. 109).

In 1907 Huntington and McClure (25), studying the develop-
ment of lymphatic vessels in their relation to the veins in embryos
of the cat, found that ‘‘the lymphatics begin as extra-intimal
spaces along the course of the primitive embryonal veins. They
subsequently become confluent and form continuous vascular
channels” (p. 42).

Huntington, in 1908 (14), while retaining this view of the gene-
sis of the systemic lymphatic vessels, as distinguished from the
jugular lymph sacs, defined the latter as the connecting links
between the hemal vascular system and the general system of
the lymphatic vessels, which ‘‘arise, not by transformation of
veins, but by the formation of spaces lying outside the intimal
lining of the veins, which spaces, becoming confluent, form the
general lymphatic channels of the body” (p. 25).

THORACIC DUCT IN THE CHICK 133

In the same year (1908) McClure (21) abandoned the view
previously held jointly by him and Huntington as regards the
development of the thoracic and right lymphatic ducts in cat
embryos, and states:

The anlages of the thoracic and right lymphatic ducts consist of a
series of independent outgrowths which first appear along the common
jugular and innominate and then along the azygos veins exactly in the
line subsequently followed by these ducts; these outgrowths are sub-
sequently split off from the veins, by a process of fenestration, in the
form of a series of isolated, more or less spindle-shaped spaces which
later become confluent with one another and with a process of the
jugular lymph sac to form a continuous system disconnected from the
Wells. aerate. (py 542):

In 1909 Sabin (7) reiterates her original view, based on the study
of injected pig embryos, stating that the “presumption seems to
lie on the side that the thoracic duct develops in the same manner
as all other ducts,” namely, “from endothelial sprouts from the
sacs”’ (p. 58).

McClure in 1910 (23), after further studies of critical stages
in lymphatic development, retracted his former view and stated:

The venous line . . . . along which the cat’s thoracic duct develops
is topographically replaced by the lymphatic channel, not directly, as
assumed by me (in 1908), but secondarily by extra-intimal lymphatic
space development, . . . . the ‘extra-intimal theory,’ as originally out-
lined by Huntington and myself (in 1906), establishes a fundamental
principle of development for the main systemic lymph channels in
mammals (p. 105).

Sabin, in a later article (11) (22), states that “the thoracic
duct develops in part as a down growth of the jugular sac and in
part, especially its dilated portion or cisterna chyli, as a direct
transformation of the branches of the azygos veins” (p. 424). This
expression of opinion seems to be a correction of her earlier state-
ments and a partial adoption of Lewis’s view. In place of her
former concept of an uninterrupted centrifugal lymphatic growth
from the sacs, she now appears to hold that in addition a portion
of the thoracic duct develops as the result of direct transformation
of azygos venous tributaries into lymphatics.

134 ADAM M. MILLER

Huntington again in 1911 confirmed and elaborated his for-
mer view by extensive observations on reptiles (2) and the eat,
stating specifically on page 13 of the first number of the Memoirs
of The Wistar Institute (3) that ‘‘the entire extensive system of
lymphatic vessels proper of the adult animal, including the tho-
racic and right lymphatic ducts and their tributaries, is formed by
the confluence of the extravenous intercellular mesodermal spaces,”
and that ‘“‘these spaces are lined by a lymphatic vascular endothe-
lium which is not derived from the hemal vascular endothelium,
but develops independently of the same,” and giving his summary
and conclusions in remarkably clear terms on pages 153-171 of
the same publication. He also points out that the systemic lym-
phatic development in the mammalian embryo is ‘‘by no means
confined to the immediate environment of degenerating embryonic
veins. The same field, which shows the above described histo-
genetic processes in the development of extra-intimal lymphatic
spaces surrounding and replacing a decadent venule, will at the
same time contain numerous equivalent lymphatic mesenchymal
clefts and spaces which continue to develop independently of
any association with retrograding veins” (p. 49).

Sabin in 1912 (24) still maintains that ‘‘the thoracic duct (in
the pig) arises in part as a downgrowth from the left jugular
sac and in part from a plexus of lymphatics which buds off from
the veins of the Wolffian body”’ (p. 336).

Recently Kampmeier (15), after studying serial sections of both
uninjected pig embryos and one of Sabin’s injected specimens,
concluded that

the actual genesis of the thoracic duct is initiated by the appearance of
blind mesenchymal lymphatic spaces either around or not immediately
in contact with the venous derivatives, or veno-lymphatics, which become
detached from their venous trunks and break up into degenerating seg-
ments . . . . During their inception and growth the walls of the dis-
continuous thoracic duct anlagen are composed of mesenchymal cells

: Injected specimens of the early lymphatic stages certify the
reality of blind uninjectible anlagen beyond the farthest points to which
the injecta have penetrated, demonstrating that discontinuities in a
developing lymphatic channel are not ‘appearances’ found only by the
study of uninjected embryos (pp. 463-464).

THORACIC DUCT IN THE CHICK 135

Stromsten (16) in his account of the development of the tho-
racic duct in turtles arrived at a like conclusion, stating that
“the development of the peri-aortic lymphatic plexus in the
loggerhead turtle is immediately preceded by the formation
of isolated, independent spaces. They (the spaces) cannot be
injected . . . .” (p. 354). “The intercellular spaces thus
formed enlarge and fuse together to form lymphatic lacunae. At
a later stage the lacunae acquire an endothelial lining and become
the isolated anlagen of the thoracic duct” (p. 356).

As regards the thoracic duct in the chick, itgwill be my object
in this article (1) to demonstrate its origin by confluence of inter-
cellular spaces in the mesenchyme, independent of the veins, and
to reconsider the significance of the mesenchymal cords described
by Sala in their relation to the developing lymphatics; (2) to dis-
cuss the establishment of the morphological drainage line of the
thoracic duct and the means by which this duct and the jugular
lymph sacs communicate; and (3) to show that the organization
of the avian thoracic duct corresponds in type with that estab-
lished in reptiles and mammals.

Il. MATERIAL

Embryos of the domestic fowl (Gallus gallus) have been used
chiefly on account of the certainty in procuring the critical
stages. Some embryos of the English sparrow (Passer domesticus)
have also been used. Of this material I have examined thirty-two
individual embryos in serial sections, comprising twenty-seven
chicks and five sparrows. Four of the chicks were injected with
India ink through the umbilical vein (table 1).

Unfortunately the sparrow embryos, of which series nos. 123,
124, 126, 154 and 509 were examined, were not measured and
could be judged as to stages of development only by comparison
with the chicks.

The embryos were fixed in vom Rath’s, Bouin’s, or Zenker’s
fluid (some in Zenker-formol), Bouin’s fluid giving the best
results with the least shrinkage. The sections were cut in paraffin
and stained on the slide by one or the other of the following
methods. After fixation in vom Rath’s mixture, the staining was

136 ADAM M. MILLER

TABLE 1

CHICK EMBRYOS SERIES NO. Bun CONS es iN CRBADBSD LENG ZH aN MM.,
415 96 7.0
371 108 6.75
336 120 9.0
355 ? 120 15)
356 | 120 12.0
326 130 12.5
411 132 9.0
410 132 9.25
370 132 9.5
357 | 132 10.75
412 | 145 10.75
340 145 11.0
428 | 145 11.0
414 145 11.25
339 lis 145 13.5
426 | 160 SES
465 | 165 14.0
463 | 166 16.0
464 166 E 16.5
520 171 injected |
521 192 injected
522 192 injected
519 206
523 216 injected
320 230
524 240
483 260

done with a much diluted Delafield’s hematoxylin, followed by
a weak solution of picric acid in alcohol. After all the other
fixatives the sections were overstained in Weigert’s hematoxylin,
decolorized in water acidulated with HCl, and counterstained in
a weak solution of Orange G in distilled water. The blood cells
are clearly differentiated by either method; the cytoplasm of
those containing even a trace of hemoglobin shows some tinge of
yellow. Developing muscle tissue and nerve fibers also are yellow.
Other elements are stained by the hematoxylin, the delicate
processes of the irregular mesenchymal cells showing especially
well.

THORACIC DUCT IN THE CHICK 137
III. HISTOGENESIS

Sala, in his account of the development of the thoracic duct in
the chick (10), states that the anlagen of this lymph channel
appear as isolated mesenchymal spaces which become clothed
with endothelial cells derived from the mesenchyme, and which
subsequently coalesce to form continuous vessels. He thus
implies a denial that the lymphatics in this region arise from
veins and that the lymphatic endothelium is derived from hemal
vascular endothelium. He also calls attention to accumulations
of mesenchymal cells which contrast clearly with the surrounding
tissue. They appear to consist in large part of elements ex-
hibiting all the characteristics of young connective tissue cells,
with roundish forms, no processes and large intensely colorable
nuclei. Among these elements appear red corpuscles in larger or
smaller numbers. The accumulations or clumps of cells develop
first on the mesial aspect of the superior vena cava, and then
extend caudad to the level of the celiac artery. Without describing
the histogenesis of these masses of cells, Sala states further that
within them in large part the anlagen of the thoracic duct are
excavated or ‘hollowed out.’ The details of the ‘hollowing’ process
are not given.

In the main I can confirm the results of Sala’s observations so
far as he has carried them. As for the isolated mesenchymal
spaces which he describes as the anlagen of the thoracic duct
my study of their histogenesis in the closely graded series of chick
embryos leads to the conclusion that they do arise independently
of the veins and that their endothelial lining is derived from the
indifferent mesenchymal cells bordering upon them. The details
of the process I shall attempt to demonstrate in the following
pages. After tracing the development of the accumulations of
cells in the mesenchyme and their subsequent history in relation
to the developing thoracic duct, it seems to the writer that a
different and greater significance can be attached to them than
was given by Sala. Since the cells composing them correspond
so closely in their history to the developing blood cells as described
by Dantschakoff (17) in the extraembryonic area of the chick,

138 ADAM M. MILLER

undoubtedly they should be considered as collections of intra-
embryonic developing blood cells.

These clumps of cells, as noted by Sala, develop in the vicinity
of the aortic arches, especially the sixth, and along the dorsal
aortic roots and the aorta as far as the exit of the superior mesen-
teric artery. The early stages in the formation can be clearly
seen in embryos of 100 to 110 hours.

Some of the stellate elements of the mesenchymal reticulum
(syncytium) become differentiated from their neighbors. Their
processes are retracted and separated from the general reticulum,
the cells thus becoming rounded (figs. 1 and 2). The cytoplasm
acquires a more strongly basophilic character, increases in amount
and becomes more homogeneous than that of the true mesen-
chyme. The nuclei contain a relatively small amount of chro-
matin and one or two, usually two, distinct nucleoli. Mitotic
figures are occasionally seen (fig. 2). While the cell contours are
generally regular, there are sufficient irregularities to denote an
ameboid character. These cells appear to be identical with the
large mononuclear cells which Dantschakoff describes as differen-
tiating from the blood islands in the area vasculosa of the blasto-
derm and later in the capillaries of the yolk sac, and which she
calls lymphocytes.

The cells here under consideration increase in number not
only by their own proliferation but also by continued differen-
tiation from the mesenchymal syncytium (fig. 2). For the most
part they lie in compact groups, which gradually increase in
size as the cells increase in number, but some cells usually appear
in the vicinity of the groups (figs. 4, 5, 7, 8 and 10, 16) and not
infrequently at some distance from them. After their differentig-
tion and separation from the mesenchymal syncytium all the—
cells, both members of the groups and isolated, lie free in the spaces
among the stellate elements of the mesenchymal tissue (figs. 3
and 4). No blood vessels or lymphatics are present in the imme-
diate vicinity of the cell groups when the latter first develop and
consequently the cells are extravascular (figs, 1, 2, 3 and 4).
It will be shown later that after the lymphatics develop in this
region the cells for the most part are included within them and
thus become intravascular elements.

THORACIC DUCT IN THE CHICK 139

The application of the term ‘blood islands’ to the accumulations
of cells has been avoided because there is no real condensation
of the mesenchyme whereby the differentiating elements fuse to
form a solid protoplasmic mass. Each stellate element differen-
tiates individually (figs. 1 and 2) and condensation occurs only
as a subsequent aggregatron of the differentiated cells. The result-
ing large mononuclear cells, however, so closely correspond to the
first lymphocytes that develop in the area vasculosa of the blasto-
derm, the subsequent histories of the cells arising in the two
localities being identical, the conclusion is warranted that the
differentiated mesenchymal elements in question serve as an
intraembryonic source of blood cells.

While the present investigation has not been of such a nature
as to determine whether the mononuclear basophilic cells which
have been described give rise to granular leucocytes, they certainly
give rise to red blood cells. The cytoplasm of some of the cells
gradually loses its basophilic character and acquires a stronger
affinity for the plasma dyes, at the same time becoming quite
homogeneous. These changes are concomitant with the addition
of hemoglobin. The nuclear changes comprise an increase in the
amount of chromatin, its arrangement into a rather heavy retic-
ulum and the disappearance of the nucleoli (fig. 2).

Cells thus modified and containing a moderate amount of
hemoglobin, as indicated by the reaction to plasma stains, are
erythrobalsts. Without further visible structural changes these
acquire more hemoglobin until eventually they are indistinguish-
able from the red blood cells in the general circulation. ‘They have
therefore become erythrocytes. It is probable also that they are
definitive rather than the larger primitive erythrocytes described
by Dantschakoff. The changes described above occur both in
the cells composing the groups and in the isolated cells, so that
fully developed red blood cells as well as the earlier develop-
mental stages are seen not only in the groups but also in the mesen-
chymal spaces more or less remote from the groups.

Among the developing erythroblasts, especially in the later
stages, are many small cells which apparently go through the same
processes of differentiation as the red blood cells themselves and

140 ADAM M. MILLER

resemble the latter in every respect except size. These are un-
doubtedly microcytes, the earlier stages being microblasts.

After the middle of the fifth day the masses of developing
blood cells increase rapidly in size by proliferation of the com-
ponent cells and continued differentiation of the branching ele-
ments of the mesenchymal syncytium. -They increase in number
also by the same processes of differentiation in other localities.
Up to about the beginning of the sixth day all the developing
blood cells in question remain extravascular, that is, free in the
intercellular mesenchymal spaces which are not lined by endo-
thelium.

By the end of the seventh day the aggregations of cells reach
the height of their development. At this time they extend in
two main lines from the level of the aortic arches along the dorsal
aortic roots to the confluence of the latter to form the aorta (figs.
6, 20 and 22, 1/6). At this level, or a little further caudal, the two
main lines unite to form a single line which extends about to the
level of the superior mesenteric artery (figs. 7, 8, 10, 20 and 22, 76).
At their greatest development the larger groups together form an
almost continuous mass of the cells which in places is greater in
diameter than the aorta. Usually there are also numerous smaller
outlying groups which belong to the same general line (figs. 7
and 8).

While the main lines above described are established in all
the embryos examined, yet there is a wide range of variability
in the form of the groups and their arrangement in the lines.
In most of the embryos examined a large mass of cells or a collec-
tion of smaller groups had developed in the region dorsal to the
aortic roots and esophagus (figs. 6 and 22, 16a). One of the most
interesting and one of the most important features of the masses _
of developing blood cells is the fact that the main lines in their
general arrangement correspond with the lines of the thoracic
duct (figs. 20, 22 and 24, 16, 17 ,17a).

To anticipate, it may be stated here that as the multiple anlagen
of the thoracic duct develop the masses of developing blood cells
are in a large part included within them, and thus become strictly
intravascular (see fig. 10, 16, 17). The details of this process will

THORACIC DUCT IN THE CHICK 141

be considered in the subsequent description of the formation of
the lymphatic spaces and channels. After the seventh day, as
the lymphatic channels in this region develop and establish com-
munication with the jugular lymph sacs, the intravascular masses
of blood cells decrease and, by about the eleventh day, are reduced
to a few groups scattered through the lymphatic plexus (ef. figs.
24, 27 and 28). A considerable number of extravascular groups
persist, however, until the fourteenth or fifteenth day, or even
later. In the later stages the vast majority of the cells in these
groups are practically mature erythrocytes lying free in the mesen-
chymal spaces in the vicinity of the lymphatics (fig. 11).

Inasmuch as the reduction in the masses of blood cells in the
lymphatics follows the coalescence of the lymphatic spaces to
form continuous channels and the establishment of communica-
tion between the latter and the jugular lymph sacs, and since
the lymph sacs open into the great veins, the blood cells in question
eventually reach the general circulation by way of the thoracic
duct and jugular lymph sacs. The thoracic duct at one period
acts, therefore, as conveyor of the erythrocyte series of hemal
cellular elements which have developed from the indifferent
mesenchyme along the line of the duct.

A consideration of the histogenesis of the lymph channels
constituting the anlagen of the thoracic duct leads on to contro-
versial ground. As stated earlier in this paper, the controversy
hinges upon the question whether the lymphatics, exclusive of
the lymph sacs or hearts, arise as sprouts or outgrowths from
pre-existing vascular channels or de novo from the intercellular
mesenchymal spaces.

It is the opinion of the writer that in the chick the lymph
channels which constitute the anlagen of the thoracic duct arise
through enlargement and coalescence of intercellular spaces in the
mesenchymal tissue, and that the endothelial lining of these chan-
nels is derived directly from indifferent mesenchymal cells? that
chance to border upon the spaces. In the material studied there

2 It should be understood that when ‘mesenchymal cells’ are spoken of, they
are considered as the irregularly stellate masses of protoplasm the processes of
which anastomose to form the mesenchymal syncytium, or reticulum.

142 ADAM M. MILLER

is no evidence of any growing out, budding or sprouting from the
endothelium of pre-existing blood vessels, the tissue in which
the thoracic duct develops being non-vascular. The lymphatics
in question, and their endothelial lining, arise independently of
pre-existing vascular channels.

Prior to the appearance of any specialized spaces or channels
in the mesenchyme in the region of the future thoracic duct, the
mesenchymal syncytium consists of irregularly stellate proto-
plasmic elements the slender processes of which anastomose freely
with like processes of neighboring elements. The cytoplasm is
finely granular and the nuclei, while relatively large and vesic-
ular, contain little chromatin and one or two distinct nucleoli.
Among the protoplasmic components of this tissue are the cor-
respondingly irregular interstices or spaces which also are con-
tinuous with one another. These are called the mesenchymal
intercellular spaces. The tissue as a whole might be compared
with a sponge, the anastomosing protoplasmic parts representing
the parenchyma of the sponge and the intercellular spaces the
pores. While some of the protoplasmic elements during this
time differentiate into blood cells, as previously described, our
conception of the general syncytium and its spaces is In no way
invalidated.

The first changes in the mesenchyme leading toward the for-
mation of definite channels occur during the latter half of the
sixth day of incubation. These changes, instead of involving
the mesenchyme generally, begin in several localities. In one of
the localities, for example, the intercellular spaces increase in
size and coalesce, most of the protoplasmic elements of the syn-
cytium being pushed farther apart or broken. In this manner a
considerably larger space is formed out of a number of the original _
smaller mesenchymal spaces (fig. 9, 1/7). For the most part the
smaller spaces of the surrounding tissue open freely into the larger
space, although in places along the edge of the latter the proto-
plasmic elements lose some of their stellate character and become
flattened on the side toward the larger space.

The phenomena in general indicate the accumulation of the
fluid filling the mesenchymal spaces, the cells and their processes

THORACIC DUCT IN THE CHICK 143

being subjected to pressure and friction incident to the flow of
the interstitial substance. Thus there arises in the mesenchyme
a space larger than the original interstitial spaces but derived
directly from them by their enlargement and coalescence (cf. figs.
12, 13 and 14). So far as there is any definite lining for the new
space, it is formed by the partially or wholly flattened mesen-
chymal cells upon which the fluid in the space impinges (fig. 13).

The further changes in one of these larger spaces consists in
the main of its elongation through the enlargement and addition
to it of other intercellular mesenchymal spaces, a progressive
flattening of the cells along its sides, and an approximation of
the edges of the flattened cells to form a definite lining of endo-
thelium. There is thus formed a distinct channel in the mesen-
chyme. For the most part it is lined and its lumen is separated
from the surrounding interstitial mesenchymal spaces by a layer
of endothelial cells which represent metamorphosed stellate ele-
ments of the mesenchyme (figs, 15 and 16). At or near its ends
the channel opens freely into the adjoining mesenchymal tissue
spaces which in the further course of development are added to
the lumen of the channel and thus become a part of it (figs. 15,
16, 17 and 18). The endothelial lining already formed merges
near the ends of the channel with the mesenchymal syncytium
which in turn, as the channel elongates, gives rise to more endo-
thelium by differentiation of certain of its elements (figs. 15, 16,
17 and 18).

As stated in a previous paragraph, the larger spaces appear
in several localities in the mesenchyme. Consequently the chan-
nels resulting from their further development are for a time iso-
lated; that is, they are not directly connected with one another
or with the jugular lymph sacs or any part of the hemal vascular
system. These isolated spaces and channels constitute the mul-
tiple anlagen of the thoracic duct (fig. 21).

In succeeding stages each of the channels in question increases
in size, especially in length, until it meets and coalesces with its
neighbors. The increase in length is due in the main to the addi-
tion of more mesenchymal tissue spaces to its ends and the con-
comitant transformation of more stellate mesenchymal cells into

144 ADAM M. MILLER

endothelial cells. There is probably also some proliferation of
the endothelial cells, although in the study of the sections mitotic
figures were not seen.

The coalescence of the originally unconnected lymphatics
results in a network or plexus of channels (ef. figs. 21, 22 and 24).
This is constantly being augmented by the coalescence of other
independently formed spaces and channels with one another
and with the previouly established plexus. Most of the vessels
composing the plexus lie longitudinally in the embryo along the
line of the aorta and dorsal aortic roots, and out of this plexus is
eventually crystallized the main drainage lines of the thoracic
duct. The establishment of these lines is best considered, how-
ever, in the section on morphogenesis.

The correlation of groups of developing blood cells in this
region, the formation of which has already been described, and
the lymph spaces and channels constituting the anlagen of the
thoracic duct now remains to be discussed. As stated earlier in
this paper, the blood cells that are differentiated from the mesen-
chymal syncytium, whether they are arranged in groups or isolated,
lie free in the tissue spaces. In case the tissue spaces enlarge and
coalesce in the region where the blood cells are situated the latter
are then allowed to become free also in the larger space resulting
from the enlargement and coalescence. The larger space becomes
lined with endothelium, in the manner previously described, to form
a definite vessel or channel. The blood cells, therefore, which
were originally free in the tissue spaces are included in any of the
lymphatics developing in that particular locality (fig. 10, 16, 17).

In that manner some of the blood cells become intravascular
elements during the earliest stages of lymphatic development.
Occasionally an entire group of cells is included in a lymph
channel; in other cases only part of a group or a few scattered
cells. It is true also that a great many lymphatics develop in
the mesenchyme quite apart from the blood cells (figs. 15, 16 and’
7)

In the earlier stages of lymph vessel formation there are great
numbers of blood cells, in various degrees of differentiation, in
the mesenchymal tissue spaces in the vicinity of or more or less

THORACIC DUCT IN THE CHICK 145

remote from the lymph spaces and channels. In part at least these
cells become intravascular when new lymphatics are formed out
of the tissue spaces in which they lie and join the general plexus
of previously formed vessels.

Thus far, therefore, the admission of the blood cells to the
lymph vessels depends merely upon the topographical relation-
ship in the development of the two sets of structures. There are,
however, other factors which in all probability enter into this
process. Two are of especial interest and importance in the case
under consideration.

It has been pointed out by Dantschakoff that the primitive
blood cells in the area vasculosa of the blastoderm and the large
mononuclear cells derived from them are capable of ameboid
movement. The developing blood cells in the region of the tho-
racic duct anlagen are demonstrably of the same type as those in
the area vasculosa. Hence it is reasonable to conclude that some
of the developing blood cells pass from the tissue spaces into the
vessels by virtue of their ameboid character.

The other factor has hitherto, so far as the writer is aware,
been considered only in the study of living tissues. It seems
justifiable, however, to extend the conclusions drawn therefrom
to fixed tissues. In their study of chick blastoderms in vitro,
McWhorter and Whipple (18) have observed the to and fro move-
ment, synchronous with the heart-beat, of blood cells not only in
the isolated, endothelium-lined spaces which eventually coalesce
to form blood vessels but also in the tissue spaces. Further-
more, they have observed the entrance of blood cells into the
general circulation following their to and fro movements in the
tissue spaces. These phenomena certify the pulsation of the fluid
substance in the tissue spaces in response to the heart-beat. It
is not unreasonable, therefore, to conclude that some of the blood
cells in the region of the developing thoracic duct are driven or
sucked into the lymph spaces or channels which, as pointed
before, open freely into the mesenchymal tissue spaces.

As a corollary to the phenomena mentioned in the preceding
paragraph, an additional factor in the formation of endothelium
might be suggested. Granting that the blood cells lying free in

146 ADAM M. MILLER

the tissue spaces move to and fro in response to the heart-beat,
as has been clearly observed in the living chick blastoderm, the
assumption is justifiable that their movement, with the resultant
friction and pressure upon the adjacent protoplasmic elements of
mesenchymal tissue, assists in the flattening of these elements
and the consequent formation of endothelial cells.

The hydrodynamic factors of pressure and frietion of the inter-
stitial fluid substance upon the protoplasmic elements of the
mesenchyme, which were first discussed by Thoma (19) in con-
nection with blood vessel formation and have already been noted
in this particular case of development of lymphatics, together with
the additional factor of pressure and friction of oscillating blood
cells would, in the opinion of the writer, afford adequate mechanical
means of changing the irregular plastic elements of the mesen-
chymal syncytium into the endothelial cells of the vessel wall.

IV. MORPHOGENESIS

Up to this point we have been considering the histogenetic
changes which occur in the mesenchymal tissue, resulting in the
formation of lymph channels and their endothelial lining and of
blood cells. It has been demonstrated that aggregations of the
developing blood cells, identical with the mesenchymal ‘cords’
described by Sala, appear along the lines of the future thoraeic
duct. It has been shown also that the rudiments of the thoracic
duct develop as isolated spaces and channels in the mesenchyme,
that the endothelial lining of the channels is derived directly
from the mesenchymal cells forming their borders and that the
channels coalesce to form a plexus. It is our object, under the
head of morphogenesis, to trace the subsequent history of the
isolated channels and the plexus formed therefrom.

Chick embryo of six days and sixteen hours, 13.5 mm. (Columbia
Embryological Collection, series no. 426). Reconstruction, ventral
view. Figure 20. The incipient stages of thoracic duct develop-
ment are shown about this time. The lymph spaces (/7) are three
in number, two on the right side and one on the left, situated
in the mesenchyme ventro-lateral to the aorta (7) about midway
between the exits of the celiac (5) and superior mesenteric arte-

THORACIC DUCT IN THE CHICK 147

ries. They are isolated, for absolutely not any direct connection
with any other vessel can be traced even with high powers of
magnification. They open freely into the surrounding inter-
cellular spaces, as show in figures 12, 13 and 14, which are photo-
graphic reproductions of three successive sections of the embryo.

The spaces fit the previous description, given in the section
on histogenesis, of accumulations of an intercellular fluid in the
mesenchyme. The objection that they may be shrinkage spaces
is nullified by the fact that the preservation of the tissue is
practically perfect, and that in this and in other stages the more
nearly perfect the preservation the more clearly defined are the
spaces. Furthermore, in cases of poorer preservation where there
are obvious shrinkage spaces in the mesenchyme the boundaries
of such spaces are almost invariably ragged and irregular and
do not anywhere exhibit a smooth endothelial lining.

The masses of developing blood cells in this particular embryo
(six days and sixteen hours) are perhaps unusually extensive
(fig. 20, 16). They extend in irregular groups from about. the
level of the superior mesenteric artery forward along the ventral
and ventro-lateral aspect of the aorta (1) to the level of the celiac
artery (5), with a tendency to cluster around the last named
vessel (cf. fig. 7, 16). They then divide into two general lines,
one on each side, which bend laterad and extend forward along
the mesial aspect of the ducts of Cuvier (12) and precardial veins
(10), ending rather abruptly in a large mass which lies at the level
of the sixth aortic arches (3) and extends across the mesial line
ventral te the dorsal aortic roots (2).

The three spaces representing the first anlagen of the thoracic
duct (17) bear no particular relation to the large groups of devel-
oping blood cells (16), although a few isolated blood cells lie in
the tissue spaces around the larger rudimentary lymph spaces.

Chick embryo of six days and twenty-one hours, 14mm. (Columbia
Collection, series no. 465). Reconstruction, dextro-ventral view.
Figure 21. In this embryo there is a considerable increase in the
size and number of lymph spaces and channels which constitute
the early anlagen of the thoracic duct (17). They are situated
for the most part in the mesenchymal tissue ventral and ventro-

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 2

148 ADAM M. MILLER

lateral to the aorta (/) near the exit of the celiac artery (4).
Other spaces and channels of a similar nature are seen along the
dorsal aortic root (2), thus continuing the same general line of
lymphatics toward the jugular lymph sac (/5) with its thoracic
duct ‘approach’ (15a). Some of these early lymphatics are of
considerable length and have acquired a distinct endothelial
lining. Others are simply enlarged spaces in the mesenchyme
exactly like those described in the preceding stage (figs. 13 and 14).
There is not yet any plexiform arrangement of the channels.

The principle underlying the formation of the longer channels
is in the main the enlargement and coalescence of two or more of
the original smaller spaces or channels. For instance, two isolated
spaces or channels lying near each other in the mesenchyme
increase in size until eventually they flow together to form a
single space or channel. Or, the same process occurs in several -
spaces or channels in a discontinuous series until all the members
of the series flow together and thus form a continuous channel.

There is also in all probability some proliferation of the endo-
thelium lining the channels.’ In fact there is no valid reason for
not assuming that, after the inception of a given vascular channel,
either hemal or lymphatic, along with its increase in size there
is a concomitant proliferation of its endothelial cells. The crucial
point, however, is the orzgin of the channels. The evidence at
hand points clearly to the origin of the lymphatics constituting
the rudiments of the thoracic duct directly from the mesenchymal
interstitial spaces in the manner previously described, the endo-
thelium of the lymphatics representing mesenchymal cells which
are modified in accordance with the new conditions of pressure
and friction.

In this embryo (series no. 465) the rudiments of the thoracic
duct (fig. 21, 17) are isolated. There is no connection between
these and the hemal vascular system; nor on the other hand is
there any communication with the jugular lymph sacs (15). The
thoracic duct ‘approach’ of the lymph sac, which the duct even-
tually joins, is well developed at this stage (15a) but a considerable
distance intervenes between the ‘approach’ and the rudimentary
duct.

THORACIC DUCT IN THE CHICK 149

The masses of developing blood cells in this embryo are un-
usually scarce. A few small groups (16) are situated in the mesen-
chymal tissue ventral to the aorta (7), and three larger groups
(also marked 16) are seen between the dorsal aortic root (2) and
the jugular vein (10).

Chick embryo of six days and twenty-two hours, 16 mm. (Columbia
Collection, series no. 463). Reconstruction, ventral view. \ Figure
22. In this embryo the lymphatics (/7) are in approximately
the same stage of development as in the preceding embryo. They
are situated for the most part along the ventral aspect of the
aorta (1). A few spaces are situated ventro-lateral to the dorsal
aortic roots (2).

The masses of developing blood cells (16) are much more ex-
tensive than in the preceding embryos of about the same stage,
thus exhibiting the variability of the structures. The groups
associated with the rudimentary thoracic duct (1/7) le ventro-
lateral to the aorta (1), with some tendency to cluster around
the celiac artery (5). The rest of the groups extend in a continu-
ous mass on each side along the ventro-lateral aspect of the dorsal
aortic roots (2) and along the mesial aspect of the jugular veins
(10) (ef. fig. 6, 16) as far forward as the fourth aortic arch (4).
A large aggregation of developing blood cells (16a) also lies
between and somewhat dorsal to the aortic roots (2) and the arches
(3, 4) (ef. fig. 6, 16a), being connected with the left lateral groups
by cords extending ventral and dorsal to the roots and arches.
Associated with this large mass are a few lymphaties (1/9) (ef.
fig. 6, 19) which subsequently join the thoracic duct and may also
communicate with the cephalic end of the jugular lymph sac.

One of the most interesting features of this particular stage is
the well developed thoracic duct ‘approach’ of the jugular lymph
sac (fig. 22, 15a). A reconstruction to show this structure was
made on a larger scale and is illustrated in figure 23. The lymph
sac iself (15) lies dorsal to the jugular vein (10) and fits into the
angle between the latter and the subclavian vein (1/1). The tho-
racic duct ‘approach’ (15a) is situated on the mesial aspect of the
jugular-subclavian angle and thence extends a short distance
caudad along the mesial side of the precaval vein. It lies for the

150 ADAM M. MILLER

most part between a mass of developing blood cells (76) and the
vein (10) and ends blindly (see also fig. 19, 76, 15a). A short
distance farther caudad are two spaces in the mesenchyme which
represent the extreme cephalic end of the rudimentary thoracic
duct (fig. 23, 17). These spaces are isolated, for with high magni-
fication there cannot be discerned any connection between them
and the thoracic duct ‘approach’ (15a) or the other rudiments of
the thoracic duct lying still farther caudad.

The thoracic duct ‘approach’ of the jugular lymph sac in the
chick is without doubt the homologue of a similar structure
described by Huntington and McClure (9) in the cat. Like the
lymph sac itself, it is of venous origin in the chick as in the mam-
mal, and forms an integral part of the sac. It arises from some
of the more mesially and caudally situated components of the
early venous plexus in the region of the jugulo-subclavian angle.
When fully formed it extends caudad and mesad for some dis-
tance along the mesial aspect of the precaval vein, and terminates
blindly (figs. 19 and 23, 15a). Subsequently when it is joined
by the thoracic duct, it serves as the portal of entry of this duct
into the lymph sac.

Chick embryo of seven days (Columbia Collection, series no. 512).
Reconstruction, ventral view in figure 24; view from left side, figure
25. In this embryo there is considerable advance in the devel-
opment of the rudiments of the thoracic duct. The spaces and
channels, which in the preceding stage were unconnected, have
here coalesced to form an extensive plexus (/7) of channels
extending from the level of the junction of the dorsal aortic roots
(2) nearly to the exit of the superior mesenteric artery. The
plexus lies for the most part ventral to the aorta (1), but a few
of its components extend around on the lateral aspect of this
vessel. Most of the channels in the network are much larger
than the original spaces and channels, leaving but small areas of
mesenchymal tissue among them; in part they have even fused
to form irregular sinuses. The celiac artery (5) in most of its
longitudinal course penetrates the plexus (1/7). The plexus as a
whole is still isolated, being connected neither with any part of
the hemal vascular system nor with the thoracic duct ‘approach’
of the jugular lymph sac (14a).

THORACIC DUCT IN THE CHICK 151

Along the dorso-lateral aspect of the aorta (7) a number of
spaces and channels have also developed (fig. 25, 20). In addition,
a few smaller lymphatics have appeared along the lateral aspect of
the aorta. The dorsal aorta is thus almost completely encircled
by a group of spaces and channels comprising the large ventral
lymphatic plexus described in the preceding paragraph and the
dorsal and lateral sets of lymphatics.

These encircling lymphatics are in the aggregate homologous
with the dorsal set of peri-aortic sinuses in reptiles, as described
‘by Huntington (2). They are also homologous, in all probabil-
ity, with the azygos portion of the thoracic duct in the cat, as
described by Huntington (3), and in Tragulus, as described by
Tilney (20). A comparison with Stromsten’s (16) description
and figures indicates, too, that the lymphatics associated topo-
graphically with the dorsal aorta in the chick are collectively the
homologue of the peri-aortic network of lymph vessels in the
loggerhead turtle, or, more specifically, with that portion of
the network surrounding the dorsal aorta. In addition, the lym-
phatics around the dorsal aorta in the chick, as previously
described, may be placed in the same phylogenetic line as the
posteardinal and supracardinal divisions of the thoracic duct in
the pig, as recently worked out by Kampmeier (15).

Returning to a further consideration of conditions in the chick
at this stage (seven days, fig. 24), it is seen that a few small
lymph spaces (17a), also isolated, have appeared along the ventro-
lateral aspect of the dorsal aortic roots (2) in the interval between
the plexus previously described (17) and the thoracic duct
‘approach’ of the jugular lymph sac (15a). These small spaces
represent the beginning of the connection between the thoracic
duct ‘approach,’ on each side, and the unpaired portion of the
thoracic duct itself, here composed of the large plexus (/7).

The conditions in the chick thus correspond so closely to those
in other forms that it is possible to draw a clear homology between
the lymphatics joining the thoracic duct ‘approach’ to the un-
paired portion of the duct itself in the chick and the preazygos
segment of the thoracic duct in the cat (Huntington) and Tragu-
lus (Tilney), with the cephalic portion of the peri-aortic lymph

152 ADAM M. MILLER

plexus in the loggerhead turtle (Stromsten), and with the pre-
cardinal division of the thoracic duct in the pig (Kampmeier).

In the mesenchyme between and dorsal to the aortic roots
and arches in this embryo (fig. 24) a number of lymph spaces
have developed. Some of these lie within the masses of develop-
ing blood cells in this region (/6a) while others are situated at
some distance from them. A few have coalesced to form a distinct
channel (19). The subsequent history of these lymphatics will
be given in the discussion of later stages.

A few isolated lymph spaces are also found in and near the
root of the dorsal mesentery at the level where the celiac artery
enters the mesentery (figs. 24 and 25, 21). These belong to the
category of mesenteric lymphatics, but do not yet communicate
with the developing thoracic duct.

The masses of developing blood cells in this embryo are exten-
sive (figs. 24 and 25, 16, 16a). Those associated with the main
portion of the developing thoracic duct lie either in the meshes
of the lymph plexus (1/7) or are already included within the vessels
composing the plexus. A plate-like mass extends over the mesial
aspect of the pleural cavity on the right side. The blood cells
still, as in preceding stages, tend to cluster about the celiac artery
(5). There is no connection in this stage between the masses
just mentioned and those which lie farther forward along the
mesial aspect of the great veins (10, 12), and which are associated
with the thoracic duct ‘approach’ of the jugular lymph sac (14a).
In the region between and dorsal to the dorsal aortic roots (2)
and aortic arches (3) there is seen a large mass (16a) with which
certain lymphatics (19), previously referred to, are associated.
Situated farther caudad are also several masses associated with
the lymphatics which lie along the dorso-lateral aspect of the
aorta (fig. 25, 16a).

A feature shown in this reconstruction, and not in any other, is
a portion of the splanchnic plexus of veins (figs. 24 and 25, 14).

3 Splanchnic venous plexus is the name given by Dr. A. J. Brown, in his yet
unpublished work on the development of the pulmonary veins, to the network
of venous capillaries in the wall of the alimentary tube in the earlier stages of
development.

THORACIC DUCT IN THE CHICK 153

This is seen near the cephalic end of the large ventral lymph
plexus (1/7) but has no connection with the latter.

At this point it may be well to state that in the chicks of the
Columbia Collection I have found no evidence of extra-intimal or
perivenous origin of the lymphatics that make up the thoracic
duct, such as described by Huntington in the cat and Kampmeier
in the pig. This mode of development appears to be a strictly
mammalian specialization. In fact Huntington (3), speaking of
the extra-intimal replacement of veins by developing lymphatics,
states (p. 155):

The association of these (lymphatic) channels, in the mammalian
embryo, with certain embryonal venous lines is purely a secondary,
mechanical and topographical relationship, expressed by the condensed
term of ‘extra-intimal’ development of mammalian systemic lymphatic
vessels, and absolutely devoid of genetic significance. This is, without
reference to other vertebrate classes, proved by the development within
restricted areas in the mammalian embryo of systemic lymphatic chan-
nels through the direct confluence of intercellular mesenchymal clefts,
not related topographically or in any other sense to the embryonal
veins. It is true that in the mammal this independent lymphatic gene-
sis is extremely limited, and that the majority of the lymphatic vessels
develop in close association with embryonal veins, as products of the
confluence of perivenous extra-intimal spaces. But this is merely, as
shown by comparison with other amniote embryos, the expression of the
peculiar relations obtaining in the mammal between the venous and
lymphatic circuits of the vascular system, developed independently of
each other. .

Further, Huntington (2), describing the independently formed
system lymphatic channels of the reptilian embryo, by confluence
of intercellular mesenchymal spaces, states concerning the latter:

They are not complicated by close topographical relations to adjacent
temporary embryonic venous plexuses, as in the mammal, but develop
independently by themselves in mesenchymal territory not occupied by
hemal vascularelements. . . . . Inboth lacertilian and chelonian em-
bryos the greater part of the enormously enlarged systemic lymphatic
channels develop without any reference whatever to embryonic veins, in
mesenchymal areas where the latter are extremely scanty or entirely
wanting (pp. 271-273).

Stromsten (16) has reached similar conclusions in studying the
development of the prevertebral peri-aortic sinuses in chelonian
embryos.

154 ADAM M. MILLER

These finding in reptiles, and my own results in the bird,
warrant the conclusion that the sauropsids agree absolutely
genetically with the mammals in the development of the main
axial lymphatic lines by confluence of independently formed
intercellular mesenchymal spaces, but that the latter are charac-
terized by close topographical association of these spaces with
temporary embryonic veins which they in large part replace,
while the former present no such association. In them the prog-
ress of thoracic duct development is not, as is the case in the mam-
mal, complicated by the presence of an extensive azygos venous
system, and axial lymphatic development, especially in the reptile,
occurs chiefly by confluence of mesenchymal spaces surrounding
the main arterial trunks.

Chick embryo of eight days (Columbia Collection, series no. 513).
Reconstruction, ventral view. Figure 26. The next important
change in the lymphatics constituting the thoracic duct comprises
the further development of certain isolated spaces situated along
the ventro-lateral aspect of the dorsal aortic roots in the interval
between the cephalic end of the large ventral plexus and the
thoracic duct ‘approach’ of the jugular lymph sac. The incipient
stage in the formation of these spaces was illustrated in figure 24,
17a. In the embryo now under conside-ation they have enlarged
and coalesced to form a continuous channel (figure 26, 17a).
-This in turn has united with the ventral unpaired portion of the
thoracic duct (17) and with the thoracic duct ‘approach’ of the
lymph sac (15a). There is thus established a direct and free
communication between the ventral lymph plexus (1/7), which
had arisen as an independent and isolated structure, and the
jugular lymph sac (15). Therefore, as clearly shown in figure 26,
the thoracic duct ‘approach’ (15a), which was previously described
as an integral part of the jugular lymph sac (see fig. 23, 15a),
serves as the portal of entry of the thoracic duct into the lymph
sac (fig. 26, 15).

The writer has in a previous article (1) shown and will here
again point out in a later stage that in the chick, as in reptiles
and mammals, a communication is established between the jugular
iymph sac and the great veins in this region through one or more

THORACIC DUCT IN THE CHICK os

taps. And inasmuch as the thoracic duct opens into the lymph
sac, the latter serves as the portal of entry of the systemic lym-
phatics into the venous system, a point upon which emphasis
has already been laid by Huntington (2) (8) in his work on rep-
tiles and the cat.

In the embryo of eight days the large ventral lymph plexus of
the preceding stage (cf. fig. 24, 17) has undergone further coales-
cence of its component channels to form a large irregular sinus
with a few fenestrae (fig. 26, 17). The sinus lies ventral to the
aorta (1) and extends from the junction of the dorsal aortic roots
(2) to the level of the exit of the superior mesenteric artery (9).
At its cephalic end it branches off into the two slender trunks,
one on each side, which extend cephalad and laterad to join the
‘approaches’ (14a) of the lymph sacs (15). These trunks, which
are the last components of the lymphatic drainage line to develop,
constitute in the bird, according to the anatomical terminology,
the right and left thoracic ducts.

The lymphatics (19) situated between and dorsal to the aortic
roots (2) and arches (3, 4) are here seen to comprise a plexus and
a few outlying isolated spaces and channels (cf. fig. 24 and 26,
19). This plexus now communicates with the chain of lymphatics
(20) lying dorso-lateral to the aorta (7), which in’ turn commu-
nicate with the large ventral plexus threugh channels formed by
coalescence of spaces lateral to the aorta (cf. fig. 25, 20, 17).
The entire group of lymphatics in the region of the dorsal aortic
roots and the dorsal aorta as far back as the superior mesenteric
artery, with the exception of the mesenteric lymphatics (fig. 26,
21), which have not yet joined the thoracic duct, drain into the
jugular lymph sacs.

The masses of developing blood cells were omitted from the
reconstruction of the eight day stage (fig. 26). A careful study
of the serial sections showed that they were fewer and smaller
than in the preceding stage. This may be due to the variability
characteristic of the masses, or it may be due to actual reduction
of the masses since the blood cells, as stated in in the section on
histogenesis, now have access to the jugular lymph sacs through
the recent connection established between these structures and

156 ADAM M. MILLER

the chain of lymphatics with which the blood cells have for the
most part been associated.

Chick embryo of nine days and fourteen hours (Columbia Collec-
tion, series no. 320). Reconstruction, ventral view and view from left
side. Figures 27 and 28. At this stage the main features of the
adult thoracic duct have been established. The large plexus (17)
ventral to the aorta (1) now drains through the right and left
thoracic ducts (17a) into the ‘approaches’ (15a) of the jugular
lymph sacs (15) and then through the sacs into the great veins
(10, 12).

The ventral plexus (/7), the isolated anlagen of which were
the first lympathics to develop in the thoracic duct line, is rela-
tively smaller and the component channels less dilated than in
the preceding stages. This is probably due to the outflow of the
contents into the lymph sacs and veins through the more recently
formed channels which have been called the right and left tho-
racic ducts (17a). The mesenteric lymphatics (21) have increased
in size and for the most part coalesced to form sinus-like chan-
nels. Between these and the thoracic duct chain no connection
can be detected at this stage, although little tissue intervenes.
Farther cephalad another isolated group of lymphatics (22) is
associated with the esophagus.

The right and left thoracic ducts (17a) are longer than in the
preceding stage (cf. fig. 26, 17a). The left is considerably greater
in diameter than the right. Each opens into the corresponding
‘approach’ (14a) of the jugular lymph sac. The ‘approach’ on
each side is still patent as a branch of the main portion of the sac.

There is now free communication between the lymph sacs
(15) and the great veins through recently formed taps, of which
two are present on the left side of the embryo and one on the
right. Of the two on the left side, one is situated on the dorso-
mesial side of the superior vena cava (12) just below the level of
the jugulo-subclavian juncticn (10, 11); the other is situated
farther forward on the mesial side of the jugular vein (1/0). The
tap on the right side of the embryo is located on the dorso-mesial
side of the superior vena cava at the level of the jugulo-subclavian
junction.

THORACIC DUCT IN THE CHICK Lear

In a previous article on the development of the jugular lymph
sac in birds (1) the writer stated, in the description of tap forma-
tion, that ‘‘it is not improbable that a study of later stages will
reveal a homologue of the common jugular tap in the mammal”
(loc. cit., p. 486). There is little doubt that the tap on the mesial
side of the left jugular vein, referred to in the preceding paragraph,
fulfils the requirement. Moreover, the other tap on each side
near the jugular-subclavian junction in the chick is in all proba-
bility homologous with the jugulo-subclavian tap in the mammal
(26), although it is slightly different in position.

The previously described lymphatics situated dorsal to the
aortic roots and arches and dorso-lateral to the aorta here con-
stitute a long chain of continuous channels reaching from the
level of the jugular lymph sac to the level of the celiac artery
(figs. 27 and 28, 19, 20; cf. figs. 24, 25 and 26, 19, 20). At the
extreme cephalic end one of the channels of this series opens into
the right lymph sac. The probable significance of this opening
will be considered in the subsequent discussion of the masses of
developing blood cells. The plexus (19) in the region of the aortic
arches is considerably reduced as compared with preceding stages.
The portion of the dorsal plexus (20) associated with the dorsal
aorta communicates with the large ventral plexus (17) through
small channels which curve around the lateral aspect of the
aorta. It is seen, therefore, that the entire group of lymphatics
associated with the aorta and dorsal aortic roots, with the excep-
tion of a few still isolated spaces and channels, can now discharge
the contents of the channels into the great veins.

The most interesting and, in fact, from the standpoint of the
hemophorie‘ function of the thoracic duct, the most important
feature of this stage is the great reducticn in size and number of
the masses of developing blood cells. It has been shown in the
foregoing pages that the differentiating blood cells associated
with the developing lymphatics are admitted or gain access to
the lymph spaces and channels and that when the communication
is established between the thoracic duct and the jugular lymph
sacs the blood cells can thus reach the sacs. Now since the taps

4 Hemophoric—blood bearing or carrying—is a term suggested by Dr. Schulte
as a codrdinate with hemopoietic—blood producing.

158 ADAM M. MILLER

between the lymph sacs and great veins have been formed, the
blood cells are admitted to the hemal vascular system.

In this connection there are two aspects of the question which
are especially worthy of consideration. In the first place there
are few blood cells within any of the channels forming a part of
the thoracic duct system, while in earlier stages, before the tho-
racic duct had open communication with the lymph sacs and
great veins, many of the lymph channels were filled with the
hemal cellular elements. In view of this, and the fact that there
are no other means of egress for the blood cells formerly contained
in the lymphatics, it must be concluded that the blood cells reach
the hemal vascular system wa the thoracic duct, the jugular
lymph sacs and the recently formed taps between the sacs and
the great veins.

The other feature is the great reduction in the size and number
of the extravascular masses of developing blood cells. The dim-
inished masses are shown in yellow in figures 27 and 28, the con-
ditions in which should be compared with those in figures 22 and
24. A few fairly extensive groups are found in the mesenchymal
tissue among the channels composing the large ventral plexus
(17). These are for the most part quite closely associated with
the lymphatics. A few small groups still are found in the region
of the thoracic duct ‘approach’ (fig. 28, 15a). Theextensive
masses associated in earlier stages with the lymph plexus dorsal
to the aartic arches have almost wholly disappeared. This plexus
itself is considerably reduced, and it is not unreasonable to assume
that the connection between the cephalic end of the plexus and
the jugular lymph sac, previously alluded to, is in some way
associated with the discharge of the numerous blood cells differen-
tiated in this region inte the lymph sac. .

In view, therefore, of the intimate relationship between the
developing thoracic duct and blood cells in the same general
region, and of the sudden and marked reduction in number of
these blood cells, both intravascular and extravascular, following
the establishment of communication between the duct and the
jugular lymph sacs and great veins, the importance of the tho-
racic duct as a carrier of hemal cellular elements for a period of
embryonic life in the bird can scarcely be doubted.

THORACIC DUCT IN THE CHICK 159

V. SUMMARY

Prior to the appearance of lymphatics in the region of the future
thoracic duct, namely, along the aorta and dcrsal aortic roots,
the mesenchyme comprises a syncytium of irregular strands with
correspondingly irregular interstitial spaces. The tissue is non-
vascular.

The initial change in conditions is manifested in the appear-
ance of distinct lacunae in the mesenchymal tissue along the
ventro-lateral aspect of the aorta at the level of the celiac artery.
The lacunae are bounded by unmodified protoplasmic elements
of the mesenchymal syncytium, and open freely into the adjacent .
intercellular—interstitial—spaces. Obviously the lacunae repre-
sent enlarged intercellular spaces, and the inference is justifiable
that they are filled with the intercellular fluid.

In a slightly advanced stage of development in general there is
a greater number of lacunae in the same region in the embryo
and also an increase in size and a difference in the appearance of
some of the lacunae. The increase in size certainly depends in
part upon actual dilatation and in part upon addition of more of
the adjacent mesenchymal intercellular spaces, for every possible
gradation can be seen between the smallest and the largest. The
difference in appearance is observed to be due to the presence
of flat cells which form a distinet boundary or wall, although
not usually complete. Morphologically these cells are equiva-
lent to endothelial cells. Inasmuch as they shade by invisible
gradations into the unmodified mesenchymal cells bordering upon
the rest of the lacuna, we conclude that they are derived directly
from the indifferent mesenchymal cells. The differentiation, we
may also infer, is due in part to pressure and friction incident
to the flow of the tissue fluid. Another factor in the differ-
entiation may also be pressure and friction incident to the to
’ and fro motion of blood cells in the tissue spaces and lacunae in
response to the heart-beat, a phenomenon observed in living
blastoderms.

Studies of later stages show these isolated lacunae to be the
rudiments of the thoracic duct, and the conclusions that they are
direct derivatives of the mesenchymal intercellular spaces and

160 ADAM M. MILLER

that the flat cells forming their endothelial walls are differentiated
in situ from the mesenchymal cells are based upon prolonged and
thorough studies, with high magnification, of serial sections of
chick embryos in practically perfect states of preservation. The
writer is therefore forced to ally himself unequivocally with the
advocates of the view that the thoracic duct originates independ-
ently of the veins and lymph sacs.

Studies of subsequent stages also show that the numerous
isolated lacunae, or rudiments of the thoracic duct, enlarge still
further, principally in a longitudinal direction, and coalesce with
one another to form a plexus of lymph channels which lies ven-
tral to the aorta. Other similar isolated lymphatics develcp along
the dorso-lateral aspect of the aorta and in the region dorsal to
the aortic roots and arches, and then coalesce to form plexuses.
Eventually all the plexuses intercommunicate.

In the meantime a connection is established between the large
ventral plexus and a branch of each jugular lymph sac known as
the thoracic duct ‘approach.’ All the components of the tho-
racic duct system thus drain into the jugular lymph sacs. Com-
munications, or taps, are established between the lymph sacs and
the great veins, and the thoracic duct then drains into the hemal
vascular system, the lymph sacs serving as portals of entry.

In the region of the developing thoracic duct, namely, along
the aorta and dorsal aortic roots, and also in the region dorsal to
the aortic arches, a great number of blood cells arise. The gene-
sis of these cells is indicated by certain changes in some of the
irregular elements of the mesenchymal syncytium, comprising a
marked increase in the basophilia of the cytoplasm, a rounding
of the cell body and a separation from the general mesenchymal
reticulum. The resulting cells thus lie free in the interstitial .
spaces, and structurally are similar to the large mononuclear
cells (lymphocytes of Dantschakoff) in the area vasculosa of the
blastoderm. They increase in number both by mitosis and by
constant differentiation from the mesenchymal syncytium.

Many, at least, of these basophilic cells are transformed
into erythrocytes through the addition of hemoglobin to the
cytoplasm and certain nuclear modifications comprising the

THORACIC DUCT IN THE CHICK 161

disappearance of the nucleoli and the rearrangement of the
chromatin into a heavy reticulum.

These developing blood cells, at first scattered in the mesen-
chymal intercellular spaces, become aggregated, following the
increase in their number, into extensive masses which lie along
the line of the thoracic duct and also in the region dorsal to the
aortic arches.

When the lymphatics comprising the rudiments of the thoracic
duct develop, some of the developing blood cells, even some of
the smaller groups, are seen to be contained within them, while
others are still free in the mesenchymal intercellular spaces, that
is, extravascular. Subsequently more and more of the cells are
observed to be intravascular. It may be concluded that the
developing blocd cells become intravascular by simple inclusion
as the lymph channels develop, or by virtue of their ameboid
character, or as a result of their motion to and fro in the tissue
fluid in response to the heart-beat.

The blood cells that are admitted to the lymph channels con-
stituting the thoracic duct system during its development rapidly
diminish in number after communication is established between
the thoracic duct and the jugular lymph sacs and between the latter
and the great veins. It can be inferred then that the blood cells
which develop along the line of the thoracic duct reach the blood
stream wa this duct and the lymph sacs. Considering the vast
number of hemal cellular elements, especially erythrocytes, aris-
ing in this region and the probability that they reach the general
circulation wa the thoracic duct, this duct assumes an additional
phase of importance in the chick in that it performs a hemophoric,
or blood carrying, function.

In conclusion, I wish to thank Dr. Huntington and Dr. Schulte
for their valuable criticism and suggestions, Dr. McWhorter for
his painstaking work in making the photomicrographs, and Mr.
Petersen for his careful execution of the color plates.

162 ADAM M. MILLER

REFERENCES

(1) Mituer, A. M. 1912 The development of the jugular lymph sac in birds.
Amer. Jour. Anat., vol. 12.

(2) HuntrneTon, GrorceS. 1911 The development of the lymphatic system in
reptiles. Anat. Rec., vol. 5.

(3) 1911 The anatomy and development of the systemic lymphatic vessels in
the domestic cat. Memoirs of The Wistar Institute of Anatomy and
Biology, no. 1, May.

(4) Hoyer, H. 1905-1908 Untersuchungen iiber das Lymphgefiisssystems der
Froschlarven. Bull. de Vacad. des sciences de Cracovie, classe des
sciences math. et nat., I Theil, 1905; II Theil, 1908.

(5) KNowrer, H. McE. 1908 The origin and development of the anterior
lymph hearts and the subcutaneous lymph sacs in the frog. Anat. Ree.,
vol. 2.

(6) Lewis, F. T. 1905 The development of the lymphatic system in rabbits.
Amer. Jour. Anat., vol. 5.

(7) SaBin, FLORENCE R. 1909 The lymphatic system in human embryos, with
a consideration of the morphology of the system. Amer. Jour. Anat.,
vol. 9.

(8) Mrerzesewsky, L. 1909 Beitrag zur Entwicklung des Lymphgefisssystems
der Voégel (vorliufige Mitteilung). Extrait du bull. de l’acad. des
sciences de Cracovie.

(9) Huntinaeton, G.S., anpD McCuiure, C.F.W. 1910 Theanatomy and devel-
opment of the jugular lymph sac in the domestic cat. Amer. Jour. Anat.,
vol. 10.

(10) Santa, L. 1900 Sullo sviluppo dei cuori limfatici e dei dotti toracici nell’
embryone di pollo. Ricerche fatta nel Laboratorio di Anatomia Nor-
male della R. Univ. di Roma, vol. 7.

(11) Sasrn, FLrorence R. 1902 On the origin of the lymphatic system from
veins, and the development of the lymph hearts and thoracic duct in
the pig. Amer. Jour. Anat., vol. 1.

(12) Lancer, C. 1868 Uber das Lymphgefiisssystem des Frosches. Sitz. d.
Akad. d. Wissensch., Bd. 57.

(13) Ranvier, L. 1897 Morphologie et developpement des vaissaux lympha-
tiques chez les mammiferes. Arch. d’anatomie microscopique, tome 1.

(14) Huntinaton, GrorceES. 1908 The genetic interpretation of the develop-
ment of the mammalian lymphatic system. Anat. Rec., vol. 2.

(15) Kempmeter, Orto F. 1912 .The development of the thoracic duct in the
pig. Amer. Jour. Anat., vol. 13.

(16) StrRomsTEN, FRANK A. 1912 On the development of the prevertebral (tho-
racic) duct in turtles as indicated by a study of injected and uninjected
embryos. Anat. Rec., vol. 6.

(17) DantscHakorr, W. 1908 Untersuchungen iiber die Entwickelung des
Blutes und Bindegewebes bei den Végeln. I. Die erste Entstehung der
Blutzellen beim Hiihnerembryo und der Dottersack als blutbildenes
Organ. Anatomische Hefte, 113 Heft (37. Band, Heft 3).

THORACIC DUCT IN THE CHICK 163

(18) McWuorter, J. E., AnD WuippPLe, A. O. 1912 The development of the
blastoderm of the chick in vitro. Anat. Rec., vol. 6.

(19) THoma, R. 1893 Untersuchungen iiber die Histogenese und Histomechanik
des Blutgefiisssystems. Stuttgart.

(20) Tinney, F. 1912 The development of the veins and lymphatics in Tragu-
lus meminna. Amer. Jour. Anat., vol. 13.

(21) McCuureg, C.F. W. 1908 The development of the thoracic and right lym-
phatic ducts in the domestic cat. Anat. Anz., Bd. 32, nos. 21 and 22.

(22) Sasrn, FroreNce R. 1911 A critical study of the evidence presented in
several recent articles on the development of the lymphatic system.
Anat. Rece., vol. 5.

(23) McCuureg, C. F. W. 1910 The extra-intimal theory and the development
of the mesenteric lymphatics in the domestic cat (Felis domestica).
Anat. Anz., Erganz.z. Bd. 37.

(24) Sasrin, FLoRENCE R. 1912 On the origin of the abdominal lymphatics in
mammals from the vena cava and the renal veins. Anat. Rec., vol. 6.

(25) Huntineton, GEO. S., anp McCuure, C.F. W. 1907 The development of
the main lymph channels of the cat in their relations to the venous sys-
tem. Anat. Rec., vol. 1, no. 3.

(26) McCuure, C. F. W., AND SiILvEesTER, C. F. 1909 A comparative study of
the lymphatico-venous communications in adult mammals. Anat. Rec.,
vol. 3.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 2

PLATE 1
EXPLANATION OF FIGURES

1 From a section of a chick embryo of 108 hours, 6.75 mm. (series no.
371; slide II, section 80). Outlines drawn under Edinger projection apparatus,
1500; reduced to X1000. In the upper left corner is a portion of the aorta with
a few erythrocytes. Outside of the aortic wall is an area of mesenchyme in which
some elements show stages of differentiation leading toward large basophilic cells,
two of the latter lying free in the tissue spaces at the lower left corner of the
figure. These are apparently identical with the developing blood cells (lympho-
cytes) in the area vasculosa of the blastoderm as described by Dantschakoff.

2 From the same embryo as figure 1 (slide II, section 97). Outlines drawn
under Edinger projection apparatus, 1500; reduced to X 1000. A small part of
the aorta is shown in the upper right corner. Some of the protoplasmic elements
of the mesenchymal syncytium are shown in various stages of differentiation lead-
ing toward the free basophilic cells (lymphocytes of Dantschakoff). In the lower
left corner is seen one of these cells in the anaphase of mitosis. At the right of
this is a practically mature erythrocyte lying free in a mesenchymal intercellular
space.

3 From a section of a chick embryo of 132 hours, 9 mm. (series no. 411;
slide IV, section 16). Outlines drawn under Edinger projection apparatus, X
1500; reduced to < 1000. This figure, taken just ventral to the aorta, shows a
number of the rounded basophilic cells lying free in the mesenchymal intercel-
lular spaces.

164

THORACIC DUCT IN THE CHICK PLATE 1
ADAM M. MILLER

PLATE 2
EXPLANATION OF FIGURES

4 From a section of a chick embryo of 5 days, 12 mm. (series no. 336; slide
IV, section 7). Photomicrograph, 166.

aorta 10, precardinal vein

il
3, aortic arch Vi : 16, group of developing blood cells

aortic arch IV

*

5 From a section of a chick embryo of 6 days, 13.5 mm. (series no. 339;
shde VIII, section 35). Photomicrograph, x 166.

2, dorsal aortic root 16, groups of developing blood cells
3, ‘aortic arch VI 27, vagus nerve

166

9

PLATE

x

THORACIC DUCT IN THE CHICI

MILLER

ADAM M.

PLATE 3
EXPLANATION OF FIGURES

6 From a section of a chick embryo of 7 days (series no. 512; slide III,
section 15). Photomicrograph, S80.

2, dorsal aortic root 19, lymphatics dorsal to aortic arches
3a, pulmonary artery and esophagus
10, precardinal vein 27, vagus nerve
16, groups of developing blood cells esophagus in center of figure
along precardinal vein bronchi at lower border of figure

16a, groups of developing blood cells
dorsal to esophagus

7 From a section of a chick embryo of 6 days and 16 hours, 13.5 mm
(series no. 426; slide XI, section 21). Photomicrograph, x 133.

1, aorta 23, celom (pleural cavity)
5, celiac artery 25, mesonephros
8, dorsal somatic artery 26, lung anlage

16, groups of developing blood cells

168

THORACIC DUCT IN THE CHICK PLATE 3
ADAM M. MILLER

PLATE 4

EXPLANATION OF FIGURES

8 From a section of a chick embryo of 6 days and 22 hours, 16.5 mm.
(series no. 464; slide XXII, section 22). Photomicrograph, 133.

7, aorta 16, groups of developing blood cells
5, celiac artery 23, celom (pleural cavity)
8, dorsal somatic artery 24, sympathetic nerves

9 From a section of a chick embryo of 6 days and 16 hours, 13.5 mm.
(series no. 426; slide XVIII, section 3). Photomicrograph, X 233.

1, aorta 17, lymph spaces, rudiments of thoracic
16, developing blood cells duct
23, celom (abdominal cavity)

170

THORACIC DUCT IN THE CHICK PLATE 4

ADAM M. MILLER

PLATE 5
EXPLANATION OF FIGURES

10 From a section of a chick embryo of 7 days (series no. 512; slide VIT,
section 1). Photomicrograph, * 233.

f, aorta: 17, lymphatics, rudiments of thoracic
5, celiac artery duct
16, groups of developing blood cells 23, celom (abdominal cavity)

11 From a section of a chick embryo of 14 days (series no. 518; slide
XIII, section 9). Photomicrograph, * 133.

1, aorta 17, lymphatics (part of thoracic duct
16, blood cells, mostly mature erythro- system)
cytes, In tissue spaces

172

PLATE 5

THORACIC DUCT IN THE CHICK

ADAM M. MILLER

~) 7%; Tr, ! ere

» ? -

‘

PLATE 6
EXPLANATION OF FIGURES

12, 13, 14 From three successive sections of a chick embryo of 6 days
and 16 hours, 13.5 mm. (series no. 426; slide XII, sections 9, 10 and 11). Photo-
micrographs, X 350.

In figure 13 the arrow (17) points to a lymph space in the mesenchyme. At
its right side the lymph space is seen clearly to open into the adjacent mesenchy-
mal intercellular spaces. The preceding section in the series (fig. 12) shows no
space (the arrow points to the same locality as in fig. 13). In figure 14 the arrow
points toa light area in the mesenchyme which represents the opening of the space
of figure 13 into the adjacent mesenchymal intercellular spaces; this can be seen
much more clearly with the microscope by changing the focus. The lymph space
shown in figure 13 is the same one represented in figure 20, 17 at the right of the
aorta. Aorta, 1; celom (abdominal cavity), 23.

174

THORACIC DUCT IN THE CHICK PLATE 6
ADAM M. MILLER

PLATE 7
EXPLANATION OF FIGURES

15, 16, 17 and 18 From four successive sections of a chick embryo of 7 days
(series no. 512; slide VII, sections 21 and 22; slide VIII, sections 1 and 2). Photo-
micrographs, X 250.

In figure 15 four lymph spaces, in part lined with endothelium, are seen at the
left of the aorta (1). The one nearest the aorta, while in part lined with endothe-
lium, opens freely into the adjacent mesenchymal intercellular spaces. The larg-
est space also opens below in asimilar manner. In figure 16, from the succeeding
section in this series, only one of the spaces appears, opening above into tissue
spaces. The others have simply become continuous with tissue spaces and there-
fore do not appear as distinct lacunae; this can be clearly demonstrated with the
microscope by changing the focus on the rather thick sections (20 micra). In
figure 17 the arrow points to the termination of the distinct lacuna of figure 16;
the free communication with the tissue spaces is quite obvious. In figure 18 there
are no lacunae, all those of the preceding sections having opened into the mesen-
chymal intercellular spaces. Aorta, 1; part of sympathetic nervous system, 24.

176

THORACIC DUCT IN THE CHICK PLATE 7
ADAM M. MILLER

t
4

ey

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PLATE 8
EXPLANATION OF FIGURE

19 From a section of a chick embryo of 6 days and 22 hours, 16 mm. (series
no. 463; shde XVI, section 9). Photomicrograph, X 160.

2, dorsal aortic root 15, jugular lymph sac
3a, pulmonary artery 15a, thoracic duct ‘approach’
12, precaval vein 27, vagus nerve

13, vertebral vein

178

PLATE 8

THORACIC DUCT IN THE CHICK

MILLER

ADAM M.

179

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 2

PLATE 9
EXPLANATION OF FIGURE

20 Drawn from a reconstruction of a chick embryo of 6 days and 16 hours, 13.5
mm. (series no. 426). Ventral view.

1. aorta 11, subclavian vein

2, dorsal aortic roots 12, duct of Cuvier

3, aortic arch VI 13, vertebral vein

3a, pulmonary artery 15, jugular lymph sac

4, aortic arch IV 16, groups of developing blood cells
5, celiac artery 17, first rudiments of thoracic duct

10, precardinal vein

180

PLATE 9

THORACIC DUCT IN THE CHICK

ADAM M. MILLER

181

PLATE 10

EXPLANATION OF FIGURE

21 Drawn froma reconstruction of a chick embryo of 6 days and 21 hours, 14

mm. (series no. 465).

1, aorta

2, dorsal aortic roots
3, aortic arch VI

3a, pulmonary artery
5, celiae artery

6, notocord

10, precardinal vein
11, subelavian vein

Ventro-mesial view.

12, duet of Cuvier

13, vertebral vein

15, jugular lymph sac

15a, thoracic duct ‘approach’ of jugular
lymph sac

16, groups of developing blood cells

17, rudiments of thoracic duct

182

THORACIC DUCT IN THE CHICK PLATE 10
ADAM M. MILLER

PLATE 11

EXPLANATION OF FIGURE

«

22. Drawn from a reconstruction of a chick embryo of 6 days and 22 hours, 16
mm. (series no. 463). Ventral view.

1, aorta 15a, thoracic duct ‘approach’ of jugular
2, dorsal aortic root lymph sae

3, aortic arch VI 16, groups of developing blood cells
3a, pulmonary artery 16a, groups of blood cells dorsal to
4, aortic arch IV aortic roots and arches

5, celiac artery 17, rudiments of thoracic duct

10, precardinal vein 18, lymphatics along aortic arches

11, subclavian vein 19, lymphatics dorsal to aortic roots
12, duct of Cuvier and arches

13, vertebral vein 21, mesenteric lymphatics

15, jugular lymph sae

184

THORACIC DUCT IN THE CHICK PLATE 11

ADAM M. MILLER

PLATE 12
EXPLANATION OF FIGURE

23. Drawn from a reconstruction of a chick embryo of 6 days and 22 hours, 16
mm. (series 463). Mesial view.

10, precardinal vein 15a, thoracic duct ‘approach’ of jugular
11, subclavian vein lymph sac

12, duct of Cuvier 16, groups of developing blood cells

15, jugular lymph sac 17, rudiments of thoracic duct, extreme

cephalic end

LS86

THORACIC DUCT IN THE CHICK PLATE 12
ADAM M. MILLER

187

PLATE 18

EXPLANATION OF FIGURE

24 Drawn from a reconstruction of a chick embryo of 7 days (series no. 512).
Ventral view.

1, aorta 16, groups of developing blood cells

2, dorsal aortic roots 16a, blood cells dorsal to aortie roots
8, aortic arch VI and arches

3a, pulmonary artery 17, thoracic duct, ventral plexus (homo-
5, celiac artery logue of azygos segment)

7, carotid artery 17a, thoracic duct (homologue of pre-
10, precardinal vein azygos segment)

11, subclavian vein 18, lymphatics along aortic arches

12, duct of Cuvier 19, lymphatics dorsal to aortic roots
14, part of splanchnic plexus of veins and arches

15, jugular lymph sae 21, mesenteric lymphatics

15a, thoracic duct ‘approach’ of jugular
lymph sac

THORACIC DUCT IN THE CHICK PLATE 13

ADAM M. MILLER

189

PLATE 14
EXPLANATION OF FIGURE

25 From same reconstruction as in fig. 24. View from left side.

1, aorta 14, part of splanchnic plexus of veins
3, aortic arch VI 15, jugular lymph sae

5, celiac artery 16, groups of developing blood cells
7, carotid artery 16a, blood cells dorsal to aortic roots
8, dorsal somatic arteries 17, thoracic duet, ventral plexus

10, precardinal vein 18, lymphatics along aortic arches
11, subclavian vein 20, lymphaties dorso-lateral to aorta
12, duct of Cuvier 21, mesenteric lymphatics

13, vertebral vein

190

THORACIC DUCT IN THE CHICK PLATE 14

ADAM M. MILLER

PLATE 15

EXPLANATION OF FIGURE

26 Drawn from a reconstruction of a chick embryo of 8 days (series no. 513).

Ventral view.

1, aorta

2, dorsal aortic roots

8, aortic arch VI

3a, pulmonary artery

4, aortic arch IV

5, celiac artery

6, notocord

7, carotid artery

9, superior mesenteric artery
10, precardinal (jugular) vein
11, subclavian vein

12, duct of Cuvier

15, jugular lymph sac

15a, thoracic duct ‘approach’ of jugular
lymph sac

17, thoracic duct, ventral plexus (homo-
logue of azygos segment

17a, thoracie duct (homologue of pre-
azygos segment)

18, lymphatics along aortic arches and
duct of Cuvier

19, lymphatics dorsal to aortic roots
and arches

20, lymphatics dorso-lateral to aorta

21, mesenteric lymphatics

192

THORACIC DUCT IN THE CHICK PLATE 15
ADAM M. MILLER

PLATE 16

EXPLANATION OF FIGURE

27

(series no. 320). Ventral view.

, aorta

, dorsal aortic roots

3, aortic arch VI

3a, pulmonary artery

, aortic arch IV

, celiac artery

7, carotid artery

9, superior mesenteric artery
10, p ecardinal (jugular) vein
11, subelavian vein

12, duct of Cuvier

15, jugular lymph sae

1
2

/
4
d
y

15a, thoracic duct ‘approach’ of jugular

lymph sac

194

Drawn from a reconstruction of a chick embryo of 9 days and 14 hours

16, groups of developing blood cells

17, thoracic duct, ventral plexus (homo-
logue of azygos segment.

17a, thoracic duct (homologue of pre-
azygos Segment.

18, lymphatics along aortic arches

19, lymphatics dorsal to aortic roots
and arches

21, mesenteric lymphatics

22, lymphatics associated with esoph-
agus

THORACIC DUCT IN THE CHICK PLATE 16
ADAM M. MILLER

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 2

PLATE 17

EXPLANATION

OF FIGURE

28 From same reconstruction as figure 27. View from left side.

1, aorta

2, dorsal aortic roots

3, aortie arch VI

7, carotid artery

8, dorsal somatic arteries

9, superior mesenteric artery
10, precardinal (jugular) vein
11, subclavian vein

12, duct of Cuvier

13, vertebral vein

15, jugular lymph sac

196

15a, thoracic duct ‘approach’ of jugular
lymph sac

16, groups of developing blood cells

17, thoracic duct

18, lympathies along aortic arches

19, lymphatics dorsal to aortic roots
and arches

20, lymphatics dorso-lateral to aorta

21, mesenteric lymphatics

22, lymphatics associated with esoph-

agus

THORACIC DUCT IN THE CHICK PLATE 17
ADAM M. MILLER

- i
‘

ee. ot) AD | eR ae ee Sh eee” Ae a ‘end :

ON THE PRENATAL AND NEONATAL LUNG

WILLIAM H. F. ADDISON AND HAROLD W. HOW

The Anatomical Laboratory of the University of Pennsylvania
BIGHT FIGURES
INTRODUCTION

Of the various changes occurring in the lung at birth, we have
studied especially the increase in size of the spaces within the
respiratory lobules, and the change in the character of the con-
tents of these spaces. Whenever this portion of the lung’s his-
tory is considered, the statement is made that, with the first
pulmonary inspirations after birth, the spaces become distended,
but we have found no data expressing the amount of this dis-
tention. As to the question of the existence of fluid contents in
the spaces of the prenatal lung, it is seldom mentioned in current
literature, although it would seem to be of importance, especially
in connection with the beginning of breathing.

In making comparisons, we have used material from experi-
mentally controlled animals, in order to obtain specimens of pre-
natal and neonatal lungs, which would be comparable in all
respects. This was done by using animals of the same litter,
of which some had breathed, and others had not breathed.

LITERATURE

Measurements made on the minute parts of the lung have
been recorded by a comparatively small number of investigators.
Friedrich Merkel, in Bardeleben’s Handbuch (’02), reproduces
from Rossignol (’46) a table of sizes of lung alveoli at different
ages. The first item in the list gives the mean size of alveoli
of newborn children which have not breathed, or have breathed

199

200 WILLIAM H. F. ADDISON AND HAROLD W. HOW

only a few hours. It is exactly between these two classes of
individuals that we have sought to study the differences. K6lliker
made numerous lung measurements but apparently none directly
contrasting the two conditions seen in the prenatal and post-
natal lung. Miuller’s (95) table of sizes of the various air-pas-
sages In dogs is from material of one stage only. Concerning
the phenomenon of expansion from a more general point of view,
there are many observations. For instance, the fact that all
parts of the neonatal lung do not expand equally has been recorded
a number of times. Dohrn (’91) says that at least two days
are required to unfold the lungs completely. Jalan de la Croix
(’83), found in the lung of a child which lived seven days groups
of alveoli which persisted airless. In dealing with the lung of
children which have survived only a few days after birth, there
is the possibility, however, of the organ being premature or
pathological, and either of these conditions would affect its expan-
sion. The question of the contents of the spaces in the fetal
lung has for the most part been referred to only incidentally, by
observers interested in premature respiratory movements. The
experiments recorded by Preyer (’85) in his ‘“‘Specielle physio-
logie des embryo,” are the most definite we have found, and
these will be referred to later.

A complicating factor in studying the appearance of the nor-
mal fetal lung is the possible occurrence of intrauterine respira-
tory movements. These have been described in the study of
living human fetuses by a whole series of clinical observers (for
instance, Ferroni 799) and have even been recorded by means of
tracings. The current opinion as expressed by Howell (11) is
that the mammalian fetus under normal conditions makes no
respiratory movements while in utero. Ballantyne (’02) in his
‘Antenatal pathology and hygiene’’ accepts the results of the
clinicians, but says it is doubtful if such movements are strong
enough to draw liquor amnii into the lungs. It would also seem
from our experiments that if these movements are really similar
to respiratory movements, they are not to be compared in point
of intensity with the postnatal efforts, and do not affect the
structure of the lung to any extent.

PRENATAL AND NEONATAL LUNG 201
METHOD OF PRESERVATION OF MATERIAL

In order to preserve, as nearly as possible, the normal relations
of the lung to surrounding structures, and to prevent its collapse,
all animals were injected with 5 or 10 per cent formalin, either
through one of the umbilical vessels, or through the aorta, with-
out opening the thoracic cavity. After the lungs had hardened
in situ, portions were removed, embedded in paraffin or celloidin
and sectioned.

CONTENTS OF THE SPACES WITHIN THE FETAL LUNG

When a living fetus, near full-term, is exposed within the
uterus, with the amniotic sac still unruptured, it can readily be
stimulated to make respiratory movements. The angles of the
mouth begin to twitch, and are drawn slightly upwards, the
abdomen enlarges, evidently due to the descent of the diaphragm,
and almost simultaneously the nostrils dilate, and the mouth
shghtly opens in a yawning manner. The result is the drawing
into the respiratory tract of the amniotic fluid. We found that
the mere manipulation necessary to expose the fetus is sufficient
to bring about these movements, if the animal is very near the
end of gestation. A surer method is to clamp the umbilical
cord, but neither method will act if the animal is not sufficiently
advanced in its development. Several have previously recorded
the above or similar observations. Winslow is quoted by Preyer
as having written in 1787, ‘ Liquorem amnii respirare videntur.”’
Leclard (15) clamped the neck of a still living fetus, and on
opening the trachea found there a fluid analogous to amniotic
fluid. When a colored fluid had been injected previously into the
amniotic fluid that in the bronchi was likewise colored. Preyer
(85, p. 148) repeated and verified Leclard’s experiment with a
guinea-pig near the end of gestation. He found that the fuchsin
which he injected into the amniotic sac not only colored the
lips, tongue, palate and all the pharynx, but also the lungs and
the inside of the stomach. Geyl (’80) added to the experiment
in the following manner. With all aseptic precautions, he in-
jected aniline blue into the amniotic sacs of seven fetuses of a

202 WILLIAM H. F. ADDISON AND HAROLD W. HOW

rabbit, which was nearly three weeks pregnant. Three days
later the seven young were born, three dead and four alive. The
three former had their lungs colored blue, as had also one of the
latter. While there is no doubt that liquid is present within
the lungs after these inspirations of amniotic fluid, none of the
observers have directed their attention especially towards see-
ing the contents before such inspirations had taken place. One
can easily deduce that liquid is present all the time during the
development of the fetal lung, but in order to obtain, if possible,
direct evidence several simple experiments were performed. The
first series was with large sheep fetuses, obtained from an abat-
toir, with membranes and uterus intact. In two cases the fol-
lowing procedure was followed. The uterus was opened, and
the fetus, 35 em. in length, exposed within the unruptured amni-
otic sac. By means of a needle carefully passed through the
amnion, a strong ligature was drawn through the tissues of the
neck of the fetus, behind the trachea, and out of the amnion
again at the point of entrance. With the head of the fetus
covered by the amniotic fluid, the ligature was tied tightly and
the trachea constricted. Our special aims were to prevent liquid
escaping from the amniotic sac and air from entering it. The
trachea and lungs were carefully dissected out, without injuring
the visceral pleura, and placed in a large jar of water, from which
all air-bubbles had been previously removed. After they had
been allowed to sink to the bottom of the jar they were agitated
in order to remove adherent air. Different parts of the lung
were then cut with scissors and crushed. <A yellowish-red fluid
escaped from the crushed masses of tissue and diffused through
the water but no bubbles were seen to escape.

In a third sheep fetus the trachea was ligated at its upper
and lower ends and then dissected out. The closed segment of
trachea was carefully cleaned and dried, before being opened over
a dry glass plate. It was found to contain a faintly yellowish
slightly viscid fluid, which was pressed out on the plate. When
this fluid was tested with acetic acid there was a reaction show-
ing the presence of mucin. The bronchi contained a similar fluid,
and when the lungs were compressed this fluid was forced out.

PRENATAL AND NEONATAL LUNG 203

In order to secure histological material for a study of the
normal appearance of these lungs, another fetus of approximately
the same size was injected through the umbilical vein with 10
per cent formalin. Later the thorax was opened and after the
relations of the lung were observed, pieces of the lung were taken
for histological sections. The appearance as evidenced by char-
acters to be described later, was that of a lung which had not
made premature respiratory movements.

The second series of experiments were repetitions of the fore-
going, performed on a litter of living dog fetuses lying within
the uterine horns of the mother. The parent animal was anes-
thetized and the young carefully exposed one at a time without
unnecessary manipulation. In order to prevent the entrance of
amniotic fluid, due to premature respiratory movements, the
trachea of each fetus used was clamped with an artery forceps
as soon as it was exposed and before the amnion was ruptured.
As a result of this stimulation the animal at once began to make
violent efforts to breathe, but if the trachea was well clamped
these were ineffectual and the animal soon died of asphyxiation.
The lungs were removed as described for the sheep fetuses, and
experiments carried out in a parallel way. The results were
similar to those of the first series, so it may be concluded that
normally in the fetal trachea, bronchi and lungs there exists a
liquid which resembles the amniotic fluid in appearance. The
question arises as to what becomes of this liquid when breathing
begins. From observations on fetuses removed from the uterus,
the first act in breathing is always inspiration. As the lung
enlarges, due to the contraction of the diaphragm and other re-
spiratory muscles part of the liquid in the upper parts of the tract
is drawn downwards and part remains distributed along the walls
of the air-passages. When the fetus inspires amniotic fluid before
its removal from the sac, it is always much handicapped. It
does not breathe so strongly, and its efforts become weaker, and
farther apart until they cease altogether. If in these circum-
stances, as we tried with young dogs, the animal be held with
head downwards, and the thorax compressed at short intervals,
the difficulty in breathing is often relieved, and the animal im-

204 WILLIAM H. F. ADDISON AND HAROLD W. HOW

proves and lives. As is known from both clinical medicine and
experimental studies, the lung has a marked capacity for the
absorption of liquids, and the quantity of fluid present in normal
birth is readily disposed of.

MATERIAL. FOR BREATHING AND NON-BREATHING LUNG

In order to secure examples of the breathing and non-breath-
ing lung which would be strictly comparable, we used pregnant
dogs near the end of the gestation period. By the manner de-
scribed in experiment on the contents of the prenatal lung, some
of the fetuses were removed and not allowed to breathe. Others
were removed from the membranes as quickly as possible, before
any attempt at breathing had been made. After the umbilical
cord was tied and severed, they were laid in a warm place. They
soon showed signs of activity, crying and crawling about. These
were etherized at the end of an hour, and injected with formalin.
Fetal material was also obtained by etherizing the parent ani-
mal after several fetuses had been taken out and allowing the
remainder of the young to die before removing them. For com-
parison with the new-born, pups two days old were used, and
these were injected with formalin through the abdominal aorta.

STUDY OF PRENATAL LUNG

The appearance of sections from the fetal lung obtained in
the above described manner is shown in figures 1 and 2. The
texture of the organ is quite gland-like, with the mesenchyme
framework present in large amount. The spaces are variable in
size and shape on account of the structures being cut in different
planes of section, but the distribution of the spaces is fairly
uniform. In order to study the relative area occupied by lung
tissue and the intervening open spaces, drawings were made with
the Edinger drawing apparatus, on cross-ruled millimeter paper.
Various magnifications were used, varying from 60 to 220. The
results of a series of such drawings showed that the percentage
of area occupied by lung tissue was 70 to 80 per cent and by the
intervening spaces 20 to 30 per cent. In view of this condition
it does not seem correct to call the fetal lung ‘solid,’ as do some

NEONATAL LUNG

AND

PRENATAL

Ae.

X 62.

ation.
_ at end of gestation, with the granular reticular

, at end of gest
substance in the spaces, and two of the free mononuclear cells.

Fetal lung of dog

Fig. 1

Fetal lung of dog

Oe

Fig.

280.

x

206 WILLIAM H. F. ADDISON AND HAROLD W. HOW

of the present-day text-books of physiology and pathology. On
the other hand, it is very easy to increase the size of the spaces
artificially. In J. M. Flint’s paper on the development of the
lungs (’06), he has an illustration of the lung of a fetal pig 27
em. long, which is a stage shortly before birth. For the pur-
pose of preservation the lung was injected intra-tracheally with
the fixing fluid and this fact is responsible for the appearance of
the lung in section. The appearance resembles very closely that
of a lung which has breathed, as one can see by comparing it
with his next figure, no. 29, that of a two-day-old pig. This is
because the spaces were distended by the injection of the fixing
fluid and indeed, as we found with sheep fetuses, relatively little
force is required to distend the fetal lungs when introducing fluid
through the trachea. In consequence of this distention the lin-
ing cells of the respiratory lobules are artificially stretched and
flattened and no longer show the normal condition.

When the spaces are examined they are seen not to be entirely
empty, but to contain here and there light pink-staining irregular
masses of a finely granular substance. This is apparently a pre-
cipitate derived from the liquid existing within the spaces, and
was clearly seen at the period just before birth in all well-pre-
served fetal lungs not only of dog, but also of cat, rat and man.
The origin of the mucin constituent of the fluid is at least partly
from the goblet cells of the trachea, for in sections of the latter
we found them numerous and characteristically stained. Men-
tion may also be made of conspicuous large rounded cells lying
free within the spaces. They are distributed rather evenly but
not in large numbers, and usually occur singly. They measure
11 to 14 uw in diameter, and have a single nucleus which is eccen-
trically placed. The nucleus may be round, oval or indented,
and varies from 6 to 7 u in its greatest dimension. If flattened
in shape, the width is 38 to 44. These cells in the dog have a
distinctly granular cytoplasm, which stains well with eosin. They
probably belong to the same variety of cells as those present in
the air-spaces of the normal breathing lung which take part in
removing carbon particles and other foreign matter from the
alveoli. In the fetal lung they may have the similar function of

PRENATAL AND NEONATAL LUNG 207

removing cellular débris, from the liquid-filled spaces. They also
resemble the ‘heart-failure cells’ of the lung which are seen in
certain pathological conditions. Several views are held as to the
nature of these phagocytic cells in the breathing lung, but the
study of these very similar appearing cells in the fetal lung, leads
one to agree with Kolliker (02 vol. 3, p. 310), that they are a
form of ‘Wanderzellen,’ and not desquamated epithelium.

NEONATAL LUNG ONE HOUR OLD

When the respiratory muscles first begin to act at the begin-
ning of neonatal life, the thoracic cavity is enlarged. In con-
sequence of the relation between thorax and pleural sacs the
lung is likewise enlarged and inspiration is the result. With the
increase in size of the lungs to occupy a larger volume, the frame-
work of the organ is stretched and put on a tension, while the
spaces become larger. This is easily apparent when one com-
pares figures 4 and 3, showing the lung of a dog which had breathed
one hour, and a fetal lung respectively. By examining sections
across the entire lung and sections from different regions, it can
be seen that expansion does not take place equally in all parts
of the lung, nor in any particular area do all the alveoli and air-
saes increase alike. As a result it was not easy to arrive at a
quantitative estimation of the area of lung-tissue and of the air-
passages, as seen in the sections. In regions, which had not been
much inflated, and did not appear much more open in character
than fetal lung, the framework still occupied 60 per cent of the
total area. But in regions where the respiratory channels were
more dilated, it occupied only 40 per cent or in restricted areas
even less. Thus there is a variation in the ratio, but in general
it may be said that the tissue occupies 40 to 60 per cent of the
area of the cross-section. The fact that the lung does not expand
at once after birth was seen also in newborn albino rats. These
were obtained from The Wistar Institute of Anatomy with the
aid of Dr. Stotsenburg. These young were taken immediately
after birth, before they were dry and before they had begun to
feed. Examination of their lung (fig. 6) shows that some areas
have been inflated very little, in contrast with other parts, which
are quite expanded.

10.

showing texture of lung before breathing.
g.4 Lung of one-hour-old dog of same litter, showing expansion of lung after breathing one hour.

Fetal lung of dog at end of gestation,

x 40.

PRENATAL AND NEONATAL LUNG 209

A eareful examination shows that the granular reticular sub-
stance may still be found usually lying in contact with the walls
of the air-spaces. Its presence here indicates how the fetal liquid

Fig. 6 Lung of albino rat, one-half hour after birth, showing partial expan-
sion. X 25.

is disposed of when breathing commences. When the lung ex-
pands and the fluid in trachea and bronchi is drawn downward,
part of the fluid remains adherent to the inside of the walls.
In many sections it appears that more of the granular precipitate

210 WILLIAM H. F. ADDISON AND HAROLD W. HOW

is seen in the peripheral portions of the lobes, immediately under
the pleura, as if more of the liquid had found its way to the ends
of the lobules.

The large mononuclear cells which were seen scattered about
in the fetal lung are now difficult to find, although they may
still be seen by searching. In comparing the prenatal and neo-
natal lungs of kittens and other litters of dog, this same condition
was noted.

LUNG OF TWO-DAY-OLD DOG

The structure of the lung at this stage resembles, in general,
the mature lung. Reference to figure 5 shows the texture to be
very open in character. By means of outline drawings on cross-
ruled millimeter paper the framework is found to be only 20 to
30 per cent of the entire area, the open spaces constituting the
remainder 70 to 80 per cent. As the lung enlarges with the
further growth of the animal, the spaces still further increase.
As it is generally expressed, the growth of the lungs does not
keep pace with the growth of the thorax, as a whole, and con-
sequently the framework of the lungs becomes more and more
stretched, and the spaces are thereby also enlarged.

In the study of the lungs of these prenatal and postnatal ani-
mals other points are seen. The behavior of the lungs when the
thorax is opened is different in animals which have breathed,
and those which have not breathed. In the former the lung
retracts quickly into the dorsal parts of the thoracic cavity, while
in the latter little change takes place. The best method for
comparing the positions of the lungs within the chest cavity, in
these two conditions, is to inject the blood vessels with formalin
without opening the thorax. It is true that the formalin causes
a swelling of the tissues of the organs, but the relations are well-
preserved. When one opens the thorax under these conditions,
the fetal lung is found not to extend so far forward and not to
cover as much of the heart as the postnatal lung, but in gen-
eral the relations are very similar. The differences are the result
of the increase in size of the lungs due to inspiration, while the
size of the other organs remains the same.

PRENATAL AND NEONATAL LUNG oA |

The fetal lung is opaque, and dense-looking, being often com-
pared in consistency with the thymus. Its color is dull gray-
ish-red. The breathing lung has a translucent pinkish color due
to the greatly increased flow of blood, and to the presence of
air. It floats upon water in contrast to the non-breathing lung,
and this is utilized as a medico-legal test. When the breathing
lung is moved between thumb and finger, the characteristic crepi-
tations are elicited.

In sections the postnatal lung shows congested blood-vessels,
in striking contrast with the fetal lung, which shows but little
blood. It would seem from examples of lung, which we obtained
from still-born animals, that the increased flow of blood begins im-
mediately, when the thoracic cavity is enlarged under the action of
the muscles. These examples were from kittens, which were born
at full-term. Two of them were found with the fetal membranes
intact, and had never breathed air, while one of the litter had
breathed freely. In the lungs of the non-breathing animals, the
spaces were distended slightly more than in the normal fetal lung
and the blood-vessels were much congested. The animals had
evidently tried to begin to breathe air, but had only succeeded
in inspiring the liquor amnii. It would appear that, as soon as
the negative pressure commences, more of the blood stream is
deflected into the pulmonary arteries, less going through the
ductus arteriosus, and this occurs irrespective of what is being
drawn into the lung spaces.

APPEARANCE OF LUNG OF PREMATURELY BORN ANIMAL

An important factor in the expansion of the lung is the stage
of development of the fetus at birth, that is, whether it is pre-
maturely born or not. In a kitten born one week before the
remainder of the litter, we had an opportunity of seeing this
condition. This kitten had the appearance of a healthy normal
animal, although somewhat undersized. It crawled about and
cried vigorously when disturbed but lived only about twelve hours.
Sections from the lung of this animal showed evidence of the
struggle to start and maintain respiration. The general appear-
ance is given in the outline drawing, figure 7. The bronchi are

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 2

ADDISON AND HAROLD W. HOW

WILLIAM H. F.

212

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PRENATAL AND NEONATAL LUNG 213
all much distended, but only a few of the respiratory spaces
have been affected. Those which have been distended, however,
are often over-expanded, as may be seen by comparison with
figure 8, from a kitten of the same litter, born at normal time,
and which had breathed twelve hours. The remainder of the
lung is denser than the ordinary fetal lung, and has apparently
been compressed by the over-distention of a few of the spaces.
Evidently respiration was begun before the lungs were sufficiently
developed to assume their normal function. <A point of interest
seen in examining the sections is that the alveoli which are situ-
ated directly on the bronchioles are distinctly distended. No
doubt it was these alveoli which played a large part in aerating
the blood of the animal during the period that it survived, and
indeed it must be these which function first at the beginning of
normal respiration.

We here wish to express our thanks to Professor Piersol for
helpful advice and criticism and to Mr. E. F. Faber for assist-
ance with certain of the drawings.

SUMMARY

1. During prenatal life the future respiratory passages are filled
with a liquid. With the first inspirations in air, the thoracic
cavity is enlarged by the action of the respiratory muscles, the
lung is thereby enlarged, and the liquid in the trachea and
bronchi is drawn down into the lung, and is distributed along
the walls of the alveolar and other spaces.

2. In microscopic sections of lung taken from fetal animals,
there is seen a finely granular substance (a precipitate from the
fluid present) widely scattered through the spaces; and large
mononuclear cells, probably phagocytic in function are found
uniformly distributed in small numbers.

3. In sections of lung of neonatal animals the finely granular
substance is still found, usually close to the walls of the air-
spaces and fewer mononuclear cells are seen.

4. Before breathing, the lining epithelial cells of the alveoli are
irregularly cuboidal with rounded nuclei; but after breathing,
with the increased area of the walls of the alveoli, the nuclei are

214 WILLIAM H. F. ADDISON AND HAROLD W. HOW
e

spaced farther apart, the cytoplasm is drawn out and the cells are
very thin and flat. After breathing has begun, the mesenchyme
appears denser, with its nuclei more compacted, and the blood-
vessels are distended and more conspicuous.

5. Measurements on cross-ruled millimeter paper show that the
lung tissue in sections of fetal lung of dog constitute 70 to 80
per cent of the entire area; in sections of lung of dog which has
breathed one hour, 40 to 60 per cent; and in sections of lung
of dog which has breathed two days, 20 to 30 per cent.

LITERATURE CITED

BALLANTYNE, J. W. 1902 Antenatal pathology and hygiene. William Wood,
New York.

DE LA Crorx, J. 1883 Die Entwickelung des Lungenepithels beim menschlichen
Foetus und der einfluss der Atmung auf dasselbe. Archiv fiir mikros.
Anatomie, Bd. 22.

Dourn 1891 Uber den Mechanismus der Respiration des Neugeborenen. Ab-
stract in Archiv fiir Kinderheilkunde, Bd. 13.

Frerront, E. 1899 Osservazioni e ricerche sui movimenti ritmici fetali intra-
uterini. Annali di Ostetricia e Ginecologia, T. 21.

Fuint, J. M. 1906 The development of the lung. Amer. Jour. Anat., vol. 6.

Gry, A. 1880 Die Aetiologie der sogenannten ‘puerperalen Infection’ des
Fétus und des neugeborenen. Archiv fiir Gynakologie. Bd. 15.

Howe tz, W. H. 1911. Text-book of physiology; 4th Edition. W. B. Saunders,
Philadelphia. —

K6O.LuikER, A. 1902 Handbuch der Gewebelehre des Menschen. Bd. 3, p. 310.
Edited by V. von Ebner.

Lecitarp 1815 Abstract in Intelligenzblatt of Deutsches Archiv fiir die Phy-
siologie, Bd. 1, p. 154, under heading ‘Untersuchungen, welche zu
beweisen scheinen, dass der Fétus das Schafwasser athmet.’

Merke., F. 1902 ‘Atmungsorgane,’ in Bardeleben’s Handbuch der Anatomie
des Menschen.

Miuuter, W. 8. 1893 The structure of the lung. Jour. Morph., vol. 8.

Preyer, W. 1885 Specielle physiologie des embryo. Leipzig.

THE DEVELOPMENT OF THE CEREBRAL VENTRICLES
IN SEELE, PIG

CHESTER H. HEUSER

Harvard Medical School, Boston, Massachusetts

TWENTY-SIX FIGURES

In this study of the cerebral ventricles of pig embryos three
methods have been employed—wax reconstruction, dissection,
and the making of minute casts. Wax reconstructions of the
ventricles have the advantage of large size. The volume of the
various cavities of the brain can best be estimated by immersing
portions of such reconstructions in water, and observing the dis-
placement. From these observations the actual size of the ven-
tricles can be readily calculated. But dissections and casts are
more accurate for showing details, as, for example, the neuromeral
grooves; and most of the drawings have been made from such
preparations.

Brains of pig embryos measuring from 12 to 45 mm. were dis-
sected under the binocular microscope, with finely ground instru-
ments. Some of the dissections were designed to give a median
sagittal view of the right half of the entire brain. The embryo,
fixed preferably in Zenker’s fluid, was held in a mass of cotton
wet with alcohol, between the thumb and index finger of the left
hand; and under a small stream of alcohol, a longitudinal cut was
made with a sharp safety razor blade. Very perfect cuts can
thus be prepared, such as will pass between the hemispheres and
thence backwards, sectioning the infundibulum, corpus mamillare
and other divisions of the brain almost exactly in the median
plane. Mesenchyma which covers the hemisphere, olfactory
lobe, or other portions which it is desired to expose, can be easily
removed with delicate needles and forceps.

215

216 CHESTER H. HEUSER

Casts of the ventricles, in embryos measuring from 12 to 22
mm., were made after the following method. Longitudinal
sections of the entire embryo, made slightly to the right side of
the median plane, were prepared as above described. Then,
under the binocular microscope, obstructing portions of the brain
walls were dissected off, and the ventricles were cleaned of all
precipitation with a small brush and syringe. Next the embryo
was taken from the alcohol and pinned down to a piece of cork
and with narrow pointed strips of filter paper all the alcohol was
removed from the cavities of the brain. In a large measure the
success of the cast depends upon the completeness of the removal
of the alcohol. The cast was made by filling the cavities with
black wax with the aid of a fine electrically heated needle. A
small piece of wax was carried with a cold needle to the proper
place and then melted with the electric needle. This process
was repeated until the ventricles had been filled. Then if desired,
white wax was melted over the upper surface of the dissection
for holding the parts more closely together. Moreover, the
white background thus provided contrasts sharply with the black
cast. Lastly the preparation was turned upside down and the
brain wall was carefully dissected away from the wax. For
making a permanent preparation of the cast, it is best to mount it
in a dry cell on a microscopic slide. Casts thus made show a
little more than one-half the ventricles. They can be studied
with the binocular microscope, and drawings can be made quite
as satisfactorily as from larger models. After embryos exceed
20 mm. in length the interventricular foramen is so small that it
is difficult to get the lateral ventricles completely filled, and after
24 mm. the lateral recesses can hardly be cast.

For the electric needle a piece of no. 18 copper wire is coated, except
for about 15 mm. at one end, with a thin layer of some insulating paste
which will harden and be resistant to heat. ‘The commercial ‘caemen-
tium’ is very satisfactory. The bare end is ground down to a fine point
which is the working part of the instrument. Behind this, covering a
length of about 20 mm., a piece of no. 32 German silver wire, about 20
em. long, is wrapped around the insulated copper wire, the coils being
well insulated from each other and covered with caementium. Attached
to the ends of the German silver wire are small copper wires which run

CEREBRAL VENTRICLES IN THE PIG PAW

toalamp board. The lamp board, with the sockets connected in paral-
lel, serves as a rheostat, and any desired temperature can be obtained
by turning on the proper lights. One 50 c.p. globe allows about the
desired amount of current to flow. A larger electrically heated smoother
for wax models and an electrically heated knife for cutting wax plates,
which were used in making the models, will be described at a future date.

Those who study the nervous system from the standpoint of
comparative physiology regard the ‘‘functional system of neu-
rones” as the real unit of the nervous system. Thus Herrick
(08),+in discussing the morphological subdivisions of the brain,
refers to the influence of metamerism as primitive, dominating
the subdivision of the nervous system of lower vertebrates. He
further states that transverse divisions, such as the diencephalon
and mesencephalon, are not ‘natural regions’ because the primary
metamerism has ceased to be an important factor. But the
anatomical subdivisions exist no less than the physiological, and
although their significance may not be profound, they form a
convenient basis for description. In the following account the
primary divisions of the brain will be considered in this order—
fore-brain, mid-brain, hind-brain. Reference will be made es-
pecially to the casts and models of the ventricles, but the wall
of the brain must also be studied as the changing structure which
modifies the cavities within.

FORE-BRAIN

A model of the cerebral ventricles of a 5.1-mm. embryo, the
youngest one studied, shows that the three divisions of the brain
are distinctly marked off from each other, and that the fore-brain
is already subdivided into telencephalon and diencephalon. John-
ston (’09) states that the boundary between the telencephalon and
the diencephalon is determined in mammals, as well as in lower
vertebrates, by the velum transversum above and the primitive
optic groove or postoptic recess below. This subdivision is dis-
tinct in the young pig studied as seen in figure 4. In the model
very slight lateral swellings from the telencephalon indicate the
cavities of the rudimentary’ cerebral hemispheres. The dien-
cephalon shows a division into two parts or neuromeres, the first

218 CHESTER H. HEUSER

or parencephalon, and the second or synencephalon of Kupffer
(03). The relatively large cavities of the optic vesicles extend
out from the antero-ventral part of the diencephalon. Almost
one-fourth of the length of each vesicle, representing the optic
stalk, extends nearly straight out laterally, and the remainder
bends sharply toward the mid-brain.

In an embryo of 12 mm. the lateral ventricles have expanded
considerably and they now slope outward forward and slightly
upward from the interventricular foramen. The foramen is
almost circular in outline and has an area of about 0.428 sq. mm.
In the cast (fig. 5) the rounded mass of wax filling the lateral
ventricle shows a slight concavity below, which has been pro-
duced by the developing corpus striatum. This body arises as a
thickening of the ventro-lateral wali of the hemisphere, as seen
in the dissection, figure 6. This figure, it may be noted, bears
a very striking resemblance to the reconstruction of the brain of
Anguis fragilis (40-50 somites), published by Kupffer (’03, p.
220, fig. 224). In both, the parencephalon and synencephalon
are sharply marked off from each other. Two swellings, which
correspond with these subdivisions, are seen in the cast (fig. 5).
The parencephalon is larger than the synencephalon, and its cavity
produces a more extensive swelling, which however, is quite low.

The mamillary recess and the infundibulum below it, neither
of which had appeared in the 5.1-mm. embryo, are now very
distinct. The velum transversum, a portion of which is shown
in figure 7, is well developed, producing a deep inward bend of
the anterior wall of the fore-brain above the hemisphere.

The brain of a 17-mm. pig embryo, as seen in median section
(fig. 8), shows a prominent corpus striatum, above which is the
interventricular foramen. The foramen is now crescentric, since it
has been invaded from below by the corpus striatum, but not-
withstanding the growth of the entire embryo, its area has become
actually reduced. It measures about 0.315 sq. mm. As seen in
the dissection, the lower portion of the corpus striatum is bounded
behind by a deep groove which is continuous with the optic recess.
This groove appears as a ridge upon the cast (fig. 9), and in front
of it the position of the corpus striatum is indicated by an excava-

CEREBRAL VENTRICLES IN THE PIG 219

tion. Above this hollow is seen the inferior horn of the lateral
ventricle. The outline of the ventricle is no longer round, as in
the 12-mm. embryo. It is prolonged anteriorly or downward to
form the first indication of the olfactory lobe, and posteriorly or
above, it is somewhat flattened. This upper portion terminates
in the inferior or descending horn. Fraser (’94) has referred to
this horn in an abnormal human adult brain as in ‘‘reality ascend-
ing’ and Thompson (’08) similarly states that ‘“‘from the develop-
mental point of view, this descending horn is in reality an ascend-
ing horn.”’ In pig embryos, however, as seen in figures 5 and9,
its primary direction is apparently ventral to the cerebral axis
so that the inferior horn may be said to descend, even in early
stages. The cavity of the lateral ventricle is seen laid open in
figure 10. It differs from that of the 12-mm. pig embryo (fig. 6)
since it has been invaded by a prominent chorioid fold. This fold
is a lateral extension of the velum transversum, and it consists of
vascular mesenchyma covered by the thin wall of the brain. At
this stage its ventricular surface is perfectly smooth, but in a 22-
‘mm. embryo the chorioid fold has developed a vascular fringe
which appears as a plaited frill. This is shown in figure 13.

The walls of the diencephalon in the 17-mm. embryo have
thickened considerably so that the external depression between
the parencephalon and the synencephalon has become almost
imperceptible. Their cavities however, can be distinctly seen
when the dissection of the brain is studied (figs. 8 and 9). The
mamillary recess now projects out some distance from the third
ventricle. Midway between this recess and the infundibulum
the floor of the brain has thickened to form the tuber cinereum.
The optic thalami, represented by the thickenings of the lateral
walls of the diencephalon, have developed to such an extent that
they have already partially fused with the corpus striatum. The
place of fusion appears as an interruption of the groove between
the interventricular foramen and the optic recess.

The median section of a brain of a 22-mm. embryo (fig. 11),
as compared with that of the 17-mm. specimen, shows several
new features. Along the cut edge dorsally, the pineal body
has appeared as a slight elevation. Within the third ventricle,

220 CHESTER H. HEUSER

the thalamus has enlarged, and the corpus striatum appears as
if pushed forward before it. Between the corpus striatum and
the upper part of the thalamus, the interventricular foramen is
seen as a slit, which arches up over the corpus striatum, being
widest immediately under the velum transversum. The area of
the aperture has become further reduced. Between the corpus
striatum and hypothalamus, the optic recess appears as in the
17-mm. specimen. Somewhat further back, and parallel with it,
is the recessus infundibuli, and between the two grooves made by
these recesses, is the pars optica hypothalami. This is convex
toward the ventricle. Between the hypothalamus below and the
thalamus above is the sulcus hypothalamicus (B.N.A.) or sulcus
limitans (His ’93)—a groove which continues from the mid-brain
downward and forward becoming somewhat indistinct above the
pars optica hypothalami. Reichert (’61) described this groove in
the brain of a pig embryo (also in cat and human embryos) as
extending from the foramen of Monro to the entrance of the aque-
duct of Sylvius, and he named it the sulcus of Monro. In fig.
12 an extension may be traced from the sulcus to the lower end
of the interventricular foramen; another extension proceeds to-
ward the recessus postopticus. But the main continuation is
probably that which ends in the optic recess. This accords with
the opinion of His (98, p. 177), and also of Johnston (09, p. 517).
The sulcus limitans is indicated in the embryo of 17 mm. and in
younger specimens, but it is more distinct at 22 mm.

The median section of the 22-mm. embryo shows the uncut
median surface of the right hemisphere, ending below in a rounded
olfactory lobe. Extending from the lower border of this lobe
toward the notch below the velum transversum, is a groove,
which, with the lamina terminalis, bounds a triangular portion of
the hemisphere. Smith (’03) named this triangular thickening
the ‘paraterminal body’. Herrick (10) proposes to divide this
body into a ventral component, the ‘corpus precommissurale,’
and a dorsal component, the ‘primordium hippocampi,’ but these
subdivisions do not appear externally in the 22-mm. embryo.

In the dissection of a 45-mm. embryo (fig. 14) most of the para-
terminal body together with the adjacent medial wall of the right

CEREBRAL VENTRICLES IN THE PIG PAN

hemisphere have been cut off, thus displaying the structures within
the ventricle. The corpus striatum is seen to be divided by a
vertical groove into two portions. The groove is deep below but
fades out above. Thompson (’09) has described a similar groove
in a cat embryo 20 mm. in length, and he refers to the larger
lateral and the smaller median subdivisions which are set apart
by the groove as ‘roots’ of the corpus striatum. His (’89) de-
scribed the corpus striatum as being composed of three limbs. In
the 45-mm. pig there are also three limbs, but only two are seen
in figure 14. The small median limb, which is not shown, fuses
with the lamina terminalis just below the interventricular fora-
men. The groove is shown at x in figure 18, which is a frontal sec-
tion through the head of a 45-mm. pig. The plane of this section
is indicated in figure 14. The groove is best seen in the right side
of the section, where the median subdivision may be observed
to pass down toward the paraterminal body. In a section fur-
ther back (fig. 19) the groove is quite shallow. Above the corpus
striatum (figs. 14 and 19) the chorioid plexus is seen projecting
into the lateral ventricle. The fold, surmounted by a frill,
which was seen in the 22-mm. embryo, has given place to a very
thin layer with many reduplications and subdivisions. Villi
are nowhere present, but in thin sections the smaller processes
extending out from the main folds may simulate them. Under
the binocular microscope it is clearly seen that this plexus consists
only of folds with secondary subdivisions. The plexus is attached
along a fissure measuring 2.1 mm. in length, which extends back-
ward from the interventricular foramen.

The caudal portion of the plexus—about one-fourth of the
entire length—is very much attenuated. It forms a short free
projection extending 0.18 mm. beyond the end of the fissure, and
the free portion shows no secondary folds (fig. 21). The anterior
portion of the plexus forms a larger free projection which extends
0.72 mm. past the front end of the fissure.

In the 22. mm. embryo (fig. 13) a slight invagination of the
medial wall of the hemisphere, above the chorioid plexus and
parallel with it, extends from the interventricular foramen back-
ward into the inferior horn. This represents the hippocampus,

222 CHESTER H. HEUSER

which is better developed in the 45-mm. embryo. It is seen in
figures 19, 20, and 21 (marked +).

Considered as a whole, the lateral ventricle has expanded
greatly and may readily be divided into three parts—the anterior
horn, which is in front of the corpus striatum; the body, which is
above it; and the inferior horn, descending behind it. A cast of
the left lateral ventricle is shown in side view in figure 16, and in
ventral view in figure 17. The latter shows the large excavation
made by the corpus striatum. ‘Toward the olfactory lobe this
concavity is bisected by a ridge, which lies between the large roots
of the corpus striatum, as described in connection with figure
14. Below the concavity, in figure 17, there is a ridge which is
fissured, posterior to the interventricular foramen, to receive the
chorioid plexus. This ridge separates the hollow for the corpus
striatum above, from that for the hippocampus below.

The lateral ventricle communicates with the third ventricle
by an interventricular foramen which is smaller than in the pre-
ceding stages. The third ventricle has become reduced to a slit-
like space, owing to the great thickening of the walls of the dien-
cephalon. <A portion of it has become practically obliterated.
This is where the thalami have grown against each other (figure
21). They have not yet fused, however, to form the massa inter-
media; that is the ependymal layer over each thalamus is still
uninterrupted. Along the dorsal margin of the cleft, which rep-
resents the ventricle, there is a thin lateral expansion on either
side (fig. 15). The expansion begins at the interventricular fora-
men, where it is broadest, and it diminishes backward, ending
a short distance in front of the pineal recess. Thus the expansion
is wedge-shaped when seen from above. The brain-wall which
overlies this expansion is the tela chorioidea, which has a corru-
gated surface in relation with the vascular mesenchyma. Below
and behind the thalamus is a deep groove—the sulcus limitans—
which, as already described, sends prolongations to the interven-
tricular foramen, recesses postopticus and recessus opticus. The
sulcus is seen in section in figure 20, with the thalamus above and
the pars optica hypothalami below.

CEREBRAL VENTRICLES IN THE PIG 225

The oldest embryo studied measured 260 mm. Its brain was
dissected out, embedded in celloidin, and cut into sections 0.2 mm.
thick. From these sections a wax model of the ventricles was
constructed, as shown in figures 25 and 26. The three subdivi-
sions of the lateral ventricle have become highly developed.
The anterior horn, which in the 45-mm. embryo ended in a short
and slightly pedunculated olfactory bulb, now extends through
the olfactory stalk and terminates in the expanded ventricle of
the olfactory bulb. The body of the lateral ventricle is corru-
gated above, where it is in relation with bundles of fibers in the
corpus callosum. The inferior horn not only descends, but ex-
tends forward as a slender prolongation, which ends blindly in the
temporal lobe of the brain. As seen from below, the cast of the
lateral ventricle shows the concavities for the corpus striatum and
the hippocampus respectively, separated by a ridge into which
the chorioid plexus is invaginated. The ridges bounding the cor-
pus striatum correspond with those seen in the 45-mm. embryo.

In the 260-mm. specimen, the interventricular foramen has
expanded considerably, and its area was found to be 6.92 sq.
mm. Extending backward from the foramen, between the region
occupied by the thalamus below and the tela chorioidea above,
there is a tubular expansion of the third ventricle, somewhat
flattened dorso-ventrally. This corresponds with the much
smaller expansion, T-shaped in section, which was described in
the 45-mm. embryo. In the 260-mm. embryo there is a long
caudal extension of the dorsal part of the third ventricle which
curves slightly upward and forms the suprapineal recess. It
measures about 5.6 mm. in length and extends backward with
quite a uniform diameter of 1.4mm. This conspicuous feature
is not represented in the 45-mm. specimen. The cast of the cay-
ity (fig. 25) is deeply corrugated on all sides by longitudinal folds.
These accomodate branches of the medial cerebral vein. A large
branch of the vein on either side produces a deep groove along
the dorsal expansion of the third ventricle. Between the su-
prapineal recess above and the pineal recess below there is a well
marked lateral compression of the ventricle, produced by the hab-

224 CHESTER H. HEUSER

enular ganglion. The conspicuous ridge at the lower margin.
of this habenular concavity continues caudally into the pineal
recess. Between the pineal recess and the groove for the pos-
terior commissure there are two slight projections of the third
ventricle (infrapineal recesses). The very extensive fusion of
the thalami, forming the massa intermedia, has produced, a hole
in the model of the third ventricle. It is oval in outline, measur-
ing about 4.6 mm. by 3 mm.

MID-BRAIN

It is well known that in young embryos the cavity of the mid-
brain is very large, forming the middle cerebral vesicle. In the
adult it is generally described as ‘“‘reduced to a narrow slit—the
aqueduct of Sylvius.”” Cunningham states that the mid-brain
“is tunnelled by a narrow passage, the aqueduct of Sylvius,
which extends between the fourth and third ventricles,” and Bens-
ley (’10) writes: ‘‘The mesencephalon is noteworthy in a mam-
mal as lacking a ventricle. Its cavity is a narrow canal, the
aqueduct of Sylvius . . . .” But Retzius (’00) described
a spindle-shaped expansion in the middle portion of the cavity
of the adult human mid-brain, and named it ‘ventriculus mesen-
cephali.’ It will be shown that a distinct cavity exists in the
mid-brain of the adult pig, comparable with that found by Ret-
zius in the human brain.

In the 5.1-mm. pig (fig. 4) a slight constriction separates the
caudal part of the diencephalon from the cavity of the mid-brain.
Another slight constriction marks the position of the isthmus
between the mid-brain and the hind-brain. The angle formed
by a line passing through the axis of the diencephalon with an-
other through the isthmus and cavity of the medulla, is very acute
(30°) in this stage. In the adult it is very wide.

The cavity of the mid-brain continues to expand and becomes
sharply marked off from that of the hind-brain at the isthmus,
as shown in the 12-mm. embryo (fig. 5). Between the mid-brain
and fore-brain, however, the cavity shows only a slight constric-
tion, which is limited to the lateral surface. As seen in the cast

CEREBRAL VENTRICLES IN THE PIG 225

neither the mid-dorsal nor the mid-ventral line is indented at
this point.

Figure 9, of a 17-mm. embryo, shows that the dorsal surface
of the mid-brain has become flatter than before and its caudal
end projects further toward the hind-brain. This projection, on
either side, marks the position of the future inferior colliculus.
The ventricle of the mid-brain is somewhat quadrilateral in cross
section, and in the cast it shows a prominent longitudinal ridge
corresponding with the line of maximum width. The groove in
the wall of the brain corresponding with the ridge may be referred
to as the lateral suleus (sulcus lateralis internus). It must not
be confounded with the sulcus limitans which, in the cast, is a
much less conspicuous ridge, more ventrally placed. The sulcus
limitans is more evident in the dissection, figure 8. It separates
a small thick-walled ventral zone from an extensive thin-walled
dorsal zone. ‘The ventral zones, on either side of the mid-ventral
sulcus, form low rounded eminences projecting into the cavity
of the ventricle which constitutes the tegmental fold of His. The
lateral sulcus passes longitudinally through the dorsal zones,
terminating on either side, in the projection which forms the in-
ferior colliculus.

In a slightly older embryo (22 mm.) the dorsal wall of the
mid-brain has become relatively thinner, and its ventricle has
expanded greatly. At this stage it forms a very conspicuous por-
tion of the whole ventricular system. Its volume is estimated
at 4.1 cu. mm. Separate recesses for the inferior colliculi can
now be recognized. In earlier stages the posterior overhanging
portion of the mid-brain ended abruptly in a straight transverse
line. In the 45-mm. embryo this border shows a deep median
cleft, and each arm of the bifurcation thus formed represents the
cavity of an inferior colliculus. In the 22-mm. specimen the cavi-
ties projecting backward from the dorso-lateral corners of the mid-
brain ventricle have just begun to develop.

In the 45-mm. embryo the third ventricle is connected with
the cavity of the mid-brain by a more elongated constricted por-
tion than in previous stages. At the anterior end of the mid-
brain ventricle there is a median dorsal extension 1.2 mm. caudad

226 CHESTER H. HEUSER

to the pineal recess. This outgrowth (figs. 14 and 15) which lies
just behind the posterior commissure is the incisura postcommis-
suralis. It is not present in the 20-mm. pig but is very distinct
in the 45-mm. embryo. The ependymal and mantle layers but
not the ectoglial layer of the brain-wall make a distinct outward
bend above it. Caudad to the incisure, the dorsal surface of the
mid-brain cavity slopes gradually upward to a height of 0.6 mm.
and then extends nearly straight backward over the body of the
eavity.. The inferior collicular recesses are very much more dis-
tinct than in younger pigs.

The length of the mid-brain, including its collicular recesses is
5.0 mm. and- the width, in the widest part is 2.6 mm. ‘The vol-
ume of the cavity is very much greater than in preceding stages
and is now about 6.9 cu. mm.

A cast of the cavities of the mid-brain in an embryo measur-
ing 110 mm. is of an irregular prismatic form as shown in figure
24. It presents a median dorsal ridge which extends from the
incisura postcommissuralis posteriorly, ending in a median depres-

‘sion between the eminences representing respectively the cavi-
ties of the right and left inferior colliculi. The outer margin of
the cavity of each inferior colliculus is continued forward as a
prominent ridge, corresponding with the sulcus lateralis. This
ridge flattens out rather sharply and completely a short distance
behind the posterior commissure. At the base of the cavity of
the inferior colliculus, a prominent ridge ascends from the isth-
mus, but it can be followed only a short distance behind the
posterior commissure into the territory of the mid-brain, where
it terminates. This ridge represents the sulcus limitans of the
isthmus. Ventrally there is another ridge which may be regarded
as the sulcus limitans of the mid-brain, although it is not con-
tinuous with the structure just described as the sulcus limitans
of the isthmus. It arises beneath the latter in or near the median
ventral sulcus. It then passes laterally, diverging from its fel-
low on the opposite side. The point of widest separation is
soon reached, after which the two sulci gradually converge, com-
ing together in the mid-ventral line beneath the incisura post-
commissuralis. The surfaces between the ridges on the cast are

CEREBRAL VENTRICLES IN THE PIG 220

all concave; and in the central part of the mid-brain there are
five surfaces—two dorso-lateral, bounded by the median dorsal
ridge and the lateral ridges; two ventro-lateral, extending down-
ward to the sulci limitantes; and a ventral surface between these
sulci.

The form of the mid-brain ventricle in the embryo measuring
110 mm. has been described at length, since subsequently it
undergoes only slight modifications. This was determined by
modelling the ventricles in the 260-mm. pig (figs. 25 and 26)
and by making dissections of the adult. The ventricle of the
mid-brain continues to increase in size but it does not keep pace
with the growth of the adjoining cavities. The grooves remain
as described, and the ventral surfaces, between the sulci limi-
tantes, are always subdivided posteriorly by a forward extension
of the median ventral sulcus.

HIND-BRAIN

In the 5.1-mm. embryo the cavity of the hind-brain or third
cerebral vesicle is elongated and quite straight. Behind the ven-
tricle of the mid-brain (fig. 4) a well marked constriction indi-
cates the future isthmus. Caudad to this, the cavity, or fourth
ventricle, widens somewhat, and then slopes gradually through
the medulla to the spinal cord. The low ridge which follows the
line of greatest width corresponds at this stage with the line
of attachment of the thin roof-plate, and not with the sulcus
limitans. Along the ventral surface of the cast there is a sharp
median ridge which represents the sulcus medianus of the rhom-
boid fossa.

Seven neuromeral grooves can be identified. The first pro-
duces a low but broad ridge on the cast anterior to the widest
part of the rhombencephalon. Dorsally it flattens out before
reaching the line of maximum width and ventrally it does not
extend quite to the median sulcus. The second neuromeral
groove is situated opposite the widest portion of the fourth ven-
tricle. The next four are about equally spaced. They are very
prominent in the ventral zone and some of them appear to reach
upward a short distance into the dorsal zone. The last one is

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 2

228 CHESTER H. HEUSER

represented on the cast by a prominent mound, but the eleva-
tion does not extend as far dorsally or ventrally as do those
immediately preceding it. Caudally it blends with the sulcus
limitans. Bradley (04 and ’05) described seven neuromeres in
a nineteen-day embryo.!

The pontine flexure soon becomes definitely established as
shown in figure 5, which is a cast from a 12-mm. embryo. In
this embryo the median ventral edge of the cast is very sharp
around the flexure and backward toward the spinal cord. At
the pontine flexure there are six neuromeral grooves. The rela-
tion of the grooves to the median sulcus is clearly shown in the
dissection, figure 7, The former seventh groove has been taken
up by the sulcus limitans so that it is no longer recognizable.
Above the grooves the ventricle has become quite wide. In
dorsal view the body of the ventricle slopes out laterally quite
rapidly behind the isthmus, attaining its maximum width in the
region of the future lateral recesses, which become distinct in
embryos of about 15 mm.

In the 17-mm. embryo the isthmus leading from the mid-
brain to the body of the fourth ventricle is diamond-shaped in
cross section; the ventral zones are here more extensive than the
dorsal, which is the reverse of the condition in the mid-brain.
The ventral median sulcus is narrow and deep, so that in the
cast there is a sharp edge on the ventral surface of the fourth
yentricle extending around the pontine flexure, and onward cau-
dally to the spinal cord. There are now five neuromeres in the
hind-brain. The body of the fourth ventricle has expanded a
great deal since the last stage described, and the lateral recesses
are now well indicated. Slight concavities on either side of the
body of the ventricle between the isthmus and the lateral re-
cesses, in the dorsal zones, are caused by the thickening of the
brain in this region to form the lateral portions of the cerebellum.
Behind the lateral recesses the thin roof of the hind-brain has
become deeply invaginated to form the chorioid plexus. About
100 villi are developed on the fold.

1 According to Keibel’s Normentafel, pig embryos of nineteen days measure
from 4.5 to 8 mm.

CEREBRAL VENTRICLES IN THE PIG — 229

In a 22-mm. embryo (figs. 11 and 12) the fundamental arrange-
ment of parts has not been altered. The cerebellar thickenings
have enlarged considerably, producing correspondingly greater
depressions in the dorsal-zones along the body of the fourth
ventricle. The sulcus limitans, for the same reason, has been
made very much more evident. The lateral recesses extend fur-
ther to each side and are comparatively much narrower than in
the younger stage considered. The chorioidallaminaextends out
to the ends of the lateral recesses, and the number of villi spring-
ing from it has increased to about 150. Caudad to the chorioid
plexus the dorsal part of the cavity is slightly distended or puffed
up dorsally, thus forming the ‘caudal protrusion’ (Blake ’00).
In advanced embryos of dogs, cats, pigs, sheep and also the
chick, according to Blake, there is a marked caudal protrusion.
He wrote, ‘‘This protrusion is completely closed and resembles
the finger of a glove.” It can hardly be described in the pig
as ‘finger-like’ since, as shown in figure 11, it forms a rounded
dome.

Taken as a whole, the fourth ventricle in the 45-mm. pig
(figs. 14 and 15) has the same parts as seen in the 22-mm. embryo,
The cavity of the isthmus has increased in dorso-ventral diame-
ter from 0.75 to 0.85 mm. As seen by comparing figures 11
and 14, it has become relatively low, but laterally it has become
further expanded. Its thin lateral edge, as seen in the cast (fig.
15), extends backward and becomes continuous with the body
of the ventricle a short distance in front of the lateral recess.
Where this thin edge fuses with the body there are three small
wrinkles—the lateral remnants of the neuromeral grooves. The
two halves of the cerebellum have become very much thicker
and meet at an angle of about 130°. The medial part of the
cerebellum is also thicker than before, so that all together the
broad median fissure of younger stages has been appreciably
reduced. The lateral recess, while absolutely larger than before,
appears much slimmer. The floor and roof of the recess have
almost come together, the cavity being largely filled with the
chorioid plexus. In figure 23, in the left recess, is seen a long
stretch of the chorioid fold. Sections a short distance caudad

230 CHESTER H. HEUSER

to this one show the fold to be continuous from one recess to
the other. The marked villous character of the chorioid plexus
is illustrated well in this section (fig. 23). It shows also the
rhomboid fold projecting into the cavity of the hind-brain below
the chorioidal lamina. The rhomboid fold appears in the model
(fig. 15) as a sharp rim, extending along the upper part of the
body of the ventricle behind the lateral recess. It extends to
the caudal protrusion of the fourth ventricle, which has expanded
to a very great size; its volume is more than one-half that of the
remainder of the ventricle. This protrusion, from above and
from behind, appears almost spherical. The posterior boundary
slopes downward making almost a right angle with the narrow
cavity of the caudal part of the medulla. When the body of
the fourth ventricle is viewed from below, the ventral median
sulcus and the two sulci limitantes are seen converging towards
the cervical flexure. The ventral median groove is nearly straight
and produces with the slim cavity of the cord an angle of about
PANS

In the 260-mm. embryo (figs. 25 and 26) the cavity of the
isthmus is very broad and flat. On the ventral surface a deep
ridge—the median ventral sulcus—is continuous with the similar
ridge of the mid-brain ventricle. On account of the extensive
growth of the cerebellum the dorsal surface of the body of the
ventricle has become deeply concave. Only a very slight caudal
protrusion is now present. Bradley (’05) described the first ap-
pearance of openings in the lateral recesses—foramina of Luschka
—in embryos of about 100 mm. Because of these openings and
on account of the growth of the chorioid plexus the cavities of
the lateral recesses, as seen in the model, have been reduced to
slim irregular bodies.

MEASUREMENTS OF THE BRAIN

In order to show the change in the volume of the cavities
during development, measurements of wax models were made
by the immersion method. The results of these measurements,
reduced to cubic millimeters, are given in table 1.

CEREBRAL VENTRICLES IN THE PIG Zor

TABLE 1

Volume of the cerebral ventricles in cubic millimeters

Bae | eee eb ae | iy

mm. | | | fe rf ’
5.1| 0.0948 | 0.022 0.206 | 0.3227
APE) sol 7 16 Lote Ostai2 0.357 3.38 | 5.453
20 Olli 2 143 eee 2.481 2.96 3.407 13.477
24 9.893 Pace | 3.768 5.181 SHO 18.644
45 | 30.49 4.19 = ales} dlls} 6.904 5.84 43 234
260 | 711.58 | 94.6 | 308.4 | 10.07 BD) a: inlicy

From table 1 it is seen that each part of the entire ventricular
system increases in size continually during development. The
increase is relatively greater at first, and as would be expected,
it becomes gradually less toward the adult. For a time—until
embryos measure between 12 and 20 mm.—the fourth ventricle
is the largest part of the ventricular system. After the lateral
ventricles have expanded appreciably the cavity of the fore-brain

is the largest part.
~The cross-sectional area of the interventricular foramen was
determined from wax models by measuring the area of the fora-
men cut through the narrowest portion.

The cross-sectional area of the interventricular foramen be-
comes progressively greater up to about the 12-mm. stage after
which it becomes both relatively and absolutely smaller for some
time. In later stages it again expands considerably; in the
adult it is very much larger than in younger stages.

TABLE 2

Area of the foramen interventriculare

EMBRYO FORAMEN INTERVENTRICULARE
Length in mm. Area in square millimeters
12 0.42
20 0.26
45 0.18
260 6.92

The mid-brain angle in early stages is very acute. For meas-
uring the angle in the different embryos during development it

2a2 CHESTER H. HEUSER

is difficult to establish lines whose loci will be absolutely com-
parable throughout the series. The apex of the angle has been
located in the middle point of the mid-brain ventricle; one line
has been drawn from it to the optic recess, and the other to the
middle point in the cavity of the central canal opposite the
cervical bend.

Fig. 1 Outline from figure 5, showing the position of the mid-brain angle

TABLE 3

Increase in the mid-brain angle

EMBRYO MID-BRAIN ANGLE EMBRYO | MID-BRAIN ANGLE
mm. degrees \ mm. degrees
Sell 30.0 | 45 84.0
12 48.5 | 260 146.0
17 59.5 | Adult 152.0
22 68.0

The angle increases from 30° in the 5.1-mm. embryo to 152° in
the adult. The curve of growth of this angle rises up relatively
rapidly at first and slowly in later stages to the adult.

CONCLUSIONS

The well-known series of models of the developing human
brain made by His and the papers which they illustrate make
it possible to compare the conditions which have been described
in the pig with those in man. Comparisons with the embryonic
brain of other mammals cannot be made, since it appears that
no one has attempted to extend the work of His here referred
to, or even to repeat it critically. The first impression derived

CEREBRAL VENTRICLES IN THE PIG Boo

from comparing His’s models of the interior of the human em-
bryonic brain with those of the pig is that they are strikingly
similar, especially in the earlier stages; but more careful study
shows very considerable differences. The 6.9-mm. human embryo
(His, ’89, Taf. 1, Fig. 2) is somewhat more advanced than the
pig embryo of 5.1 mm., as indicated by the greater development
of the hemisphere, and of the pontine flexure. In His’s model
the cavities of the parencephalon and synencephalon are not in-
dicated and the neuromeres of the hind-brain are not shown. It
may be questioned however, ‘whether these structures are
actually absent from the human brain at this stage. On the
other hand, the model of the human brain shows a prominent
tegmental fold passing from the mid-brain toward the fore-brain,
but no such fold occurred in the pig embryo. In a model of
a pig embryo of 5 mm. Johnston (’09) has shown the neuromeres
of the fore-brain and hind-brain but there is no tegmental fold.
This fold is the portion of the tegmentum which projects dor-
sally into the cavity of the mid-brain; the fold on either side
extends from the median ventral sulcus to the sulcus limitans.
Altogether Johnston’s model of the brain of the 5-mm. pig agrees
very closely with the first model described in the present paper,
and it is evident that we have more detailed and accurate knowl-
edge of the shape of the brain in the pig embryo than in the
human embryo of corresponding stage.

His’s model of the human embryo of 10.2 mm. corresponds
quite closely with the pig embryo of 12 mm. and his 13.6-mm.
specimen is comparable with the pig of 17 mm.; the human
embryo of the third month, which completes His’s series of
models, is the stage intermediate between those dissected in pigs
of 22 and 45 mm. In all of His’s models, except the oldest, the
tegmental fold is very prominent; and in the embryo of three
months it is perfectly distinct. The sulcus limitans is accord-
ingly well defined in the mid-brain, but it does not form a dis-
tinct continuous longitudinal groove from the spinal cord to the
optic recess in any of His’s models. In fact, His described the
sulcus as becoming leveled off in the territory of the isthmus

234 CHESTER H. HEUSER

by the growth of the walls—being interrupted at the junction
of the isthmus with the mid-brain.

In the casts of the ventricles of the pig, in early stages, there
is a broad line of maximum width corresponding with the sulcus
limitans.

A B i

Fig. 2 Sections through the hind-brain of pig embryos, (A, 3.9mm.; B, 10 mm.;
C, 23.6 mm.), showing the change in position of the sulcus limitans.

This groove which is lateral in the cord becomes a ventral
groove in the medulla as described by His, and at the junction
of the isthmus with the mid-brain it comes to anend. On account
of the expansion of the roof-plate in the hind-brain the sulcus
limitans soon falls below the line of maximum width. Thus in
the diagram (fig. 2, B) the sulci are ventrally placed, and the
width of the fourth ventricle between them is less than it is
slightly further dorsally. In an older embryo (fig. 2, C) the
sulci are close together and on the floor of the ventricle. In
the mid-brain there is early a line of greatest width, which becomes
shifted so as to form the outer border of the tegmental fold, as
shown in the diagram, figure 3.

SE
A B C

Fig. 3 Sections through the mid-brain of pig embryos, (A, 3.9 mm.; B, 23.6
mm.; C, 110 mm.); showing the change in position of the sulcus limitans.

CEREBRAL VENTRICLES IN THE PIG Paya)

This shifting in the mid-brain is brought about by the expansion
in the dorsal zone to form the cavities in the colliculi. The
line of maximum width begins behind the constriction between
the mid-brain and fore-brain in the superior collicular cavity
and terminates at the caudal end of the mid-brain in the inferior
eollicular recess. Anteriorly the hypothalamic sulcus is seen to
connect with the tegmental sulcus. This connection is most dis-
tinct in the 22.mm. embryo.

His first called attention to the dorsal and ventral zones of
the brain and cord. A demarcation of the sensory from the
motor portion of the central nervous system is obviously of fun-
damental importance and Johnston (’09) has described the sulcus
limitans as the most important landmark in the brain. Diagrams
have appeared which show an uninterrupted line, extending from
the optic recess backward through the brain and spinal cord.
However, there is no real picture which shows a sulcus contin-
uous throughout the brain. It is distinct in each division of
the brain, but at the cephalic end of the isthmus it is interrupted.

The hemisphere of course is very much larger in the human
embryos than in approximately corresponding stages of the pig.
The thalamus and habenular region are also further advanced
in the human stages. The tegmental folds never form conspicu-
ous projections into the ventricle of the mid-brain as they do
in early human embryos. The olfactory lobe becomes progres-
sively more conspicuous in the pig series—the reverse of the
condition in human embryos, of later stages especially. In the
latter, the olfactory lobe is covered over by the rapidly expand-
ing pallium. The olfactory lobe in the pig always extends some
distance in front of the end of the hemisphere. The roof of the
fourth ventricle is not present in any of the His models, hence there
is no indication of a caudal protrusion of the fourth ventricle.
This protrusion is first well indicated in pig embryos about 22
mm. long. It expands very rapidly until the cerebellum grows
downward into this region. There being no foramen of Majendie
in the pig, the posterior medullary velum stretches nearly straight
across the caudal part of the fourth ventricle so that the caudal
protrusion is just recognizable in the adult.

236 CHESTER H. HEUSER

The form of the ventricles in the adult pig has apparently
never been studied by means of casts or models. Figures 25
and 26, representing the cerebral ventricles in an embryo of
260 mm., are however very similar to those which would be
obtained from an adult and they may be compared with Dexler’s
figure of the cavity of the brain in the horse, and with any one
of several figures of the ventricles in the human brain. The
first of these were published by Welcker (’78) who filled the
ventricles with wax, injecting through the infundibulum, and
published figures and a brief description of the model thus ob-
tained. Testut (97) figured and described a plaster cast of the
ventricles. Barratt (02) constructed a wooden model from
measurements obtained from thick sections (12.56 mm.). From
the method employed accuracy of detail could scarcely be ex-
pected. By far the most delicate and satisfactory figures are
those of Retzius (’00) who made casts by the use of Wood’s
metal. Harvey (10) has recently used Wood’s metal for the
same purpose with similar results.

In the human brain immediately above the pineal body there
is a backward extension of the cavity of the third ventricle which
forms the supra-pineal recess. This term was introduced by
Reichert (61, Bd. 2, 8S. 69) who described the structure and
showed its relations in median sections of the brain. In his fig-
ures the recess measures nearly 5 mm. in length. In Welcker’s
plaster cast it appears to be somewhat larger and in the cast
by Retzius it measures 10 mm. But Testut and Harvey show
only very slight protrusions. The recess may be variable in the
human brain. In Dexler’s model of the ventricles in the horse
it is a very conspicuous object, nearly 25 mm. inlength. In
the adult pig it measures 5.5 mm. In proportion to the size
of the third ventricle it is very much longer than in man but
much shorter than in the horse. The significance of this recess
has not been determined.

A very conspicuous feature of the casts of the third ventricle
is the aperture made by the massa intermedia. In the several
models of the human brain this aperture varies considerably in
size, but in no ease is it as large as in the pig or the horse. In

CEREBRAL VENTRICLES IN THE PIG 231

the pig it appears to be somewhat smaller than in the horse,
but in both these animals the opening is very large. The area
of fusion in the 260-mm. pig measures about 8.4 sq. mm., and
in the adult pig about 61.6 sq. mm.

The lateral ventricles in pig, horse and man differ very greatly.
In man there is a posterior horn which is absent in the horse
and pig. But the latter both show an extension of the ventricle
into the olfactory lobe, whereas in man, the anterior horn ends
bluntly. In the horse the pedicle of the cavity of the olfactory
stalk is very long and markedly concave dorsally, it ends in an
elongated irregular ventricle. The pedicle in the pig is nearly
straight and much shorter. It terminates in a flattened expan-
sion which in the anterior end is compressed dorso-ventrally.

In connection with the lateral ventricle it may be noted that
its chorioid plexus in the pig is not provided with villi. Meek
(07) in his discussion of the general morphology of the plexus
writes: ‘‘Villi are scarce in the chicken, duck and pigeon, but
more abundant in the hog, while they reach a considerable devel-
opment in the horse, ox, and especially among porpoises, croco-
diles, and some of the selachians (Pettit ’02-’03).”” Findlay (99)
writes similarly of this plexus: ‘‘The surface of the chorioid
plexus is beset with a large number of highly vascular villous
projections. These are of all sizes, and the largest may branch
and subdivide many times before the ultimate villi are formed.”
The study of the brain of the pig has shown that the chorioid
plexus of the lateral ventricle first develops as an extension of
the velum transversum into the lateral ventricle. The free bor-
der rapidly becomes much longer than the attached part so that
it is early thrown into folds. These primary folds increase in
number and give rise to secondary folds, but as shown by means
of the binocular microscope, villi are not developed in this plexus.

In pig embryos of 35 to 40 mm. and in all older stages, there
is a median dorsal recess behind the posterior commissure. This
postcommissural recess is shown by Retzius who labels it the
‘incisura postcommissuralis.’ It is shown also in Harvey’s lateral
view. None of the other authors have an indication of it in
their figures. In the adult pig the recess is very prominent.

238 CHESTER H. HEUSER

Finally, it should be noted that the aqueduct of Sylvius remains
as a distinct ventricle of the mid-brain in the adult pig. One
of Retzius’s figures alone gives a good lateral view of the mid-
brain of man. This he described as ‘ventriculus mesencephali.’
The cavity of the mid-brain in the horse stands out also as a
distinct ventricle. In the pig the mid-brain has throughout
development and in the adult a well defined ventricle which
constantly increases in size as long as the brain grows.

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Minot, C. S. 1892 Human embryology. New York.

NorMaN, C., AND FraAsER, A. 1894 A case of porencephaly. Journ. Mental
Science, vol. 40, pp. 649—665.

RetcHert, C. B. 1859-1861 Der Bau des Menschlichen Gehirns. Leipzig.

Rerzius, G. 1900 Die Gestalt der Hirnventrikel des Menschen, nach Metallaus-
gussen dargestellt. Biol. Untersuch. von G. Retzius. Bd. 9. N:o.
3.u. 4. Jena.

Stsson, S. 1910 A text-book of veterinary anatomy. Philadelphia.

Surry, G. E. 1903 On the morphology of the cerebral commissures in the verte-
brata, with special reference to an aberrant commissure found in the
fore-brain of certain reptiles. Trans. Linn. Soc. London. July, pp.
455-500.

Testur, L. 1897 Traité d’anatomie humaine. Paris.

Tuompson, P. 1909 Description of a model of the brain of a foetal cat, 20 mm. in
length. Journ. Anat. and Physiol., vol. 48, pp. 154-145.

Weicker, H. 1878 Wachsausguss der Gehirnventrikel. Virchow’s Archiv. f.
Pathol. Anat., Bd. 74, 8. 500-503.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 2

ABBREVIATIONS

B.olf., bulbus olfactorius N.op., nervus opticus

C.pr., caudal protrusion N.tr., nervus trigeminus

Cobl., cerebellum Par., parencephalon

Ch.op., chiasma opticum Plx., plexus chorioideus

Coll. 7., colliculus inferior R.i., recessus infundibuli

Coll.s., colliculus superior R.l., recessus lateralis

C.inf., cornu inferius R.m., recessus mamillare

C.par., corpus paraterminale . R.op., recessus opticus

C.pin., corpus pineale R.pin., recessus pinealis

C.str., corpus striatum R.post., recessus postopticus

For., foramen interventriculare R.sup., recessus suprapinealis

F .plx., fissure for plexus chorioideus R.f., rhomboid fold

Hab., habenula S.l., suleus imitans

Hip., hippocampus Syn., synencephalon

Inc.pe., inecisura postcommissuralis T.ch., tela chorioidea

I., infundibulum Th., thalamus

Ts., isthmus V.b.olf., ventriculus bulbi olfactorii

L.ch., lamina chorioidea epithelialis V.l., ventriculus lateralis

Mes., mesencephalon Vel.tr., velum transversum

N, — Nz, neuromeres V.op., vesicula optica
PLATE 1

EXPLANATION OF FIGURES

4 Wax model of the cerebral ventricles of a 5.1-mm. pig. H.E.C. series
1904. x 20 diam.

5 Cast of the cerebral ventricles of a 12-mm. pig. X 15 diam.

6 Dissection of the brain of al2-mm. pig. ™ 15 diam.

7 Dissection of the brain of a 12-mm. pig. * 15 diam. The plane of the cut
edge of this dissection is indicated by arrows in figure 5.

THE DEVELOPMENT OF THE CEREBRAL VENTRICLES IN THE PIG PLATE 1
CHESTER H. HEUSER

Ts.

PLATE 2
EXPLANATION OF FIGURES

8 Dissection of the brain of al7—-mm. pig. X 10 diam.
9 Cast of the cerebral ventricles of al7-mm. pig X 10 diam.
10 Dissection of the brain of a 17-mm. pig. > 10 diam.

1)
ING
bo

THE DEVELOPMENT OF THE CEREBRAL VENTRICLES IN THE PIG
CHESTER H. HEUSER

a

C. str. ~ Lich.

PLATE 2

PLATE 3
EXPLANATION OF FIGURES

11 Dissection of the brain of a 22-mm. pig. X 10 diam.
12 Cast of the cerebral ventricles of a 22-mm. pig. X 10 diam.
13 Dissection of the brain of a 22-mm. pig. 10 diam.

THE DEVELOPMENT OF THE CEREBRAL VENTRICLES IN THE PIG PLATE 3
CHESTER H. HEUSER

14
15
16

y
d

PLATE 4
EXPLANATION OF FIGURES

Dissection of the brain of a 45-mm. pig. X 6.5 diam.
Wax model of the cerebral ventricles of a 45-mm. pig.
Left lateral ventricle of the model shown in Fig. 15.
Ventral view of the same ventricle.

246

< 6.5 diam.

THE DEVELOPMENT OF THE CEREBRAL VENTRICLES IN THE PIG PLATE 4
CHESTER H. HEUSER

T. ch. Cpin.

Plax

Csi

Fig.16 Fig.17

PLATE 5
EXPLANATION OF FIGURES

18 to 23. Microphotographs of frontal sections through the head of a 45-mm.
pig. H.E.C. series 1826. % 5 diam. The position of section 563 is indicated by
arrows in figure 14.

18 Section 563.

19 Section 629.

26 Section 687.

21 Section 755.

22 Section 1067.

23 Section 1115.

THE DEVELOPMENT OF THE CEREBRAL VENTRICLES IN THE PIG PLATE 5
CHESTER H. HEUSER

PLATE 6
EXPLANATION OF FIGURES

24 Wax model of the mid-brain ventricle of a 110-mm. pig, viewed slightly from
below and behind. X 15 diam.

25 Wax model of the cerebral ventricles of a 260-mm. pig. 3 diam.

26 Ventral view of the same model.

bo
or
S

THE DEVELOPMENT OF TEE CEREBRAL VENTRICLES IN THE PIG
CHESTER H. HEUSER

Coll.s.

Ine. pe.

Coll.i.

R.sup.

V.buolf. -

V. b. olf.

PLATE 6

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AN EXPERIMENTAL STUDY OF THE POSITION OF
THE OPTIC ANLAGE IN AMBLYSTOMA PUNC-
TATUM, WITH A DISCUSSION OF CERTAIN EYE
DEFECTS

CHARLES R. STOCKARD
Department of Anatomy, Cornell University Medical College, New York City

NINE FIGURES

INTRODUCTION

Numerous investigations have recently been directed towards
an analysis of the processes concerned in the development of
the vertebrate eye. Both mechanical and chemical methods of
experiment have been employed and at the present time most
of the results seem open to explanation. Various theories and
speculations, however, have been advanced which will probably
form a source of contention until the experimental results are
better or more uniformly interpreted.

By way of introduction to the experiments recorded in the
present paper and a discussion of their significance, it may be
well to outline briefly the status of the main problems concerned.

The writer has previously recorded the results of experiments
bearing upon an analysis of the manner of origin and develop-
ment of the optic cup and the optic lens. It was demonstrated
that in the species studied the crystalline lens can arise entirely
independent of any influence from other eye parts. It seems also
equally clear from the many cases observed that the optic vesicle
or cup can at some stage during its development induce the forma-
tion of a lens from the ectoderm with which it comes in contact,

253

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 3
NOVEMBER, 1913

254 CHARLES R. STOCKARD

even though this ectoderm is not exactly that from which the
normal lens would have arisen. Lenses were induced to arise
only from head ectoderm and independent lenses invariably
arose in the head region, it was thus concluded that the head
ectoderm principally possessed the independent lens forming
power. |

A consideration of numerous eye abnormalities which occurred
in these experiments seemed to throw light on the earlier condi-
tions of origin of the optic anlage from the medullary tissues.
A number of the abnormal eyes appeared to lend themselves to
a common interpretation and for this reason I advanced in an
entirely hypothetical manner the view that these conditions might
be considered as arrests in eye development.

Spemann in two more recent contributions to the development
of the eye has confirmed all of my observations that were touched
upon by his experiments. In an equally general manner he has
disagreed with most of the deductions which were drawn from
my experimental results. An attempt to satisfy these disagree-
ments will be made in the body of the present paper.

It is now concluded from a study of abnormal eyes and the
experiments below that the eye anlage in the medullary plate
is primarily median and single and normally separates or spreads
into two almost equal growth regions which develop in lateral
directions reaching further and further out until finally the optic
vesicles come in contact with the ectoderm at the sides of the
head. Provided this view is correct cyclopia is then an arrest
in eye formation.

Spemann, on the contrary, holds that the eye anlagen origin-
ally arise lateral in position along the borders of the medullary
plate. The cyclopean defect according to him is due to a failure
of central medullary tissue to develop so that the lateral eye
anlagen slump towards the median plane, fuse and form a single
cyclopeaneye. Spemann, however, presents no experimental evi-
dence to show that the eye anlagen do occupy lateral positions
since all of his operations included the median medullary tissue
as well as the lateral.

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 255

The only evidence to draw upon is the interesting experiments
of Lewis, in which it was found that cyclopia sometimes resulted
in Fundulus embryos when the anterior end of the embryonic
shield was injured by pricking. Lewis also interpreted these
defects as due to a fusion of the optic anlagen, and had sug-
gested, as Spemann now does, that the chemicals used in my
experiments suppressed the development of median tissue in the
medullary plate and thus caused the eye anlagen to come to-
gether, fuse and produce cyclopia. There are a great many
strong objections to this hypothesis of Lewis and Spemann which
have been enumerated in my previous papers and to which I
shall take occasion to refer briefly in the following discussion.

An objection of primary importance to the idea of cyclopia
as a result of the coming together of lateral anlagen through a
failure of intermediate tissue to form is the fact that eyclopean
eyes are rarely in size and extent equal to the sum of the two
normal eyes combined. A cyclopean eye is, as a rule, little if
any larger than one normal lateral eye and in fact is often much
reduced or actually minute in size as compared with a normal
eye. This fact indicates most decidedly that eye material, as
such, has been injured or arrested in its development and differ-
entiation. One is then scarcely warranted in assuming that the
defect is solely due to a failure in formation of material between
the eyes. f

Spemann has found, although he locates the anlagen in the
wrong place, that not only is the eye anlage definitely localized
in the open medullary plate but actually the tapetum nigrum |
is distinct from the other retinal layers. How then could absence
of material between the lateral eye anlagen cause less eye mate-
rial to arise?

The differentiation of these prelocalized anlagen require defi-
nite amounts of energy. Any treatment that weakens the devel-
oping embryo at certain periods in a definite way renders the
eye anlagen incapable of differentiation so that they do not arise
from the brain.

The entire problem is readily open to experimental test. The
contention may resolve itself into the question: Where are the

256 CHARLES R. STOCKARD

optic anlagen originally located in the medullary plate or tube?
The present communication will present the results of experi-
ments aimed towards an answer to this question. Certain inter-
pretations put forward by Spemann regarding the origin and
development of the primary components of the eye will also be
considered.

”" MATERIAL AND METHOD

The material used in all the experiments has been the develop-
ing embryos of the salamander, Amblystoma punctatum. Am-
blystoma eggs are surrounded by masses of jelly-like material
from which they may readily be separated. The eggs live per-
fectly without the jelly mass, provided they are well covered
with fresh water.

These eggs are of sufficient size to render it possible to cut
out with fair accuracy definite regions or groups of cells from the
medullary plate or groove.

The method of experiment has been entirely by mechanical
operation. This method is particularly useful for the problem
in hand since definite areas may be removed and the results
studied. The operations were made under a binocular micro-
scope. Fine steel needles and the smallest scissors were used as
instruments.

The embryos were kept from three to five days after the oper-
ation in water which had been previously boiled. They were
killed in a mixture of one part formalin to three parts saturated
corrosive sublimate, left for three hours, rinsed in water and
put into 70 per cent alcohol with iodine. Others were fixed for
three hours in Bouin’s fluid, formalin and picroacetic. The sec-
tional embryos were stained with Delafield’s hematoxylin and
eosin.

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA oO.

CONSIDERATION OF THE EXPERIMENTS

1. Sticking the future brain portion of the medullary plate with
needles

The anterior portion of the medullary plate was stuck with
steel needles in such a manner as to disturb the cells over an
area extending from the anterior border of the medullary fold
back to the constricted portion of the plate and laterally from
fold to fold. The needle was inserted below the outer layer of
cells and raised so as to push the cells apart; this was done a
number of times with each specimen. The needles were also
swung to the right and left in the medullary tissue until the
cells were considered to be disturbed to quite an extent. The
object in such an experiment was to determine how severe an
injury to the cells was necessary in order to prevent the develop-
ment of the optic vesicles.

Twenty-three embryos were treated in this manner, and all
were killed four days after the operation. Under a high power
binocular microscope most of them distinctly showed that the
optic cups were well pushed out laterally and in contact with
the ectoderm at the sides of the head. Both eyes were clearly
seen in seventeen of the individuals, while six seemed to have
eyes yet not so well developed. These six doubtful specimens
were sectioned and studied microscopically.

Both eyes were present and apparently normal in structure
in five of the six embryos. The sixth individual showed eyes
which were slightly irregular in form and poorly developed, yet
both eyes were distinctly present.

The experiment would indicate that a disturbance, of the type
employed, of the cells constituting the eye anlage in the medul-
lary plate was not sufficient to prevent the normal development
of the eyes in these embryos.

Another point of interest might have been attacked. by such
an experiment provided the embryos had been allowed to develop:
sufficiently long after the operation. That is, whether or not
the cells destined to form the tapetum nigrum layer might be

258 CHARLES R. STOCKARD

intermixed with those cells destined to form other retinal layers,
and so produce an eye with the pigment cells scattered irregu-
larly through the retina. Spemann’s recent experiments on cut-
ting out and reversing certain areas of the open medullary plate
indicate that the tapetum cells are fully localized and separate
from the other retinal cells in certain amphibian embryos. The
embryos in my experiments had not differentiated the retinal
layers sufficiently far to determine with certainty whether there
was a persistent disarrangement of the cells, yet the general
appearance of the eyes seemed perfectly normal.

2. The median region of the anterior part of the medullary plate

cut out, reversed and transplanted in the medullary plate

This experiment is similar to those performed by Spemann
on several amphibian embryos. Spemann found that pieces of
the medullary plate when cut out and turned around continued
to develop with their original orientation undisturbed, thus indi-
cating the early prelocalization of certain future parts of the
brain and eyes. When the operation chanced to cut the eye
anlage so that part of the future eye material was anterior to
the cut and remained in position, while part was contained in
the cut-out piece which was then turned around and transplanted,
carrying the future eye cells to a more posterior position, two
eye regions developed on each side. One arose from the ante-
rior undisturbed cells and the other from the transplanted pos-
terior cells.

The reversed pieces in the present experiment were not long
enough to carry the eye back to a distant posterior position,
and the cut extended so far foreward that the eye anlage was
not divided transversely as in Spemann’s operations. The oper-
ations were done chiefly to test whether the eye anlage in Ambly-
stoma was well localized and would develop after such reversal
of tissues.

Eight embryos were studied after having had antero-median
pieces of the medullary plate cut out, reversed and transplanted.
Seven of the eight developed both eyes, many of which. showed

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 259

indications of their misplacement. One individual showed one
abnormal eye while the other was probably indicated by a doubt-
ful eye structure situated within the archenteron. The general
structure of the eyes seemed normal and only slight indication
of their reversed origin was shown by a study of these early
stages.

Thus if the eye anlage was contained within these cut-out
pieces the reversal and transplantation of the pieces did not
show any very detrimental effects on the future development of
the eyes, though in one of the eight cases one eye was abnormal
and the other was greatly misplaced and indistinctly indicated.

The experiments of sticking and disturbing the cells in the
anterior end of the medullary plate without actually removing
the cells does not prevent the subsequent development of the
optic vesicles in an apparently normal manner. Cutting out
rectangular pieces of the medullary plate which contain the eye
anlage, reversing and replanting them merely cause the eyes to
develop in misplaced positions. These two experiments demon-
strate the fact that unless the future eye material is well re-
moved by the operation the optic vesicles may later form; this
is important in estimating the value of the results in the experi-
ments to follow.

3. Lateral regions cut from the anterior part of the medullary plate

Four embryos were operated upon, as shown in figure 1. The
indicated region of the right lateral portion of the flat medullary
plate was cut entirely away with fine scissors. The area removed
did not extend quite to the median line except that in one case
it may have or probably did remove tissue beyond the median
line. The embryos were killed three days later, cut into sec-
tions and studied microscopically.

One of the four lacked both eyes or had eyes so small and
poorly formed that they could not be recognized, and the brain
showed on one side indications of the operation. Both eyes had
thus been removed by an operation which extended at most

260 CHARLES R. STOCKARD

slightly beyond the median line and certainly did not include
the left lateral medullary tissue.

The other three embryos bad both eyes present, though one
eye in one specimen and both eyes in another were abnormal or
defective. In these cases the cut did not extend close enough
to the median line to remove all of the eye substance of that
side; yet if the eye anlagen at this time had been lateral in posi-
tion, one would have been completely removed.

A similar experiment was performed on seven somewhat earlier
embryos. In these cases where the medullary plate was younger
and wider in extent the removed area was confined to a lateral
position and did not extend close to the median line, yet it ex-
tended laterally into the medullary fold (fig. 2).

Three days later the embryos were killed. On studying the
sections of the head region six of the seven embryos were found to
possess two perfect eyes of normal proportions. The seventh indi-
vidual had only one well defined eye while the other eye was
absent.

The removal of this lateral area of tissue which has been con-
sidered the position of the early eye anlage by certain investi-
gators gives no effect on the development of the future eye unless
the cut be made to extend very close to the median line. Jn
eleven operations of this type nine individuals did not suffer the
loss of either eye, while one specimen lacked both eyes and
another one had only one eye. The case in which both eyes
were absent might have been due to the fact that the cut ex-
tended a little way beyond the median line and removed cells
destined to form the material of both eyes. Experiments re-
corded below would indicate that this was the case. A second
possibility is that the operation so weakened the individual—
which may have been below normal in vigor, though apparently
perfect specimens were selected for all the operations—that it
lacked the power to differentiate the eyes from the medullary
tissue.

Six of the nine specimens which possessed both eyes showed
no effects of the removed material in either the size or form of
their eyes. The other three embryos had one or both eyes some-

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA

262 CHARLES R. STOCKARD

what smaller than usual. The cases with only one eye defective
are perhaps clearly understood on the ground that the cut en-
croached upon the material destined to take part in the devel-
opment of this eye. In the cases with both eyes defective it
scarcely seems possible that the opposite eye could have been
injured by these cuts without entirely removing the eye on the
side where the cut was so extensive. The only plausible possi-
bility is that the material for both eyes was medially located and
the two eye anlagen closely connected. Any injury to this region
might later be shown by both eyes if the injury were at all exten-
sive, and if less extensive and confined entirely to one side of
the median line only one eye might show the effect.

I realize that this explanation might be interpreted in part
as opposed to Spemann’s experiments in which he found the
future eye materials so consistently definite in the medullary
plate. When the eye anlage was cut transversely and the pos-
terior portion reversed and planted in a distant position it was
found that if the forward part of one eye was small the part
which had been cut from this and placed posteriorly was large.
This fact does not indicate entirely that the eye stuffs are sepa-
rate; only that when the eye anlage, even if it be median and
single, is cut across obliquely and the posterior part removed
and transplanted it continues development in a normal consis-
tent manner, the small eye part coming off on the side where
less material exists and vice versa.

4. The anterior lateral part of the neural fold and a portion of the
medullary wall cut from one side

These experiments were performed on embryos older than those
used above in order to test whether the eye anlagen shortly
before the coming together of the neural folds might be situated
laterally along the border of the folds. In other words, do the
eye anlagen primarily arise in the median line or region of the
medullary plate and later occupy more lateral positions?

The operation consisted in cutting away one side of the ante-
rior part of the medullary fold and also including in the removed

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 263

part some of the material constituting the lateral wall of the
medullary groove (fig. 3).

Four embryos were operated upon in this manner. Three days
after the operation they were killed and prepared for study.
A eareful examination showed that three of the four specimens
possessed both eyes, while one had no eye, or a questionable
formation, on the operated side although a perfect eye was pres-
ent on the unoperated side. Thus in three-fourths of the few
eases used, neither of the future eyes had been injured by the

operation.

Six other embryos were operated upon in a similar fashion.
These were killed two days after the operation. Studying the six
embryos in cross section it was found that three possessed both
eyes in perfect condition, one individual had two eyes, yet one
eye was small and defective on the operated side, and two of
‘the specimens had no eye on the operated side. Thus four of the
six specimens had two eyes each, and two of the six had only one
eye each.

Combining the results of both experiments it is shown that,
of the ten operated embryos seven possessed both eyes and three
lacked an eye on the operated side. One of the seven which had
both eyes present showed a defective eye on the operated side.

A comparison of these experiments with those recorded in the
preceding section in which the operation was performed on ear-
lier embryos would indicate that the eye anlage has possibly
widened out so as to extend into more lateral regions in these
later stages. The weight of evidence, however, would indicate
that the position is not directly lateral along the edge or border
of the medullary plate, as Spemann has assumed.

It must be remembered that in the folding process which con-
verts the medullary groove into a tube the original borders or
edges of the medullary plate come to meet in a middorsal line.
After the medullary tube is thus formed the optic vesicles push
out from a lateral or really ventro-lateral region and certainly
do not in any sense come from the original borders of the medul-
lary plate which are now dorso-median in position.

264 CHARLES R. STOCKARD

5. Both anterior lateral parts of the neural fold cut away just before
the folds close together

The operation consisted in clipping away with scissors the
lateral wall of the neural groove from both sides; that is, the
raised folded parts and the dorsal portion of the lateral neural
wall indicated in figure 4. The actual crest of the neural folds
is of course not future lateral material as in the closure of the
furrow to form the neural tube the crest becomes dorsomedian
as just mentioned.

Four embryos were successfully operated upon in the given
manner. Three days after the operation they were killed and
prepared for study. All of the embryos were found to possess
both eyes in well developed conditions.

Five other embryos in a similar stage of development were
operated upon so as to remove the lateral neural folds as indi-.
cated in figure 4.

Four days after the operation the embryos were killed. Study-
ing these embryos in section showed that four of the five pos-
sessed both eyes in a well formed condition giving no indication
of the injury while the fifth individual possessed only one eye.
This last specimen was evidently cut too near the median line
on one side so that the future eye material of that side was
destroyed.

Thus in nine specimens with both sides operated upon, eighteen
operations, only a single eye was missing. The other seventeen
operations did not cause any noticeable effect in either the devel-
opment or nature of the resulting eye.

These experiments clearly demonstrate that the eye anlage is
not located along the lateral edge or border of the medullary
plate or groove, as Spemann holds. The importance of such
facts in connection with opposing explanations of certain eye
abnormalities will be fully considered in a following section of
this paper.

The next experiments to be presented were performed in order
further to substantiate the fact that the eye parts do occupy a

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 265

median position in the medullary plate or groove. I have been
led to think from a study of a number of eye abnormalities that
the eye anlage is more or less median in its original position.

6. The removal of anterior median strips of cells from the wide
medullary plate

In these operations a narrow median strip was cut from the
anterior region of the wide medullary plate. The strip in all
cases was narrow, being in fact only about one-third or one-fourth
the entire width of that portion of the medullary plate lying be-
tween the medullary ridges, as shown in figure 5.

Four embryos with wide medullary plates were operated upon
in the above manner. Four days later they were preserved for
study.

Three of these four specimens were eyeless, showing that the
operation had removed the entire eye anlage from the middle
of the medullary plate. The remaining one of the four embryos
had one poorly formed eye while the other was only slightly
indicated, so that in this case almost all of the ability to develop
eyes had been lost as a result of the operation.

Five other embryos were subjected to a similar operation to
that indicated in figure 5 except that the median strip removed
was still narrower than in the preceding experiment. These
embryos were of different stages but all showed the medullary
folds far apart so that the medullary groove was wide open.
Three days after the operation the embryos were prepared and
studied.

One of the ‘specimens was eyeless, one had two poorly devel-
oped eyes, two had one well formed eye while the other eye was
questionable or absent, one had both eyes present. Thus the
removal of a very narrow median strip gave less decided effects
than the removal of somewhat wider strips in the experiment
above. Yet four out of the five embryos showed eye defects,
two having both eyes affected and two having one normal eye
and one eye questionable or absent.

266 CHARLES R. STOCKARD

Combining the results of the two experiments it is found that
the removal of narrow median strips from wide medullary plates
exerts the following influence on the future development of the
eyes: Of nine embryos thus operated upon four failed entirely to
develop either eye. Two showed two defective eyes. Two individ-
uals developed one perfect and one defective or questionable eye.
Only one of the nine embryos showed two apparently normal eyes.

Since six of the nine embryos had the development of both
eyes either entirely suppressed or decidedly affected, and two of
the remaining three had one eye affected, it seems most certain
that cells destined to take part in eye formation are located in
the median region of the medullary plate and are removed by
the operation employed. One must conclude that the median
optic anlage occupied at least one-fourth or one-third of the
width of the medullary plate in the anterior region.

A general statement of the results of certain of the experi-
ments described above may be expressed as follows: Thirty em-
bryos studied after various operations in which lateral portions
of the medullary plate were removed at slightly different devel-
opmental stages (sections 3, 4, 5) showed in twenty-four indi-
viduals, or 80 per cent of the cases, subsequent development of
both eyes, while only six specimens or 20 per cent of the cases,
showed absence of the eye. In one case the presence of the eye
is questionable, in five cases one eye and in one case both eyes
were absent. The absence of eyes in the latter cases is possibly
due to the cut having been made in a more median position than
was intended.

Nine embryos studied after having been operated upon so
as to remove a narrow median strip of cells from the anterior
portion of the medullary plate (section 6) showed in four cases,
or about 45 per cent of the specimens, entire absence of eyes.
In four other individuals the eyes were highly defective, one
specimen having one poorly formed eye while the other was
questionably present. In only one of the nine embryos did both
eyes approach the normal condition, from this specimen an ex-
tremely narrow piece had been cut away. The optic anlage in
this case might have been sufficiently wide at the time of the

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 267

operation to allow its median portiori to be removed and yet
enough material remain on either side of the cut to give origin
to the two eyes. According to the views of several investiga-
tors the removal of this median material should have caused
cyclopia, yet it did not. I shall presently attempt to show that
eyclopia is not due to the coming together of lateral material
in the median line but to a failure of median material to spread
laterally. .

When the results of these two classes of experiments are con-
trasted one must conclude that: The eye anlage in the medullary
plate occupies an antero-median position as shown by the various
abnormalities incurred when this region is cut away. The failure
to injure the development of the eyes in the great majority of cases
when the lateral portions of the medullary plate are removed by
operation indicates further that the eye anlagen do not occupy lat-
eral positions during this stage of development.

A DISCUSSION OF PREVIOUS IDEAS REGARDING THE GENESIS OF
CERTAIN EYE ABNORMALITIES IN THE LIGHT OF THE ABOVE
RESULTS

There has lately been considerable discussion regarding the
way in which the cyclopean defect occurs. The experiments
described above may serve to concentrate the case on a definite
developmental period and, to my mind, settle the question as
far as the medullary plate stage is concerned. Whether it is
possible to carry any question of vertebrate eye abnormalities,
as such, further back in development than this stage seems doubt-
ful, since here it is that the localization of the eye anlage is first
known to exist. It is certain, of course, that this anlage does
come from cells which are present before the medullary plate
has formed. Whether these cells are localized and are entirely
future eye anlagen cells, and not indifferent ectoderm cells which
might have the power of forming any portion of the brain or
central nervous system, one is at present unable to state.

As the case stands, it seems possible to explain the entire
genesis of the cyclopean defect from the earliest time at which
the optic anlage is definitely known to be localized. This as-

268 CHARLES R. STOCKARD

sumption is not made solely from the material presented in the
present paper, but from the facts furnished by these experiments
together with the observations made upon the large number of
cyclopean eyes and brains which the writer has studied during
the past several years.

Various authors have at different times thought that cyclopia
was due to a fusion of the eyes after they had arisen from the
brain. The earlier in development the fusion occurred the more
intimately associated the two eye components became. This
view has been proven incorrect by actual observation on cyclo-
pean monsters where it is found that the cyclopean condition
of the eye, whether large and hour-glass shaped or of small size
resembling a normal eye, is present from the earliest appearance
of the optic vesicle from the brain.’ In other words, the several
degrees of the cyclopean eye come off from the brain in their
final conditions.

The idea of the fusion of the eye parts was deep rooted, how-
ever, and now exists in the recent views of Spemann in a refined
form. Spemann believes, as several others had previously sug-
gested, that cyclopia is due to an absence of non-ophthalmic
tissue in the median region of the medullary plate or groove.
This lack of median tissue allows the eye anlagen which he
holds to be lateral in position, near the borders of the medullary
plate, to come together and fuse in the median plane and later
give rise to a cyclopean eye. Cyclopia, according to this idea,
occurs in a more or less passive manner, and is, after all, actually
a fusion of the eye anlagen of the two sides during development.

I am certain that this fusion explanation which has now been
forced entirely back into the medullary plate, is as false as its
bolder predecessor which assumed the fusion to take place out-
side of the brain tissues after the optic vesicles or cups had
arisen. Spemann did not advocate this late-fusion view, but
claimed from his beautiful experiments on Triton that the cy-
clopean eye arose out of the medullary tissues in its final
condition. He now, however, assumes the réle of a most ardent
supporter of the fusion of the optic anlagen within the medul-
lary plate.

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 269

The present writer’s opinion of the cause of cyclopia, which
was advanced in 1909, was to the effect that this deformity is
the result of a weakened development. Spemann has termed
this view the ‘laming hypothesis,’ since my assumption was that
the various chemical substances employed in producing cyclo-
pean defects in fish embryos have a tendency to lower the de-
velopmental energies and so reduce the power necessary to ac-
complish the processes involved in the outpushing of the optic
vesicles from the brain.

Considering the probable manner in which the cyclopean defect
occurs, Adami (’08, p. 241) has theoretically concluded that it
is due to developmental arrest or lack of vigor. While I am
unable to agree with the details in Adami’s argument of a pri-
mary growth point at the anterior tip from which is budded off
successively the paired parts of the two sides, the anterior ones
necessarily arising last after the other parts had been left in
more posterior positions, the final conelusion that a weakening
of particular developmental processes results in cyclopia is con-
firmed by all my experiments. /

The different.types or degrees of the cyclopean defect depend
upon the stage in development at which the arrests occur as
well as upon the strength or severity of the treatment employed.
I shall now attempt to defend this position with the evidence
at hand, and in so doing shall as decidedly prove the mistake
in considering the defects as the result of any failure to arise of
median medullary tissue (other than future eye tissue) and the
subsequent fusion of the lateral optic anlagen. There is no med-
ian tissue between the eye anlagen. The median tissue is the
eye anlage itself and will subsequently go to formsome portion
of the eye, either optic cup or optic stalk, depending largely
upon its position and the extent of normal development attained.

The writer had (’07, ’09, 710) recorded a number of eye con-
ditions which are considered to be different degrees of cyclopia
using the term in a general sense. At any rate, these several
conditions differ only in degree and grade perfectly into a con-
tinuous series. There is no qualitative difference between them.
Spemann has objected to including among these defects certain

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 3

270 CHARLES R. STOCKARD

modified types which are at least closely related to the series in
manner of origin, though they may not actually intergrade.
(We are now considering only the cyclopean series, not the mon-
ster monophthalmica asymmetrica which will be dealt with later. )

One finds on referring to my paper of 1909 (p. 293) that the
cyclopean series, A to G, passes from the normal individual
through different degrees of association of the two eyes to a
median cyclopean eye only as large as one normal lateral eye,
then to a cyclopean eye of smaller dimensions until it is extremely
minute and may finally be deeply buried beneath the brain as
a small pigmented vesicle, as is shown in figure 52, page 321.
Only one step further and the eyes fail to arise entirely so that
eyeless individuals exist which with a slightly greater power of
differentiation or more developmental energy might have given
cyclopean monsters. The last assumption is warranted since
these eyeless specimens actually resemble the cyclopean monsters
in other structures; for instance, the mouth is a narrow pro-
boscis similar to that in the cyclopean monster instead of the
usual laterally spread mouth of the normal embryo.

Why should every step and gradation in this series exist if
several, or any of the conditions are of a different quality or
type? It seems certain that one examining the large number
of cyclopean fish on which:my study was based would be forced
to admit the correctness of the statement that these individuals
exhibit different degrees of one and-the same defect.

The question then follows, if cyclopia were due, as Lewis,
Spemann and others assume, to a failure to develop of median
medullary tissue so allowing the eye anlagen to come together
in the median plane and fuse, why is not every cyclopean eye
equal in mass to the two normal eyes fused? Spemann does
not suggest in any place that eye material also fails to arise.
He shows in his recent experiments that the future eye is fully
laid down in the medullary plate. Not only is the eye present
in the medullary plate but the cells destined to form different
layers are distinct. Spemann found that certain cells cut out
of the medullary plate and planted in more posterior positions
formed only the tapetum nigrum layer. If the eye is thus so

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 271

definitely predetermined in the medullary plate and Spemann
believes that cyclopia is due to failure of median medullary cells
other than future eye cells then cyclopean eyes ought always to
be large or double in size.

The fact is that these predetermined eye cells in the medul-
lary plate in most cases of cyclopia are incapable of perfect dif-
ferentiation on account of insufficient energy, so they remain
in the brain, or only part of them is capable of differentiation
and thus small defective cyclopean eyes result.

The actual ‘Lihmungs’ or suppression is of the eye material
itself. Cyclopia is an eye defect, and an injury of the eye forming
material is the cause. The brain may also be defective as an
accompaning abnormality, although in some cases the brain,
with the exception of the optic tracts and parts, may be struc-
turally and functionally perfect, as is indicated by the normal
life and behavior of many of the cyclopean Fundulus embryos
as well as by the existence of the huge cyclopean ray, Mylio-
bates noctula, reported by Paolucci in 1874.

The chemical substances employed by the author in produc-
ing the cyclopean defect and a number of others with which
McClendon has obtained similar results, all tend to suppress or
arrest the development of the eye material in the brain. This
future eye material is assumed to occupy a median position.
When the arrest is complete, and necessarily taking place at
early stages, no eye parts arise from the brain nor are any dif-
ferentiated within the brain substance itself. Thus a completely
eyeless individual is produced.

Spemann states that the eye is capable of differentiation even
though it be contained within the brain substance as he has
found in amphibia and as Mencel recorded in a teleost. It must
be realized that these are exceptional cases. A certain amount
of energy is necessary for differentiation of the eye to take place
even within the brain and when only this amount of energy is
present the eye may differentiate within the brain, but when
the required energy for any reason is not available the eyes are
incapable of any differentiation. Many eyeless individuals have
been observed in my experiments which have no indication what-

22 CHARLES R. STOCKARD

ever of eye parts within the brain. Could any one ask, whethe:
this be due to the failure of non-ophthalmic parts of the medul-
lary plate to arise or, on the other hand, to a failure of the future
eye forming cells to arise, or to differentiate after they have
arisen?

It is not meant to convey the idea that all eyeless brains
are related to the cyclopean series, as this is not the case. The
future eye forming cells may in some cases have been absent.
from the start. In other specimens the future optic vesicles might
have been in positions to arise normally and laterally and for
some reason were incapable of outpushing or of differentiation.

Certain eyeless brains, however, such as those in individuals
having the proboscis-shaped mouth, do belong to the series and
must be caused in the same way as are the various cyclopean
conditions. They have merely responded to a more exaggerated
degree.

The most extreme cases of cyclopia with actual eye structures
are those in which a small pigmented vesicle arises from a ventro-
median part of the brain, as is shown in my figure 52, 1909.
This pigment layer of the retina seems to be its most persistent
portion, as it may appear when all other recognizable retinal
parts fail to arise. There is a possibility that the small tapetum
nigrum groups of cells which in some cases formed from Spemann’s
transplanted portions of eye anlagen may not be due to the fact
that only the anlagen of such cells were transplanted, but that
all other retinal cells except these were incapable of differentia-
tion when so small a piece of future eye tissue was isolated by
the operation.

The next degree of cyclopia is exhibited by an individual hay-
ing a median eye that is much smaller than one normal lateral
eye. We then have a median cyclopean eye of about the same
size as one normal lateral eye. The latter case has been termed
the ‘perfect’ cyclopean condition.

All these abnormalities are best explained as follows: The
future eye forming cells occupy a median position in the medul-
lary plate and the cells destined to form the two eyes are ar-
ranged in one group. This median group of future eye cells

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 273

normally widens or spreads laterally while two centers of active
growth become established which gradually assume more lateral
positions until they push out as the two optic vesicles. In the
degrees of cyclopia mentioned in the preceding paragraphs the
median eye anlage does not widen or spread laterally but is
arrested in its primary condition; thus the two growth centers
are not sufficiently separated and only a single center exists,
and even more than this, the arrest is to such an extent that the
entire or normal amount of optic material does not differentiate.
Hence, one finds a median cyclopean eye consisting of an amount
of eye material far below that normally present.

Other individuals are found in which greater masses of eye
material have succeeded in differentiating, and development has
been vigorous enough to allow the early eye anlagen to spread
to a greater or less degree and establish the two eye forming
centers. Such specimens finally present a cyclopean eye show-
ing distinctly its double composition, the two retinae are more
or less distinct and the eye large consisting of a greater amount
of material than a normal lateral eye, yet less bulky as a rule
than the sum of the two normal eyes of the species.

The hour-glass eye or incomplete cyclopig is commonly ob-
served in the experiments. This case is due to a later or less
complete arrest in development than those mentioned above.
Both eyes have differentiated out of the medullary tissue but
the embryo was not vigorous enough to permit their normal sep-
aration and outpushing. Thus the eyes come off from their ori-
ginal ventro-median position and remain in close contact or actual
union.

Finally one observes individuals in which the eyes are sepa-
rate and distinct yet unusually close together. Such embryos
are able to differentiate their eye material and this material is
capable of pushing out from the brain but a slight weakness or
arrest has occurred on account of which the optic stalks are
short and the optic cups are unable to take a normal position:
so that they remain unusually near together and look in an
.abnormal direction.

274. CHARLES R. STOCKARD

In the ordinary individual the future eye forming material is
first located in a median position in the medullary plate. This
material becomes more extensive or widens laterally, and two
growth centers are established, the material between the centers
finally becomes the median ventral layer of cells of the optic
stalks. Later the incipient optic vesicles begin to evaginate or
push in a ventro-lateral direction and finally turn dorsally and
laterally to reach their usual places at the sides of the head.
The optic stalks, however, still lead back to the ventro-median
position and there in the fish the optic fibers, following the optic
stalks as paths, cross and in higher vertebrates form the optic
chiasma always in the ventro-median plane below the brain
floor and from here the optic tracts proceed to their centers in
the brain.

The median position of the optic chiasma outside and below
the brain is an important structural fact in the present consid-
eration. Figure 6 is a diagram of a transverse section through
an early brain with the optic cups in their usual position. The
optic stalks connect with the brain and the median ventral cell
layer is actually part of the stalks. As development proceeds
the optic fibers arising from the cells in the retina follow along
the optic stalks to reach the brain. The investigations onthe
development of the optic nerve have shown that the optic stalks
become solid and form the supporting paths or neuroglial scaf-
folding along which the optic nerve fibers grow. The fibers from
one retina meet those from the other in the median plane below
the brain and in the fish the fibers from the two retinae cross
directly while in higher forms partial crossing takes place and
the optic chiasma is formed outside the brain. Figure 7 is a
sketch representing a cross section of the actual condition of
the early optic nerves in a fish embryo. This position of the
optic cross is only possible if the median tissue be optic stalk
tissue.

Suppose, on the other hand, that the eyes primarily originate
from lateral medullary tissues and between the two eyes other
brain tissue is present. The optic stalks are then attached to.
the lateral regions of the brain from which the optic vesicles

e

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 275

Fig. 6 A diagrammatic cross section through the brain and optic cups of an
early embryo, with all of the median ventral medullary tissue represented as
being part of the optic stalks. The future fibers of the optic nerves, shown in red,
follow the optic stalks to the median plane where they cross and afterward enter
the brain to pursue their course as the optic tracts. The optic cross is entirely
beneath and outside the brain.

Fig. 7 A sketch representing the actual outlines of a cross section through
the brain and eyes of a late fish embryo. The optic nerves cross below and out-
side the brain and are surrounded by cells derived from the original optic stalks.

276... . CHARLES R. STOCKARD

pushed out. Figures 8 illustrates diagrammatically a cross sec-
tion of this condition. In the course of development the fibers
of the optic nerve following the stalk reach the lateral position
and must enter the brain and continue within its tissue in order
to meet the nerve of the opposite side and form the cross or
chiasma. Brain tissue would lie beneath the optic chiasma and
the chiasma would necessarily be within the brain. This con-
dition is never found in any normal vertebrate.

Fig. 8 A diagrammatic cross section through the brain and optic cups in an
imaginary case in which the optic connections are lateral with median brain tissue
originally lying between the eye anlagen. The future optic fibers growing along
the optic stalks as paths reach the lateral points on the brain from which the
stalks arose and then enter the brain tissue before having formed the cross.
Continuing to grow, the optic nerves meet and cross in the median plane. The
cross is within the brain itself and lies above the median mass of tissue which
has always existed between the eyes. No vertebrate brain exhibits such a con-
dition.

One might claim that the optic fibers on reaching the brain
ran ventrally and formed the cross beneath, but no such change
in direction is seen at any stage of their development. The
structural relationships seem to depend upon a median origin
and connection of the optic stalks.

The fact that a cyclopean eye may have no well formed optic
stalk and is entirely median in position not necessitating the

Lar

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 204

absence of any other brain part is in entire accord with the
above facts.

It is believed, therefore, that the various degrees of the typi-
cal cyclopean condition from eyes unusually close together, to
median double or hour-glass eyes, to the large oval eye, to a
median round eye of usual size, to the median eye smaller than
one normal eye, and finally to complete failure of eye material
to arise from the brain are probably all due to developmental
arrest. The arrest in development is the result of some influence
which has reduced the developmental vigor below the normal
so that the energy is not available to carry out the usual pro-
cesses of differentiation and growth.

The author has figures and described other cases of eye de-
fects which do not fall exactly into the series of cyclopia as
considered above; yet these cases are modified conditions which
are closely related to the cyclopean series both in point of origin
and in their final condition. The curtain-like eyes which face
the median plane and often have a single lens between them
might be considered as delayed cases of cyclopia. The eye an-
lage widened, the eyes became separated and continued their
differentiation, yet they were unable to turn out and assume
their normal lateral positions so that they faced in a ventro-
median direction with their anterior walls closely approximated
as is shown in figure 38 (09) and figures 1, 4, 5, 6, 13, 14 (10 a).
Such eyes often excite a single stimulus upon a more or less ven-
tral ectodermal region which responds by forming a single lens
lying between the two eyes.

The writer has illustrated by a diagram (fig. 15, A and B
10 a,) the difference between these eyes with their choroid sur-
faces against the lateral ectoderm of the head and their concave
retinal surfaces facing medially, and the eyes of a normal indi-
vidual (10a p. 380). As was then stated, the experiments did
not give a definite clue to indicate the position of the optic
anlagen in the early brain, thus my explanation, or ‘laming hy-
pothesis,’ referred mainly to the pushing out and lateral develop-
ment of the optic vesicle and cup. All these cases are decidedly

278 CHARLES R. STOCKARD

of a type which would suggest lack of developmental energy
necessary to attain the normal.

Another group of eye anomalies were extremely common under
the same experimental conditions which caused the cyclopean
series. These individuals possessed one normal eye in the usual
lateral position while the eye of the opposite side in the numer-
ous specimens showed various degrees of imperfection from a
condition slightly below the normal in size to complete absence
of the eye. Such anomalies were termed ‘monophthalmica asym-
metrica’ in contrast to the symmetrical one-eyed monsters with
a median cyclopean eye.

The genesis of the asymmetrical defects is not entirely clear,
yet they also are probably due to developmental arrest or sup-
pression of the one eye. The growth centers representing the
two future eyes of an individual are rarely equally vigorous and
it is frequently noticed that one eye arises slightly before its
mate and develops at a little faster rate. It might be that at
some critical point in development one of the future eye centers
is affected after the growth centers had begun to localize in more
or less lateral positions.

In treating the eggs with aleohol a number of embryos occurred
in which both eyes were small and defective even though they
arose from the brain and attained more or less lateral positions.
One might assume this to occur as a result of an arrest in devel-
opment which affected both eye forming centers after the centers
became separate or distinct from one another. Part of the eye
forming material is suppressed or its differentiation is prevented
so that each eye is decidedly under size and defective. In some
of the cases of cyclopia mentioned above the eye was also very
small and defective, in these cases the two growth centers which
would give rise to the future lateral eyes did not become suffi-
ciently separated so that only a single median eye arose and
the reduced vigor permitted this to form only as a small and
poorly differentiated structure.

In some instances where an embryo possessed only one mem-
ber of the normal eye pair this eye was unable to attain its usual

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 279

lateral position so that it faced the median plane, and if the
lips or periphery of the optic cup failed to reach the ectoderm
the eye was without a lens.

In none of the specimens, although many were old with the
central nervous tissue highly differentiated, was I able to detect
any material within the brain which might be considered as
representing the missing eye.

Eye conditions such as these are doubtless due to the action
of some inhibitory influence which prevents the complete origin
and differentiation of eye material, or when it does arise allows
it to develop only in a weakened or defective manner. Since
monophthalmica asymmetrica occurs so persistently in the same
experiments with cyclopean individuals as Lewis, McClendon and
I have all found, it is not improbable that the cause is the same
in the two cases.

The above considerations have been entirely from the stand-
point that the ophthalmic defects under discussion originate dur-
ing the medullary plate stage. The cause of cyclopia or the
tendency to produce such a defect might of course have its ori-
gin much earlier in development. It might, in fact, go back to
the germ cells themselves or finally it might possibly occur as
an hereditary variation. All experimental cyclopia, however,
furnishes evidence directly contrary to the latter possibilities.
This point I have considered in a previous paper and have
clearly demonstrated as have several other investigators that the
condition is not due to a germinal variation but may be induced
as the result of external stimuli applied during the early devel-
opment of the eggs.

The cyclopean abnormality may be caused in Fundulus embryos
by subjecting the eggs to various chemical stimuli after they
have developed normally for as long as fifteen hours. A fif-
teen-hour Fundulus embryo has the germ ring beginning to form
and descend over the yolk sphere, the embryonic shield is scarcely
indicated but appears very soon afterwards. Embryos of later
stages subjected to the same treatment develop normally, or do
not show cyclopia, while stages younger than fifteen hours and

280 CHARLES R. STOCKARD

as early as the first cleavage are much more readily affected in
such a manner as to cause the cyclopean defect. The optic ves-
icles appear at about thirty hours after fertilization, but the
stimulus must be applied at a time sufficiently long before this —
process occurs, since a number of important steps in eye forma-
tion are doubtless taking place before the visible signs of optic
vesicles are present.

The fact that cyclopia may be produced after the beginning
of the germ ring and embryonic shield in the teleost embryo in-
dicates directly that the defect may occur in what would be the
medullary plate stage of amphibian embryos. Thus explana-
tions of the cause of cyclopia must consider it as occurring in
the medullary plate stage.

Spemann (’12 b, pp. 38-39) has taken exception to my state-
ment regarding the non-occurrence of cyclopia when eggs are
treated later than fifteen hours after fertilization (although the
optic vesicles do not arise until about the thirtieth hour) ‘since
insufficient time exists for the substances to act on the eye anlagen.”’
The paragraph following this statement in my paper (p. 388,
’10a)is: ‘The solutions are effective up to a stage in development
preceding the formation of the germ ring and embryonic shield,
and the action of the Mg on the eye anlagen probably takes place
while the embryonic shield and outline of the embryo are forming.””!

This statement seems to me perfectly direct and clear: Yet
Spemann intimates in one sentence that I mean to infer that fif-
teen hours are necessary for the substances used to penetrate the
egg membrane! He then states that I showed in 1907 that KCl
would penetrate within a few minutes and stop the embryonic
heart beat. The Mg and other solutions used may pass through
the membrane equally as rapidly, this I have not fully tested.
The fact that it does has no bearing on the fact that stimuli do
not have sufficient time to act wpon the optic anlagen to induce
cyclopia when applied to the eggs at later periods in normal de-
velopment than fifteen hours after fertilization since they must
act on the anlagen in the embryonic shield, and to act later is

1 The last clause was not originally in italics.

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 281

too late. The reason for the nonoccurrence of cyclopia when
eggs were treated at periods later than fifteen hours after fer-
tilization or fifteen hours before the appearance of the optic ves-
icles is that the optic anlagen and all other embryonic parts are
constantly changing during development and have passed beyond
the critical stage. It is not only a question of how quickly one
may stimulate but in what condition the anlagen are at the time
of stimulation. It is safe to say that cyclopia can not be produced
after the optic anlagen have proceeded to some definite stage in
their normal development. Even if Mg should penetrate all the
membranes within a few seconds its action could never induce
cyclopia in an enibryo with the two optic vesicles visibly formed.
There was no question of the time necessary for penetration, but
an important question of the embryonic condition to be acted
upon, the critical condition of the optic anlagen.

In the above connection it may be mentioned that such sub-
stances as alcohol and ether, when administered to an early embryo
may cause it to develop into a decided monster. A late embryo
or foetus may respond to the same treatment by producing an
individual exhibiting no gross morphological defects yet showing
decidedly abnormal nervous reactions, while a similar treatment
might exert little or no effect upon a fully formed individual. A
stimulus could not cause the mature individual to change into a
structural monster. The developmental period of administration
is of as high importance in determining the result as is the nature
of the stimulus used, unless of course the stimulus be entirely
destructive.

Again Spemann misinterprets a statement regarding the action
of Mg. It was remarked that the cyclopean embryos develop-
ing in the Mg solutions were, except for the eye defects, more
perfect than those arising from treatments with alcohol, ether,
and so forth. The Mg cyclops often had apparently normal
brains, could swim in normal fashion, took food and reacted to
stimuli much as normal embryos did, while those embryos treated
with alcohol and ether had various defects of the brain and cord.
In this connection it was cited as of interest that Mayer had re-

282 CHARLES R. STOCKARD

cently found in studying nerve muscle preparations that Mg
salts seemed to prevent activity by affecting the muscle directly
without apparently affecting the nerve. There is, of course, no
direct connection between these facts and cyclopia; the mention
was made merely in a general way, and most decidedly did not
intend to convey the notion that muscle contractibility and the
outpushing of the optic vesicles were phenomena of similar nature.
They are similar only in that both are dynamic processes and
require energy for their accomplishment.

There is no necessity for further discussing the fact that a
number of eggs when subjected to the same solution do not all
respond in a like manner (Spemann ’12 b, p. 37). This is a typi-
cal case of differences in individual resistance and vigor which is
observed among any one hundred individuals of any living spe-
cies. It is equally true that the two sides of a so-called bilateral
individual are rarely, if ever, identical.

Spemann is no doubt correct in stating that the relationship
between cause and effect in my chemical experiments on cyclo-
pia is not clear. Yet it seems to me that it is not entirely dark,
the entire relationship between cause and effect in biological ex-
periments is rarely if ever clear step for step.

I should like, however, to point out that the chemical experi-
ments did one thing in proving that cyclopia could be caused
from normal embryos through the action of the environment.
This fact did away with all theories of germinal origin of the
defect, one of which was strongly presented by Wilder about the
same time. The experiments also make clear the stage in de-
velopment at which cylopia may occur, and they further supply
the richest amount of material yet available for the study of this
defect. Finally, they prove to my mind that cyclopia is a de-
velopmental arrest and may be due to any cause which lowers
developmental vigor at certain critical stages in the formation
of the eye anlagen. These important points in the study of this
defect have certainly not been made clear by the mechanical
experiments though I do not deny that they might possibly
have been.

To quote again from Spemann:

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 283

Gegen diese Defekthypothese erhebt Stockard (1909b, p. 172) die
Fragen: warum sollte bei den Magnesiumembryonen gerade das Gewebe
zwischen den Augen ausfallen, und keine anderen Gewebe?; warum sind
die Riechgruben bei Cyclopie manchmal verschmolzen und manchmal
getrennt?; . . . . ist bei den asymmetrisch-eindiugigen Missbil-
dungen der einseitige Augendefekt etwa auf die Abwesenheit der einen
ersten Augenanlage zurtickzufiihren?—Darauf méchte ich mit der Gegen-
frage antworten: sind denn all diese Tatsachen verstindlicher bei Zugrun-
delegung der Stockard ’schen Lihmungshypothese?

I should answer now, as it was inferred above, that they most
decidedly are, and an attempt to show this fact in some detail
has been made in the preceding pages.

The title of the recent paper by Spemann “Zur Entwicklung
des Wirbeltierauges”’ might better have been ‘‘The development
of the vertebrate lens,” as little or almost no attention is given to
other parts of the eye. The problem of the lens formation is
very fully considered.

‘““Cyclopia of the lens’’ is discussed from the same standpoint
as cyclopia of the optic cups. In the case of the lens median
ectodermal cells may be missing so that the two normally lateral
lenses fuse in the median line. It seems to me that this is the
one straw too much for the ‘Defekthypothese.’ The imagina-
tion which pictures the falling out of just the exact amount of
median ectodermal tissue which would allow the primary lens
forming cells of the ectoderm to fuse towards the center and
keep proper pace with the movements and final position of the
various cyclopean eyes which my Fundulus material presents must
be most vividly active. An even more plausible possibility out
of this imaginary dilemma is to consider the lens anlage as a
single median group of cells that divides into two parts which come
later to lie in lateral positions. This view would at least have
the advantage that in cyclopia of the eye ‘‘cyclopia of the lens’’
would maintain the lens forming cells in a fairly median region,
and in normal development the lens cells would have a more or
less definite path to follow and place to reach. The pineal eye
in many forms possesses a fairly definite lens which must have
arisen medially and the present lens may have been somewhat
more anterior yet also median. These suggestions are of the

284 CHARLES R. STOCKARD

most speculative nature and are intended merely as such. In
my experiments many of the embryos which possess supernum-
erary lenses show, as Spemann has called attention to, that the
accessory lens may actually lie more anterior than the eye. Nev-
ertheless, others, show free lenses in lateral positions.

The presence of a lens in the cyclopean eye is explained by the
fact, well established for several species, that an optic vesicle
or cup possesses the power to stimulate lens formation from any
region of the head ectoderm with which it comes in contact.
There would be no necessity of imagining a condition of ‘‘cyclo-
pia of the lens” even though a median lens should be observed
in an anophthalmous monster. Normal lateral lenses have been
observed in anophthalmic monsters (see figs. 1 and 5, and fig.
3, plate II, 1910 b).

The embryo from which my figure 4 (’10 b), was taken is of
new interest in connection with a hypothetical case called for
by Spemann (p. 81712 a). He states as a possibility that

der cyclopische Defekt nur die Epidermis betrifft, so dass Riechgruben
und primare Linsenbildungszellen median zusammenriicken, wahrend die
Augenbecher, wie normal! seitlich gelegen, sich ihre Linsen aus der dor-
tigen Epidermis bilden. Durch solche Faille wiirden in der Tat an einem
und demselben Kopf beide Fahigkeiten demonstriert, die der Linse zur
Selbstdifferenzierung und die des Auges zur Linsenerzeugung. Bis jetat
liegen aber derartige Fille nicht vor.

The case however, was, recorded at the time and seems to, fill
the requirements set forth. The independent origin and differ-
entiation of the lens is demonstrated in a median position slightly
more anterior than the eyes, and the more or less lateral eyes have
derived lenses from the ectoderm with which they came in con-
tact as is shown by figs. 9 and 10, 1910 b, yet this ectoderm is
part of the usual region from which an optic cup has the power
to derive a lens. The power to form lenses, as Speman and I
have claimed, is possessed by the ectoderm of the head.

According to Spemann’s assumption, the embryo (fig. 4) pre-
sents true ‘‘cyclopia of the lens.’”? The condition, however, may
better be interpreted as an illustration of the high lens-forming

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 285

capacity possessed by the ectoderm at the anterior tip of the
head.

Lens-forming power seems to diminish from the anterior tip
of the head backward until trunk ectoderm no longer possesses
the capacity to form a lens, as Spemann found in transplantation
experiments. For this reason independent lenses arise, as a rule,
far anterior to and often in front of the more or less lateral eyes
(my figs. 3, 4, 7, 9, 10, 11, 12, 21, etc. °10 b). In fewer cases
independent lenses are found in more posterior lateral regions
approximately in the normal lateral eye position (figs. 1 and 2

Fig. 9 Two diagrams indicating the primary lens-forming power of various
portions of the head ectoderm. The lens-forming tendency is considered to be
greatest at the anterior end and gradually decreases towards the trunk ectoderm
until the ability to form a lens is lost where the trunk begins. The rows of circles
indicate the magnitudes of lens-forming tendency in different regions and do
not signify the size of the lenses. Posterior lenses may be as large as anterior
ones, yet they occur less frequently as independent structures. Free lenses
usually occur near the anterior tip.

and 3, plates I and II 710 b). Figure 9 may serve to illustrate
diagrammatically the extent and gradation of the lens-forming
power possessed by the head ectoderm. This rather diffuse locali-
zation of lens-forming cells in the general head ectoderm as
demonstrated by numerous experiments seems sufficient to ac-
count for all phenomena of lens formation in ecyclopia, as well as
the supernumerary lenses which the writer has reported.

The median position in cyclopia of normally bilateral organs
involves one other part. The nose or nasal pits in the cyclopean

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 3

286 CHARLES R. STOCKARD

fish are sometimes median and single, or occasionally bilateral
and more or less normal in position. I might also add that the
nasal pits are often absent. Again the ‘defect hypothesis’ must
expect median cells to fail and so allow the two nasal pits to
fuse medially. In this case the evidence is still stronger for the
primary median origin of the ectodermal anlage. Dohrn’s stud-
ies on Ammococtes have shown the median position and relation-
ship of the nasal and hypophyseal invaginations. The question
of monorhiny in the cyclostomes is not fully determined but evi-
dence is certainly available to indicate a median nose anlage.
These, however, are phylogenetic considerations which would
only serve to prolong the present discussion.

The experimental results presented by Spemann relative to
questions of lens formation agree almost entirely with the con-
clusions which were presented in my paper of 1910 b. He dis-
agrees, however, with many of my interpretations, yet I believe the
disagreement is not as complete as it often seems.

I had suggested that the power of the ectoderm to form a lens
without the presence of an optic cup was less vigorous or efficient
than when the optic cup combined its stimulus with the tendency
to lens formation possessed by the ectoderm. For this reason
when the ectoderm was injured by many of the mechanical op-
erations which have been employed in the study of lens formation
the injured ectoderm was unable to form independent lenses al-
though normally it would have had such power. Attention was
called to the different results of Lewis and Miss King on Rana
palustris.

Spemann (’12 a, p. 49) rejects this idea although he produces
evidence in his paper to prove its very probable correctness.
Compare the results he obtained in the origin of independent
lenses after cutting out the optic anlagen from medullary plates
with glass needles, in which case the ectoderm in the primary
lens-forming region was uninjured, with the results following a
_ burning out of the eye anlagen with hot needles, in which case
neighboring tissues were necessarily injured. After the latter
operation only one well formed lens occurred in five cases. A

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 287

further comparison between the glass needle operations on early
stages with open medullary plates and later stages where the ecto-
derm was raised and the optic vesicles cut out from beneath it
show a difference in the response of the ectoderm in producing
free lenses in favor of the less injured or less disturbed ectoderm.

A few of the early lenses figured by Spemann are at least ques-
tionable. For example, figure 44 (’12 a) shows on the operated
side an irregular thickening within a single cell layer. Such a
thickening might readily have resulted from the disturbance to
which the portion of ectoderm had been subjected. ‘This speci-
men is cited as evidence that the accessory or supernumerary
lenses in my experiments may have arisen as buds from a com-
mon origin. The possibility of early lenses constricting or bud-
ding into two or more is freely admitted. Figures 14, 17, 18 and
20 (10 b) show constricted or double lenses. If the constriction
had been carried further two lenses might have resulted. Yet the
relative positions occupied by several other of the lenses figured
are difficult to account for on the above basis.

Finally, there is no question of the fact that in numbers of
the fish monsters which I have figured and described small and
ill-formed eye vesicles or fragments are associated with large
well-formed lenses. The fact of the constant association of such
optic structures with lenses whenever the optic parts chance to
lie near the ectoderm makes it practically certain that the defect-
ive eyes have stimulated the lenses to arise in these positions.
The size of the lens in Fundulus is not regulated by the size of
the optic cup. This is further proven by the small lenses in
large eyes and by large protruding lenses in rather well-formed
but small eyes. The error in logic which Spemann (’12 a, p. 81)
claims to exist on page 405 of my 1910 b paper in discussing these
eye fragments I am unable to detect and several other embry-
ologists have been unsuccessful in pointing it out.

288 CHARLES R. STOCKARD

SUMMARY

1. Experiments in which certain regions are removed by mechan-
ical operations from the medullary plate of Amblystoma punctatum
seem to show that the earliest optic anlage ts median in position.

Thirty embryos from which lateral portions of the medullary
plate and the anterior lateral part of the medullary fold were
removed at slightly different stages gave in twenty-four cases,
or in 80 per cent of the individuals, subsequent development of
both eyes. In five individuals one eye was absent and in one
specimen both eyes failed to arise. The absence of eyes in the
latter cases was probably due to the cut having been made in a
more median position than was intended.

Nine individuals were operated upon so as to remove narrow
strips of cells from the anterior median portion of the medullary
plate. Four of these cases, or about 45 per cent of the specimens,
failed entirely to develop eyes. According to Spemann and
others they should have given some degree of cyclopia. Four
other individuals possessed highly defective eyes, one embryo
having one eye poorly formed while the other was questionably
present. Only one of the nine specimens so operated upon was
capable of developing both eyes to an extent approaching the
normal.

2. When the cells in the anterior portion of the open medullary
plate are disturbed by being stuck and scraped in various ways
with steel needles they do not loose their power of giving rise
to optic vesicles and cups which are normal in appearance during
the early stages, later stages were not studied.

3. If the optic anlage be cut out of the medullary plate and re-
versed in position and then transplanted in the medullary plate
it still retains the power to give rise to optic vesicles and cups
which are abnormal in position to an extent depending upon the
distance the anlagen were shifted by the operation.

The facts furnished by these experiments are considered in
connection with recent views regarding the genesis of certain
ophthalmic defects.

POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 289

LITERATURE CITED

Apami, J. G. 1908 Principles of pathology, vol. 1, p. 241, Lea and Febiger,
New York.

Dourn, A. 1883 Studien zur urgeschichte des Wirbelthierkérpers III. Die
Entstehung und Bedeutung der Hypophysis dei Petromyzon planeri.
Mitt. Zool. Stat. Neapel., Bd. 4, pp. 172-189.

Kine, H. D. 1905 Experimental studies on the eye of the frog. Archiv f.
Entw.-MVech., Bd. 19.

Lewis, W. H. 1909 The experimental production of cyclopia in the fish embryo
(Fundulus heteroclitus). Anat. Rec., vol. 8, pp. 175-181.

Mayer, A. G. 1909 Rhythmical pulsation in Scyphomedusae. Carnegie Insti-
tution Pub., no. 102, pp. 113-1381.

McCuenpon, J. F. 1912 An attempt toward the physical chemistry of the pro-
duction of one-eyed monstrosities. Am. Jour. Physiol., vol. 29, pp.
289-297.

Mencu, E. 1903 Ein Fall von beiderseitiger Augenlinsenausbildung wirhrend
der Abwesenheit von Augenblasen. Arch. f. Antw.-Mech., Bd. 16.
1908 Neue Tatsachen zur Selbstdifferenzierung der Augenlinse. Arch.
f. Entw.-Mech., Bd. 25.

Paouuccr, L. 1874 Sopra una forma mostruosa della myliobatis noctula. Atti
della societa Italiana di Sc. Naturali, tom. 17, pp. 60-63.

SpeMAnn, H. 1912a Zur Entwicklung des Wirbeltierauges. Zool. Jahrb., Bd.
32. Abt. f. allg. Zool. u. Physiol., pp. 1-98.
1912 b Uber die Entwicklung umgedrehter Hirnteile bei Amphibien-
embryonen. Zool. Jahrb., Suppl.15. (Festschrift fiir J. W. Spengel,
Bd. 3) pp. 1-48.

Strockarp, C. R. 1907 The artificial production of a single median cyclopean
eye in the fish embryo by means of sea-water solutions of magnesium
chloride. Arch. f. Entw.-Mech., Bd. 23.
1909 The development of artificially produced cyclopean fish, ‘‘The
magnesium embryo.” Jour. Exp. Zodél., vol. 6, pp. 285-338.
1910 a The influence of alcohol and other anaesthetics on embryonic
development. Am. Jour. Anat., vol. 10, pp. 369-392.
1910 b The independent origin and development of the crystalline
lens. Am. Jour. Anat., vol. 10, pp. 393-423.

Witper, H. H. 1908 The morphology of cosmobia. Am. Jour. Anat., vol. 8,
pp. 355-440.

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THE BREEDING ‘HABITS, MATURATION OF EGGS
AND OVULATION OF THE ALBINO RAT

W. B. KIRKHAM anp H. 8S. BURR
Sheffield Biological Laboratory, Yale University

EIGHTEEN FIGURES
INTRODUCTION

The present work was started by the senior author under the
supervision of Professor W. R. Coe in the spring 1907, and in
1908 Professor Coe published in Science a brief statement of
what had been found. In the summer of 1911 the junior mem-
ber, Mr. Burr, took up the work. In the interim the literature
of the subject had been enriched by three papers, and since then
two additional ones have appeared. Lantz in 1910 contributed
to a United States government report, on the economic impor-
tance of the rat, a short paper on the natural history of the
animal. The author describes the different species of rats, their
distribution, and general habits, but pays little attention to the
details of their reproduction.

Sobotta and Burckhard (’10) made a careful study of the
maturation and fertilization of the egg of the albino rat, and
they describe and figure the ovarian egg in the stages of the
first polar spindle, and first polar body with the second polar
spindle, and the tube egg in the stages of the second polar spin-
dle, fertilization, second polar body, and the pronuclei. Ovula-
tion is stated to occur independent of pairing within thirty-six
hours after the birth of a litter, and the eggs fertilized nine to
twelve hours after copulation. Sobotta and Burckhard found
the mature rat egg, in the ovary, to measure in preserved mate-
rial 0.06 to 0.065 mm. in diameter; practically the same as the

291

292 W. B. KIRKHAM AND H. S. BURR

mouse egg. These investigators never saw a definite first polar
body associated with an egg in the tube.

Newton Miller’s paper (’11) on reproduction in the brown rat
is based solely upon observations of the living animals. He
found that both sexes become sexually mature ‘‘at least by the
end of the fourth month,” that the litters contain from six to
nineteen young apiece, and that these animals breed the year
round.

Mark and Long (712) devote most of their contribution to an
extended description of the elaborate warm chamber they have
devised for the study of living mammalian eggs. When it comes
to the results obtained with their apparatus they have, at pres-
ent, little to say. Living eggs of rats and mice obtained in a
manner similar to that described by one of us (Kirkham ’07)
were placed on the stage of the microscope in the warm chamber
and spermatozoa added, the mouse eggs underwent no change,
but the rat eggs within five minutes to two hours began the
formation of the second polar cell. Cleavage has never been
observed, and after twelve hours the eggs begin to degenerate.

The latest contribution to the literature on the subject of
rat breeding is by Helen Dean King (’13) who records for the
albino rat somewhat the same phenomena previously observed
by Daniels (’10) in mice. The normal period of gestation for
the albino rat, according to Miss King, is twenty-one to twenty-
three days. If six or more young are being carried while a pre-
vious litter of five or less are still suckling the period of gesta-
tion may be prolonged, while if more than six young are suckling
the period is always prolonged, regardless of the number being
carried. Unlike the mouse, the albino rat appears not to exhibit
any exact relation between the number of young either suckling
or borne and the extent of prolongation of the gestation period.
This paper also contains evidence that the eggs of a given oestrus
cycle in the albino rat may be discharged from the ovaries in
two sets, with an interval of two to three days, and also that
in very rare instances this interval may be extended to two
weeks. Miss King would like to interpret the latter cases as
instances of a distinct oestrus cycle occurring during pregnancy.

BREEDING HABITS OF ALBINO RAT 293

The present paper has as its object the filling in, as far as
possible, of such stages as have not previously been described,
and the presentation of evidence regarding the time relations
in the development of individual eggs. The authors’ thanks
are due to Prof. W. R. Coe for the use of the notes and drawings
of the eggs of the brown rat, and to Dr. T. B. Osborn of the
Connecticut Agricultural Experiment Station for the designs of
the cages used and for the animals with which the work was
started.

BREEDING HABITS

About 150 albino rats were under observation at different
times during the investigation. One large cage was used for all
rats not at the time under special care. For individuals two
types of cages were employed, one, a cylindrical cage ‘of wire
netting of sufficient size to accommodate two rats at a time,
and the other a much larger, rectangular cage of galvanized iron,
with wire netting only on the front and bottom. This second
type of cage was designed primarily as a breeding cage and was
large enough: to house a mother rat and a litter of the largest
size until the latter were sexually mature.

The food of the animals consisted of oats, corn, wheat, sun-
flower seeds, and dog-biscuit, together with bits of lettuce, string
beans, bread, and various kinds of cooked meat and fish.

All cages were kept as clean as possible, but except when
absolutely necessary litters less than two weeks old were never
disturbed. At the times when we were inspecting them the rats
were encouraged to come out of their cages and run about the
room, and to this familiarity with us as well as to the additional
exercise thus secured we attribute much of our success in rear-
ing large litters without their being maimed or eaten by the
parents.

Usually females were isolated in breeding cages as soon as
they were seen to be pregnant, but in the few instances when
males were left with such females until several days after the
birth of the litter no mortality occurred. This fact leads us to

294 W. B. KIRKHAM AND H. S. BURR

agree with Miller (11) and King (13) that mother rats, unless
they are in an unhealthy condition, or have been frightened in
some way, rarely if ever kill or maim their young.

Albino rats give birth to young in all seasons of the year,
but it is only from April to October that ovulation as a rule
occurs within 48 hours after parturition; during the remaining
months they are apt to skip oestrus cycles, ovulation not occur-
ring until some three weeks after parturition.

The senior author showed in 1910 that the albino rat ovu-
lates regardless of whether pairing has previously taken place,
and when males are continuously present copulation may occur
before the ripest eggs in the ovaries have formed the first polar
spindles. On several different occasions we have seen the actual
pairing. It differs markedly from the condition described by
Sobotta (95) for the mouse, since the male albino rat is not
prostrated by the sexual act, but walks slowly away. When
a previously isolated female who is in heat is placed in a cage
with several males they will all pair with her in rapid succession.

The period of gestation in the albino rat is twenty-two days
when the female is not nursing a previous litter, in which event
the period may be lengthened as found by King (713). The
litters varied in number from four to twelve and the birth usu-
ally took place in the late afternoon or the early evening, although
probably it may occur at any hour of the day, since we have
observed it at noon. The process of parturition is briefly as
follows: The female in order to aid in the expulsion of the foetus
flattens herself against the bottom of the cage while a series of
wave-like muscular movements pass posteriorly along the body
starting just behind the shoulder. As soon as the young rat is
free from her body, the female rises up on her haunches, seizes
in her forepaws the button-like placentas; which is still attached
to the offspring by the umbilical cord, and devours first it and
then the cord, cutting off the latter as close to the body of the
young animal as she can get with her teeth. The female then
again flattens herself out against the bottom of the cage pre-
paratory to the appearance of the next young rat. The process
is repeated until all have been brought forth. Then, and not

BREEDING HABITS OF ALBINO RAT 295

before, does the mother assemble the young, cleaning them up
with her tongue, after which they lie close together under her
to keep warm. From this time on until the young are able to
crawl around by themselves the mother never leaves the nest
until she has carefully covered her litter. On returning she
always looks around for any that may have rolled or crawled
out in her absence, and such offenders are quickly seized in her
jaws and hauled back into the nest.

The albino rat becomes sexually mature, at least in some cases,
as early as fifty-five days after birth, since in one instance a
litter was born to rats that were only seventy-seven days old.

MATURATION AND OVULATION

The paper by Sobotta and Burekhard (’10) on the matura-
tion and fertilization of the albino rat is by far the most com-
plete account of the subject that has so far been published.
However this report left a number of things to be cleared up.

Working with material from 81 rats we have attempted to
investigate and make clear the following points: (1) the early
development of the egg previous to the formation of the first
polar spindle, (2) the formation of the first polar body, (3) the
condition of the egg at ovulation, (4) the process of fertilization
and second polar body formation.

At first the rats were watched and killed at short intervals
up to forty-eight hours after pairing. This gave no data that
could be depended upon for determining the stage which either
the ovarian or the tube eggs had reached. However, by relat-
ing the time of killing the female to the time of parturition it
was found that the approximate development of the egg could
be predicted without much difficulty. We say approximate be-
cause even though the exact hour of parturition be:known it is
impossible to say that at a given interval of time the eggs are
in @ given stage of development.

The parturition of a female caged with a male having been
observed, she was killed twenty-four hours later. This female
yielded unfertilized tube eggs, indicating that ovulation had re-

296 W. B. KIRKHAM AND H. S. BURR

cently occurred. Cases such as this show that ovulation usually
occurs about twenty-four hours after parturition. The individ-
ual variation is so great that any complicated apparatus for
determining the exact date of parturition is valueless. We have
obtained the best results by killing the females at half hour inter-
vals, beginning in the later afternoon and continuing through the
early evening. By doing this practically all the stages of matu-
ration can be obtained.

In a number of instances the senior author dissected out the
Fallopian tubes, and after placing them in warm salt solution,
by slitting the tubes he was enabled to obtain two eggs fertilized
but unsegmented, three eggs in the two cell stage and three
eggs so obscured by follicle cells as to prevent any exact infor-
mation as to their condition. The technique of this operation
is so simple, requiring only a binocular microscope, two needles,
some warm physiological salt solution, and a female rat that
has given birth to a litter at least twenty-four hours before and
not more than five days previously, that we recommend the rat
as highly as the mouse for obtaining live mammalian eggs for
class demonstration.

In all other instances the ovaries (and also the tubes, wher-
ever ovulation was thought to have occurred) were fixed in either
Zenker’s ‘fluid or in a strong solution of Flemming, imbedded
in paraffin, cut serially into sections 0.010 mm. thick and stained
in Delafield’s haematoxylin. Such sections as were found on
subsequent examination to be worthy of detailed study were ©
later decolorized with acid alcohol and restained with Heiden-
hain’s iron-haematoxylin.

A study of the ovaries of the above rats showed that there
is a progressive development of the egg until it is ready to leave
the ovary at ovulation. The developing eggs of any adult ovary
can be readily divided into six groups. The first of these (fig. 1)
includes all those eggs that are in the resting condition. These
vary considerably in size, as do also their follicles. The earlier
stages show a small egg with a follicle consisting of from one to
three layers of radially arranged follicle cells with scattered cells
lying between the layers, the later stages lie in larger follicles

BREEDING HABITS OF ALBINO RAT 297

with many more layers of cells. The egg nucleus presents a con-
stant appearance, a clearly defined nuclear membrane, scattered
chromatin and a deeply staining nucleolus.

The second group includes those eggs which differ from those
of Group I only in their size and in the fact that they lie in
much larger follicles, the latter consisting of a large number of
cells closely packed but showing no radial arrangement except
in the layer immediately surrounding the egg. Such an egg is
shown in figure 7. |

The third group includes a much smaller number of eggs which
lie in follicles similar to the preceding, except that the cells lying

Fig. 1 Normal resting follicle. X 630

in or near the center of the follicle show a marked tendency to
separate, leaving a clear space. This condition may, however,
be found in follicles belonging to eggs of Group II, for the factors
governing the growth of the follicle are not, according to our
observation, constant, since growth may set in when the egg has
reached the stage of development included in either Groups II,
III or IV (figs. 2,3 and 4). The nuclei of the eggs of this third
group show a marked change. The nuclear membrane is still
distinct, but the chromatin is less scattered and the nucleolus
has become partially vacuolated, since it shows much less affinity
for the stain. Figure 8 shows an egg of this group.

The fourth group shows further modifications. It is at this
point that the maximum growth in the size of the follicle takes

298 W. B. KIRKHAM AND H. S. BURR

Fig. 2 Earliest observed maturation phenomena—increase in size of follicle.
X 90.

Fig. 3 Follicle of egg with first polar spindle shown in figure 10 of Plate II.
X 90.

Fig. 4 Follicle of egg with first polar body and second polar spindle shown
in figure 11 of Plate III. x 90.

BREEDING HABITS OF ALBINO RAT 299

place. While growth may have started in either of the two
preceding groups, the greatest growth occurs with the egg in
this stage of development. Eggs of this group have been ob-
served with follicles similar to those of the two preceding groups,
and also with follicles of nearly the maximum size. The nuclei
of these eggs show a diminution in the amount of chromatin
present and a complete vacuolization of the nucleoli, the latter
showing no affinity whatever for the stain. Such an egg is shown
in figure 9.

The fifth group consists of the eggs with first polar spindles.
The follicles here are typical, showing a slight tendency to be
thinner in the region where the follicle is nearest to the surface
of the ovary. The nucleus of the egg has disappeared, and in
its place lies the first polar spindle (fig. 10). ‘

The sixth group shows no change in the size of the follicle.
The first polar body has been extruded and a second polar spindle
formed (fig. 11).

In all the above divisions, with the exception of the sixth group,
wherever a distinct zona radiata can be seen, very fine proto-
plasmic bridges can readily be distinguished crossing from the
follicle cells to the egg. The presence of these very distinct
filamentous processes of the follicle cells seems to have been
entirely overlooked by previous investigators.

One striking thing is to be noted with regard to the above
divisions—never were all six found together in one ovary at a
given time. As was to be expected, Group I, since it included
all the resting eggs, was present in all ovaries. Group II, on
the other hand, was seen to drop out on the appearance of Group
IV and to reappear on the disappearance of the latter. Group
V also appeared on the disappearance of Group IV. When
Group V dropped out, Group VI appeared. Group III was found
in all ovaries.

From the fact that perfectly normal eggs of Groups II and
III were found in the ovary just at, and also just subsequent
to ovulation, it was evident that more than one oestrus cycle
was necessary for the development of the egg from the resting
stage to the stage of the first polar body and second polar spindle,

300 W. B. KIRKHAM AND H. S. BURR

at which stage the egg leaves the ovary, for, if the above changes
occurred in one oestrus cycle, aii normal eggs in the above
condition would go out of the ovary at ovulation, leaving only
Group I eggs in the ovary. This condition was not seen. Hence
we were forced to find some other explanation of the facts.

Figure 5 shows in the form of a table the facts described above.
The vertical readings show the groups of eggs. The horizontal
readings show the periods into which the oestrus cycle is divided.
Period a is the division of the oestrus cycle extending from
ovulation to the twenty-first subsequent day and covers a period
of time in which there is little change in the personnel of the
ovary. Period b covers the succeeding six hours; period c, the
next six, and period d, the last six hours remaining before ovula-
_ tion. The above figures are only approximate, as the individual
variation is too great to permit of any exact data.

By studying the figure it will be seen that Group IV disap-
pears at periodc. At the sametime the ovary contains Groups I,
II, III and V,IV and VI being absent. During the interval be-
tween periods c and d Groups I, II and III remain unchanged,
but Group V disappears and Group VI appears.

After ovulation we find in period a, Groups I, II and III only.
But in period b, II disappears and IV appears.

From the above data we were led to believe that the develop-
ment of an egg follows the arrows in the diagram. That is,
that Group II comes from I in period c, remains unchanged
through d and a, becomes transformed into III during period
b, remains unchanged through c, d and a, grows to IV in b, to
V inc and to VI in d, and so out at ovulation.

The above explanation of the facts rests on the assumption
that the normal rate of development is approximately the same
for all eggs. This assumption we think is warranted, for if II
developed into IV during period b instead of remaining unchanged
until the next oestrus cycle, the number of Group III eggs
found should be very small, since the change would be a rapid
one. On the other hand, if the development involved a longer
period of time—that is, if Group III became a second resting
stage—one would expect to find a comparatively large number

301

BREEDING HABITS OF ALBINO RAT

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3

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No.

302 W. B. KIRKHAM AND H. S. BURR

TABLE 1
Showing the relative number of eggs in the various stages of development at different
- periods of the oestrus cycle, as found in individual ovaries
Bees ee ee i oa. a
OVARY NO. | I | ie], aot | LV; Vv | VI | DEGENERATING
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Periodsdancer sane | 152 Sel 4] | 8 0 0 | 9 42

of such eggs in an ovary at any given time. This, however, was
not the case, the number of Group III eggs found being rela-
tively close to the number of Group I eggs.

Table I is compiled from a count of all the follicles in six
ovaries, representing each of the four periods. It shows the
relative number of eggs in each group present at the same time
in a given ovary. The count can only be an approximation,
owing to the occasional loss of a critical section and the fre-
quent difficulty in determining with accuracy whether or not
an egg was normal, but is sufficiently exact for this purpose.

We were unable to obtain any stages that intervene between
the eggs of Group IV and those with the first polar spindle, so
we cannot say whether the nuclear membrance disappears before
or after the first appearance of the first polar spindle. With
regard to this spindle, however, there are a number of details
worthy of attention. It is short and broad, with well defined
fibers which do not come to a sharp focus (fig. 10). The possi-
bility of centrioles being present was mentioned by Coe (’08),
but these are apparently lacking in polar spindies of the albino
rat. The chromosomes are numerous, crowded, and never found
in a definite equatorial plate. Most of the first polar spindles
seen are parallel to the surface of the egg, and this appears to
be the position in which the spindle waits for the stimulus that
leads to the formation of the first polar body (fig. 6a). When
this stimulus comes the spindle rotates on its long axis, coming
to lie more or less radially (fig. 6b and 6 ¢).

BREEDING HABITS OF ALBINO RAT 303

The next stage we were able to obtain is shown in figure 11.
This is an ovarian egg with the first polar body and the second
polar spindle. As in the case of the mouse, the nuclear material
is never gathered into a resting stage between the time of extru-
sion of the first polar body and the formation of the second
polar spindle.

The first polar body is rarely seen in eggs outside of the ovary,
but there is absolutely no reason to doubt that it is always
formed, since it is almost invariably present beside normal ova-

Fig. 6. Reconstructions of three spindles showing gradual rotation from the
paratangential position (a), through (b), to the radial position (c).

rian eggs, possessing a second polar spindle. Even in the ovary,
however, its protoplasm displays its characteristic tendency to
undergo rapid disintegration. In such fully matured eggs as
have failed to escape from the ovary and are just starting to
degenerate, as well as in those about to be discharged, the second
polar spindle may be sharply defined, yet a careful search fails
to reveal a trace of the first polar body. The chromatin in the
first polar body is always scattered, and when first formed this

304 W. B. KIRKMAN AND H. 8S. BURR

polar body is, in all probability, always larger than the second,
though disintegration may set in immediately upon its formation.

The second polar spindle as seen in the ovary is much longer
and narrower than the first, but resembles the first polar spindle
in having open ends and no centrioles. The chromosomes in
the second polar spindle are almost always spherical.

TUBE EGGS

The living unsegmented egg of the albino rat measures about
0.079 mm. in diameter (the exact size varies a few thousandths
of a millimeter in different specimens), and is surrounded by a
zona of transparent jelly about 0.022 mm. in thickness. The
two unsegmented rat eggs that were obtained sufficiently free
from follicle cells to be available for detailed study, both pos-
sessed two polar bodies, measuring in one specimen 0.019 and
0.0132 mm. in diameter respectively, and in the other specimen
0.008 and 0.0065 mm. These eggs while translucent were filled
with highly refracting globules scattered through the protoplasm.
In one egg there was a clear area near the center, where we
thought we could distinguish the two pronuclei lying side by
side.

The rare occurrence of the first polar body associated with
the egg in the tube is to be attributed to its rapid disintegration,
which, as already stated, begins almost as soon as it is formed,
and may lead to its complete disappearance before ovulation
occurs. A stained and sectioned tube egg, accompanied by the
' first polar body, is shown in figure 12. This polar body is very
small, contains only a little stainable chromatin scattered through
it, and its protoplasm is much denser than that of the egg.

Until after fertilization, and if this fails to take place until it
degenerates, the chromatin of the second polar spindle remains
in a clearly defined equatorial plate, but in the egg in the Fallo-
pian tubes, this spindle always appears much longer and thinner
than in the ovarian eggs.

The rat spermatozoon has an exceedingly long tail (fig. 16 a),
and like that of the mouse carries more or less of its tail with it

BREEDING HABITS OF ALBINO RAT 305

when it enters the egg, a fact mentioned by Coe, and by Sobotta
and Burekhard. As soon as the sperm head begins to penetrate
the cytoplasm of the egg the formation of the second polar body
is started.

In the albino rat the second polar body is characterized by
having the chromatin content massed, while the chromatin of
the first polar body is always scattered through the cytoplasm.
This distinction, however, does not hold for the Norwegian rat,
of which two eggs are shown in figures 17 and 18. The chro-
matin left in the egg after the formation of the second ‘polar
body rounds itself up and becomes surrounded by a membrane,
thus forming the female pronucleus. The sperm head on its
entrance swells up and likewise assumes a rounded form with
a nuclear membrane, as is shown in figure 16.

SUMMARY

1. Male albino rats rarely, if ever, are responsible for the kill-
ing or maiming of their young. Diseased condition or fright
are probably the chief causes of the destruction or injury of their
offspring by the females.

2. Albino rats give birth to young the year round, but only
from April to October do the females regularly ovulate twenty
to forty-eight hours after parturition.

3. Albino rats of both sexes are sexually mature when less than
two months old.

4. Living rat eggs are easily obtainable during the four days
following ovulation by dissection of the Fallopian tubes.

5, The maturing eggs in the ovary are joined to the surround-
ing follicle cells by very definite cell bridges.

6. The development of eggs can be traced in the ovary through
two oestrus cycles preceding their discharge.

7. The first polar spindle is short and broad, and is usually
formed less than twenty-four hours after parturition.

8. The first polar body is always formed in rat eggs, but its
protoplasm is very unstable, and disintegrative processes often
bring about its complete disappearance about the time the egg
reaches the Fallopian tube.

306 W. B. KIRKMAN AND H. S. BURR

9. The second polar spindle is long and narrow. Its appear-
ance marks the end of maturation phenomena in the ovary,
and the termination of all development of the egg unless fertili-
zation occurs.

10. In albino rats the chromatin of the first polar body is
scattered, that of the second polar body is massed.

11. The very long middle piece of the sperm tail follows the
head into the cytoplasm of the egg.

June 1913
LITERATURE
Cor, W. R. 1908 The maturation of the egg of the rat. Science, N. S., vol.
27, no. 690.

DanieL, J. F. 1910 Observations on the period of gestation in white mice.
Jour. Exper. Zo6l., vol. 9.

Donaupson, H. H. 1912 The history and zoological position of the Albino rat.
Jour. Acad. Nat. Sci., Philadelphia, vol. 15, 2d series.

Kine, H. D. 1913 Some anomalies in the gestation of the albino rat (Mus
norvegicus albinus). Biol. Bull., vol. 24.

Lantz, D. E. 1910 Natural history of the rat. Treas. Dept., Pub. Health and

. Marine Hosp. Service U. 8., Washington.

Mark, E. L., anp Lona, J.A. 1912 Studies on early stages of development in
rats and mice. No. 3. Univ. California Pub. in Zool., vol. 9, no. 3.

Miuurr, N. 1911 Reproduction in the brown rat (Mus norvegicus). Amer.
Naturalist, vol. 45.

Sonotta, J. 1895 Die Befruchtung und Furchung des Eies der Maus. Arch.
f. Mikr. Anat., Bd. 45.

Sonotta, J., U. BurcKkHARD, G. 1910 Reifung und Befruchtung des Eies der
weissen Ratte. Anat. Hefte, Bd. 42.

PLATES

Figures 1 to 4 and 7 to 18 were drawn with the camera lucida. All figures,
except 17 and 18 were drawn with Zeiss no. 4 oc. and ;’; oil immersion obj. giv-
ing a magnification of 1000 diameters. Figures 17 and 18 were drawn with a no.
6 oc. and ;'; oil imm. obj., giving a magnification of 1760 diameters. These
figures are reduced one-third, giving a magnification in the finished plate of
1174. All other figures are reproduced at the size drawn. |

PLATE 1
EXPLANATION OF FIGURES

7 Shows an ovarian egg in the resting stage. Theegg (Group II) has attained
approximately its greatest diameter. Nucleolus solid and deeply staining. Pro-
toplasmic bridges well marked. A follicle cell is shown dividing atgthe left.

8 An ovarian egg (Group III) at a slightly later stage showing the vacuoli-
zation of the nucleolus well started.

PLATE 1

BREEDING HABITS OF ALBINO RAT

W. B. KIRKHAM AND H. S. BURR

PLATE 2
EXPLANATION OF FIGURES

9 A later stage than fig. 8, showing the complete vacuolization of the nucle-
olus (Group IV). ;
10 A radial first polar spindle (Group V) showing the blunt ends.

308

BREEDING HABITS OF ALBINO RAT ~ PLATE 2
W. 8B. KIRKHAM AMD H.S. BURR

PLATE 3
EXPLANATION OF FIGURES

11 An ovarian egg (Group VI) showing the first polar body with a spindle,
and the early type of short, thick second polar spindle within the egg. The
protoplasmic bridges have at this stage disappeared.

12 A tube egg, showing the first polar body at the right, and the long slender
type of second polar spindle at the lower left-hand margin of the egg.

310

3

PLATE

BREEDING HABITS OF ALBINO RAT

W.B. KIRKHAM AND H.S. BURR

PLATE 4
EXPLANATION OF FIGURES

13. A tube egg, showing the second polar body in the process of formation.
Thé enveloping follicle cells still retain their continuity.

14 A tube egg, showing the first polar body at the top, the second polar body
in the process of formation at the lower left-hand margin of the egg, and the
sperm head at the right, with a portion of the tail.

312

BREEDING HABITS OF ALBINO RAT PLATE 4
W. 3B. KIRKHAM AND H. 8. BURR

PLATE 5
EXPLANATION OF FIGURES

15 A tube egg, showing the second polar body at the left and the sperm head

at the right.

16 A series of drawings showing the changes in the sperm head: a, a sperma-
tozoon in the tube. The head was drawn from a stained section—the tail was
added from data of a living spermatozoon, b to g, various stages in the transfor-

mation of the sperm head within the egg.

314

BREEDING HABITS OF ALBINO RAT PLATE 5
W.B. KIRKHAM AND H.S. BURR

315

PLATE 6
EXPLANATION OF FIGURES

17 A tube egg of a brown rat, showing the second polar body at the top, below
the deeply staining female pronucleus, the entrance cone to the right, the male
pronucleus with the sperm aster in the lower left-hand margin and the sperm
tail extending diagonally through the egg.

18 A somewhat later stage of the egg of the brown rat, showing in addition
to the above-mentioned points the nucleolus vacuolated in the female pronucleus.

Figures 17 and 18 were drawn by Dr. W. R. Coe, and are here published with

his kind permission.

316

BREEDING HABITS OF ALBINO RAT PLATE 6
W. B. KIRKHAM AND H.S. BURR

317

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 3

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A HUMAN EMBRYO OF THIRTEEN SOMITES

IVAN E. WALLIN
Anatomical Laboratory, Bellevwe Hospital Medical College, New York City

SEVEN FIGURES

The embryo which forms the basis of this work was given to
me by Dr. Rudolph Boencke in the spring of 1911.- It has been
placed in the collection of the Department of Anatomy at the
University and Bellevue Hospital Medical College and is called
embryo no. 4.

The embryo was aborted two weeks after the last menstrual
period. There was no record of coitus. After fixation and with
the amnion intact the embryo measured 2.3 mm. in length. It
was cut into transverse sections 5u in thickness, and stained
with iron haematoxylin. The embryo yielded 287 sections.

Wax plate reconstructions were made of the complete embryo,
the heart, the foregut, also of the caudal part of the medullary
tube with the hind-gut and the belly stalk vessels. A graphic
reconstruction was made representing the embryo cut in the
mid-sagittal plane. All the reconstructions were made at a mag-
nification of 200.

The embryo appears to be normal in every respect and the
following points of structure have been determined.

EXTERNAL FORM

In its general configuration this embryo is very similar to
Pfannenstiel III described by Low (’08). The body has a regu-
lar dorso-ventral curve and has a slight twist so that the head is
situated to the right of the mid-sagittal plane. The yolk sac
communicates with the primitive gut by means of an extensive
yolk stalk. The latter has its greatest diameter in the cephalo-

319

S20 IVAN E. WALLIN

caudal direction and its lateral width is greatest at the cephalic
end. Caudal and to the right of the yolk stalk the belly stalk
leaves the embryo passing ventrally and curving to the right
and caudad. Lateral to the yolk stalk the embryonic coelom
has an extensive communication with the extra-embryonic coelom.

The heart produces a prominent bulging of the right side of
the body immediately caudad to the head. The most prominent -
part of the bulging marks the flexure in the heart tube between
the bulbus cordis and the ventricle. The neck flexure has not
advanced to any prominent degree. ‘There are two prominences
on the dorsal surface of the head region, one at the cephalic end
of the mid-brain and the other at the cephalic end of the hind-
brain. Caudally the body curves gradually in a ventral direc-
tion. There is no distinct caudal flexure.

The medullary tube is open to the exterior at both ends. The
cephalic neuropore exhibits an unusual appearance for an embryo
of this age. It is very wide and gives a great breadth to the
head when viewed from the ventral aspect. The lateral lips of
this neuropore curve dorsally and form the ventral boundary of
a deep groove which is directed cephalo-caudally. The caudal
end of this groove runs into the stomodeum. ‘This part of the
nervous system which represents the forebrain has not kept
apace with the development of the remainder of the tube. It
apparently is a persistence of the condition which is present in
an earlier stage of development. Eternod’s (95) embryo of
eight somites and the embryo of seven somites described by
Dandy (’10) exhibit cephalic neuropores which appear to be in
about the same stage of development.

There are no indications of otic invaginations. Two pairs of
entodermal pouches are in contact with the ectoderm. The
points of contact are indicated on the surface by shallow depres-
sions. In figure 2 their positions have been indicated on the sur-
face by broken lines.

The amnion lies close on to the body of the embryo. The
head fold crosses the ventral aspect of the heart at about its
middle. The lateral folds follow the lateral lips of the coelom.
The tail fold is situated on the dorsal aspect of the belly stalk.

HUMAN EMBRYO OF THIRTEEN SOMITES ay

NERVOUS SYSTEM

The nervous system has not proceeded very far in its differ-
entiation. The brain flexures do not agree with the His models
of this stage, but correspond more to the older embryos described
by Thompson (’07) and van den Broek (’11). The most distal
portion representing the forebrain is still open and is bent almost
at right angles to the mid-brain. The long axis of the fore-
brain hes in a cephalo-caudal plane and almost parallel with the
long axis of the hind-brain. The most cephalic point of the
nervous system is thus represented by the junction of the fore-
brain and the mid-brain. Near the caudal extremity of the fore-
brain there is a thickening together with an evagination of the
brain ectoderm. This evagination is almost in contact with the
ectoderm of the stomodeum and undoubtedly represents the in-
fundibulum. Cephalad to the infundibulum and about in the
middle of the lateral expansions of the cephalic neuropore there
is a slight depression of the ectoderm on each side which repre-
sents the beginning of the optic vesicles.

The mid-brain is quite extensive as is apparent from an exami-
nation of figure 3. Its floor is smooth and exhibits a thickening
at the cephalic end. Caudally there is a flexure of the floor
between the mid-brain and the hind-brain. The floor of the
mesencephalon is thickened at its cephalic end. The trigeminal
ganglion is present as a distinct mass of cells. Its position is
represented in figure 3 by a broken circle. The hind-brain passes
gradually into the spinal cord. A distinct neck flexure is not
present.

The medullary tube has its greatest diameter at the cephalic
extremity. It diminishes gradually in size caudally. At the
caudal neuropore it exhibits a slight enlargement.

DIGESTIVE SYSTEM
The stomodeum is a broad and deep invagination of the ecto-
derm between the heart bulging and the head. It touches the
entoderm of the pharynx and forms with it the beginning of an

oral plate. There is no indication of an hypophysis. The ecto-
derm lining the stomodeum is thickened especially in the roof.

S22 IVAN E. WALLIN

The cephalic extremity of the pharynx projects beyond the oral
plate and nearly reaches the floor of the forebrain, a small
amount of mesoderm intervening.

The median thyreoid anlage is a very prominent evagination
of the entoderm of the floor of the pharynx. It projects between
the layers of splanchnic mesoderm at the arterial end of the
heart immediately caudad to the endothelial aorta and the first
aortic arches. The cephalic wall of the evagination is consider-
ably thicker than the caudal. Cephalad to the thyreoid anlage
the first branchial pouches are evaginated from the lateral wall
of the pharynx and immediately caudad to the thyreoid the
second pair of pouches are present. The first pair of pouches
are the larger. Their long axes are directed laterally, cephalad.
and slightly dorsal. Opposite the venous opening of the heart
the liver anlage is present as a thickening of the gut entoderm.
Lung buds have not developed in this stage.

A cross section of the foregut has a crescentic outline with the
coneavity directed dorsally. The tube is widest at the point
where the first pair of branchial pouches is developed. The
cephalic part of the foregut is flattened dorso-ventrally. Cau-
dally the dorso-ventral diameter increases gradually to the
end of the foregut where it becomes greater than the lateral
diamter.

The gut entoderm extending out into the yolk stalk retains
its thickness only a short distance (fig. 3).

The hind-gut is shorter than the foregut. Its dorso-ventral
diameter is comparatively large while its lateral diameter is small.
The allantois is evaginated from the ventral wall. The lumen
of the diverticulum is very small at its proximal end, but through-
out the rest of its extent it is distinct. At first the allantois
lies between the allantoic arteries. At its distal end it comes
to lie between the venous and arterial trunks or sinuses of the
belly stalk. The end of the allantois is not recurved as found
by Lewis (12) but ends as a straight tube. The hind-gut ex-
hibits a dorso-ventral constriction immediately cephalad to the
allantoic diverticulum. Caudal to the allantois the hind-gut wid-
ens out to form the cloaca. The entoderm of the ventral wall

HUMAN EMBRYO OF THIRTEEN SOMITES B25

Fig. 1 Wax plate reconstruction of complete embryo seen from left side.
The broken lines indicate the points where the entodermal pouches touch the
ectoderm. 100.

324 IVAN E. WALLIN

Fig. 2. Wax plate reconstruction of complete embryo seen from the ventral
aspect. > 100.

HUMAN EMBRYO OF THIRTEEN SOMITES BAD)

Fig. 3. Graphic reconstruction representing the embryo cut in the mid-sagit-
tal plane. The broken circle above the letter H represents the position of the
trigeminal ganglion. > 100.

A, Atrium Cl.M, Cloacal membrane L, Liver anlage
Al., Allantois F, Forebrain M, Mid-brain
Al.A, Allantoic artery FG, Foregut P, Pharynx
Al.V, Allantoic vein H, Hind-brain Th, Thyreoid

BC, Bulbus cordis HG, Hind-gut V, Ventricle
: I, Infundibulum

7

Fig. 4 Wax plate reconstruction of caudal end of the medullary tube and
hind-gut with the belly stalk vessels viewed from the side. X 100.

Fig.5 Wax plate reconstruction of a section of the heart with the endothelial
tube in position viewed from the cephalic aspect. X 100.

Fig. 6 Wax plate reconstruction of the heart viewed from the cephalic aspect.
< 100.

Fig. 7 Wax plate reconstruction of the heart viewed from the caudal aspect.
x 100.

326

HUMAN EMBRYO OF THIRTEEN SOMITES one

of the cloaca is fused with the body ectoderm and forms a thick
cloacal membrane. At the most caudal part of the cloaca there
is a thickening of the entoderm together with a slight evagina-
tion which is suggestive of a post anal gut.

NOTOCHORD

The notochord is about in the same stage of development as
the one described in a 2.5 mm. embryo by Kollmann (’90). The
notochord is intimately connected with the gut entoderm through-
out its length with the exception of the caudal end. The caudal
end, or tail bud, is cut off from the entoderm and lies imbedded
in the mesoderm between the neural ectoderm and the gut tube.
There is no distinct notochordal canal as described by Mall (’91),
Eternod (’99) and Grosser (’13). In places the cells of the noto-
chord are vacuolated and apparently in a stage of developing
acanal. The relationship of the notochord to the gut entoderm
is a very intimate one. In the region of the mid-gut the noto-
chord is composed of but a single layer of cells which appear to
be a modified part of the gut entoderm. Where the notochord
is composed of more than a single layer of cells the basal layer
is directly continuous with the single layer of cells forming the
gut entoderm. It is impossible to give any other interpretation
than that the notochord is developed from the gut entoderm.
In places the cells of the notochord are arranged in two lateral
masses giving the appearance of bilateral symmetry. - This con-
dition is undoubtedly accounted for by the arched nature of the
original notochordal plate. In the subsequent proliferation of
cells they would grow laterally and unless there were an espe-
cially active growth of cells in the central part a gap would nat-
urally intervene between the two lateral groups of cells. At the
cephalic end the notochord has more the appearance of a rod
and is almost pinched off from the entoderm. On account of
the plane of the sections it is not possible to determine with
certainty the cephalic limit of the notochord.

328 IVAN E. WALLIN

MESODERMAL STRUCTURE

There are thirteen pairs of mesodermal somites. These are
hardly discernible on the surface. The first pair is situated at
a level of about midway between the neck flexure and the hind-
brain flexure. The last pair is opposite the point where the
allantois leaves the hind-gut. A myocoele may be observed in
most of the somites. The cells of the somite are arranged in
a radial manner.

The pleuro-peritoneal coelom communicates with the extra-
embryonic coelom on the two sides of the yolk stalk. In its
cephalic portion it communicates with the pericardial coelom.
The lateral lips bounding the open part of the pleuro-peritoneal
ecoelom have a thickened edge produced by the allantoic veins
which run cephalad in this position. ’

The septum transversum is present as a single layer composed
of the cephalic wall of the yolk stalk fused with the caudal part
of the pericardium.

The excretory system is represented by pronephric tubules.
The morphological details of these, as far as I have studied them,
agree with the description given by Felix (12).

VASCULAR SYSTEM

The heart tube is composed of three parts, bulbus cordis, ven-
tricle and atrium. The atrium is situated in the mid-line of
the body immediately cephalad to the septum transversum. Its
ereatest diameter is transverse. From the left extremity of the
atrium the atrial canal runs to the left, ventral and cephalad
to the ventricle. The ventricle pursues a course from the left
to right, ventrally and somewhat caudad. At the right extremity
of the ventricle the heart tube makes a sharp bend so that its
continuation, the bulbus cordis, comes to lie parallel with the
ventricle. The bulbus cordis has a fairly constant size up to
its cephalic end where it diminishes slightly. It ends in the
mid-line of the body.

The ventral wall of the cephalic end of the bulbus cordis is
continuous with the pericardium as is also the case with the

HUMAN EMBRYO OF THIRTEEN SOMITES 329

caudal wall of the venous end of the heart (figs. 3, 6, 7). The
dorsal wall of the bulbus cordis has a distinct mesentery con-
necting it with the dorsal pericardium. Near the point where
the atrial canal joins the ventricle the ventricle has a mesentery
which joins the pericardium at the place where the mesentery
of the bulbus cordis joins it. Caudal to the junction of these
two mesenteries there is a small space dorsal to the atrium which
is free from mesentery and represents the future transverse sinus
of the pericardium.

From the dorsal wall of the bulbus cordis a tube-like diver-
ticulum is present (fig. 5). I have been unable to find any ref-
erences in literature to anything similar to this. The tube runs
in the mesentery of the bulbus cordis and at its distal end it
comes into close proximity to the ventricle. It is probable that
this tube represents a vestige of the space between the two
laminae in the closing up of the heart tube and the formation of
the mesocardium. Two other tubular spaces of a similar appear-
ance may be seen in the mesentery. They have no communi-
cation with the cavity of the myo-epicardium. I observed a
similar diverticulum from the bulbus cordis in a 4.06 mm. embryo
belonging to the collection of the Department of Anatomy of
Syracuse University. It may be noted that this bulbus cordis
diverticulum does not contain any endothelium. The endothe-
lial fibrillae, however, appear to extend into it.

The endothelium in no place approximates the walls of the
myo-epicardium. The caliber of the endothelial tube varies in
the different chambers of the heart, being quite constant in the
bulbus cordis, enlarged in the ventricle, and greatly reduced in
the atrial canal. In the atrium it widens out into the right and
left lateral expansions of the atrium. At its cephalic end the
endocardium is continued by the ventral aorta which immedi-
ately divides to form the first pair of aortic arches. At the
venous end of the heart the most distal part of the endocardium
represents the sinus venosus. There is no constriction between
the sinus venosus and the atrial part of the endocardium. The
endothelial fibrillae which have been observed by various authors

330 IVAN E. WALLIN
se

and to which Mall (712) ascribes the source of the intima may
be seen in connection with the endocardium in its entire length.

The blood vessels are collapsed in places so that it is not pos-
sible to trace them in their entire extent. The communication
between the first pair of aortic arches and the dorsal aortae
could not be seen. The dorsal aortae are distinct throughout
their course lying dorsal to the gut tube. There is no indication
of a second pair of aortic arches. The first pair come off at a
point cephalad to the first mesodermal somites. Vitelline ves-
sels containing blood are easily discernible in the wall of the
yolk sac and yolk stalk. Vitelline veins run dorsally in the
cephalic part of the yolk stalk to gain the caudo-ventral aspect
of the sinus venosus opposite the fourth pair of somites. The
allantoic veins (fig. 4) begin in the belly stalk as a single trunk
or sinus. As the sinus approaches the body of the embryo it
bifureates to. form the two allantoic veins which diverge and
run laterally and cephalad to gain the lateral lips of the coelom.
In this position they run in a cephalad direction to the septum
transversum where they enter the caudo-dorsal part of the sinus
venosus. The allantoic arteries leave the dorsal aortae at a point
opposite the place where the allantois is evaginated from the
hind-gut and caudal to the last pair of somites. The arteries
run ventrally on either side of the allantois in the belly stalk.
At a point more distal than the bifurcation of the allantoic ve-
nous trunk the allantoic arteries anastomose to form a single
trunk. I have been unable to find any trace of the anterior and
posterior cardinal veins. At the cephalo-dorsal aspect of the
sinus venosus on the left side there is a short bud-like diverticu-
lum which may represent the future ductus Cuvieri.

I wish to take this opportunity to thank Dr. Boencke for this
valuable embryo and Profs. H. D. Senior and F. W. Thyng for
assistance and advice in connection with this piece of work.

HUMAN EMBRYO OF THIRTEEN SOMITES Soi

BIBLIOGRAPHY

VAN DER Broek, A. J.P. 1911 Zur Kasuistik Junger Menschlichen Embryonen.
Anatomische Hefte, 44.2, 275-302.

Danpy, W. 1910 A human embryo with seven pairs of somites. Amer. Jour.
of Anat., 10, 85-109.

Eternop, A. C. F. 1895 Communication sur un oeuf humain avec embryon
excessivement jeune. Arch. Ital. de Biologie, 22

1899 Il y a un canal notochordal dans V’embryon humain. Anat.
Anz., 16, 131-148.

Perix, W.- 1912 The development of the urinogenital organs. Keibel and Mall.
Human embryology, Philadelphia.

Grosser, O. 1913 Ein Menschlicher Embryo mit Chordakanal. Anat. Hefte.
47, 653-686.

His, W. 1880 Anatomie Menschliche Embryonen, Leipzig.

KoLuMANN, J. 1890 Die Entwickelung der Chorda dorsalis bei dem Menschen.
Anat. Anz., 5, 308-321.

Lewis, F. T. 1912 The early development of the entodermal tract and the
formation of its subdivisions. Keibel and Mall, Human Embryology,
Philadelphia.

Low, ALEXANDER 1908 Description of a human embryo of 13-14 mesodermal
somites. Jour. of Anat. and Physiol., 42, 237-251.

Matt, F. P. 1891 A human embryo 26 days old. Journ. of Morph., 5, 459-480.
1912 On the development of the human heart. Amer. Jour. of Anat.,
13, 249-298.

Tuompson, P. 1907 Description of a human embryo of twenty-three paired
somites. Jour. of Anat. and Physiol., 41, 159-171.

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aE NERVE SUPP TO. THE PITUITARY BODY

WALTER E. DANDY

Hunterian Laboratory of Experimental Medicine
The Johns Hopkins University

THREE FIGURES

It is but natural that neglect of an organ itself should yield
a proportional lack of interest in its more detailed structure
and even more so, in its less important adjuncts—the blood and
nerve supply. Such has been true of the pituitary body.

The recent tremendous stimulus produced by Paulesco’s (1)
sudden transformation of the hypophysis from a structure of
vestigial curiosity to a vitally essential organ, has borne its fruit
in the rapid accumulation of co-working histological, (2) experi-
mental (3) (4) (5) (6) and clinical (7) (8) observations. Though
still very meager our information is now sufficient to have estab- .
lished a hypophyseal clinical entity, amenable in many cases to
medical and surgical treatment.

Forming as it does a link in the chain of internal secreting
glands, the hypophysis, essentially of hormone action, must be
regulated as other glands in this system, by an autonomic nerv-
ous mechanism.

Recent studies from the Hunterian Laboratory (5) by Goetsch,
Cushing and Jacobson gave evidences of hypophyseal influence
over carbohydrate metabolism. It has been shown that sugar
tolerance is dependent upon the functional activity of the pos-
terior lobe of the pituitary body. It was later shown by Dandy
and Fitz Simmons (observations unpublished) that a piqtre of
_ the hypophyseal region in rabbits produced a heavy glycosuria,
therefore giving results similar to a piqtre of the so-called Ber-
nard’s sugar center in the floor of the fourth ventricle. These
results have been amplified by Weed, Cushing and Jacobson (6).

333

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 3

334 WALTER E. DANDY

The combination of glandular or hormone activity and the
results of mechanical stimuli (presumably of nervous origin) has
suggested the possibility of a neuro-hypophyseal sugar center.

The rational interpretation of this and other physiological data
has been handicapped by the uncertainty and meager evidence
of the regulatory autonomic nervous mechanism. Accordingly
at the suggestion of Dr. Cushing under whose direction the ex-
perimental hypophyseal investigations have been conducted, the
determination of the source and distribution of the nerve supply
was undertaken.

Lying as does the hypophysis in such close proximity to the
earotid arteries with their abundant superimposed plexus of sym-
pathetic nerve fibers, it is but natural to assume that this is the
source of the hypophyseal nerve supply. Indeed evidence of
this is found in the infrequent passing reference to a nerve fila-
ment which could be traced from this plexus to the hypophysis.

BKARLY REFERENCE TO THE NERVE SUPPLY

Probably the earliest reference to a hypophyseal nerve supply
is the casual mention by Bourgery (45) that he observed sym-
‘ pathetic nerve fibers passing to the pituitary body. Further
substantiation is subsequently given in similar casual mention
by Fontona, Cloquet, Bock, Ribbes, (9) and possibly others.

In his Anatomie des Menschen (’79) Henle (9) devotes a para-
graph to the hypophyseal nerve supply and supplements this
description by a drawing of the carotid sympathetic system,
which includes a cluster of two or three twigs running from each
plexus to the pituitary body. This is the most extensive descrip-
tion of the hypophyseal nerve supply extant. He casts doubt
upon the previous discovery of nerve fibers to this gland and
concludes that on account of the inherent difficulties they have
mistaken fibrous filaments of connective tissue for nerve fila-
ments, saying, ‘‘Ohne Zweifel beruhen diese und manche Altere
Angaben auf Verwechselung fibréser Bailkchen mit Nervenfasern,
doch zeigte mir das Mikroskop in dem Netzférmigen zwischen
Carotis und Hypophyse ausgespannten Gewebe feine Nerven-

NERVE SUPPLY TO PITUITARY GLAND ood

faserbiindelchen dieselben, von denen Luschka sagt, dass sie
zwei bis drei jederseits, in den vorderen Lappen der Hypophyse
sich einsenken.”’ It is based upon this paragraph and drawing
by Henle that an occasional brief mention of hypophyseal nerve
supply is found in the more detailed and comprehensive anatom-
ies, the majority, however, passing over the matter in silence.

The internal distribution of the hypophyseal nerves was stud-
ied by Berkley (’94) (10) in a series of Golgi stained sections.
He observed numerous varicose nerve filaments in the interior
of the gland, the lobus anterior and pars intermedia in particular,
but some also in the posterior lobe. The external connections
of the nerves were not studied. On account of his inability to
observe nerve cells in the gland, he presumed they were of ex-
traneous origin and thought they probably come from the sym-
pathetic system.

MATERIAL AND METHODS

The purpose of this paper is to consider only the relatively
grosser aspects, i.e., the origin, course and distribution of the
hypophyseal nerve supply. The histological distribution and
relation of the ultimate filaments to the gland cells have not been
considered. It is analogous in character to a recent publication
(11) dealing with the blood supply of this organ.

The difficulties of deductions and the impossibility of an accu-
rate conception of the nerve supply based upon gross human
dissection have been shown (Henle) (9) by the supposedly erro-
neous observations of early investigators in mistaking connective
tissue trabeculae for the very delicate nerve filaments, which are
almost beyond the range of naked vision. These observations are _
based upon the canine and feline gland, the animals used in the
experimental investigations in the Hunterian Laboratory. The
anatomical environment of the pituitary body in these forms is
such that the difficulties of a tightly enclosed, deeply imbedded
and adherent gland encountered in man and the ape are ob-
viated. The hypophysis dangles from the brain and is readily
removed with the brain after liberation of its single point of dural

336 WALTER E. DANDY

attachment posteriorly, so that the entering nerves may be
studied in their true relations, without tearing or distortion.

We have used almost exclusively the specific methylene blue
intra vitam method of staining the nerves. For the details of
this technique we are greatly indebted to the excellent contri-
bution by J. Gordon Wilson (12). Three essentials are necessary
for the successful use of this stain: the exsanguination of the
tissues must be thorough in order to get a sharply defined picture
of the nerves, since the combination of the methylene blue with
blood presents a diffuse, indistinct picture with poorly stained
nerves; the nerves must be superficial or covered only by a thin
layer of tissue: the air must come in contact with the nerves,
otherwise no differentiation takes place.

During the final stages of bleeding the anaesthetized animal
from the femoral arteries, a 35 per cent isotonic solution of
methylene blue ‘‘nach Ehrlich” at body temperature was injected
into both carotid arteries and continued until the injecting fluid
emanated perfectly clear from the femorals. A tourniquet was
then applied around the neck below the point of injection under
a pressure sufficiently low to insure filling of the cephalic vessels
without danger of diffusion or rupture.

On account of the capricious character of this stain, litters of
very young puppies or kittens were injected at the same sitting,
so that the defects of some might be supplemented by better
staining of others. The total nerve supply then is a summation
of results, a reconstruction as it were.

After a few minutes to allow penetration of the stain, the
skull was opened and a block of tissue, including the hypophysis
with its vessels and nerves in their normal relations, was removed
from the base of the brain. The hypophysis was gently retracted
so as to allow full exposure of one side to the air. The nerves
then assume their differential blue. These specimens were imme-
diately studied under the binocular microscope. The study of
fixed specimens with post mortem staining was far less satis-
factory, because of the collapse of blood vessels, with which the
nerves are intimately associated, the more stiffened picture, and
the deficient maintenance of the blue in the nerve fibers.

NERVE SUPPLY TO PITUITARY GLAND Sl

NERVES TO THE ANTERIOR LOBE

The key to the nerve supply of the pituitary body is the ar-
terial supply to this organ. In a recent publication from this
laboratory, it was shown (11) that the anterior lobe received an
extensive blood supply from a large number of minute vessels,
most of which, even when injected, were beyond the range of
naked vision. These vessels radiate from the Willisian circle to
the hypophyseal stalk like spokes to the hub of a wheel. The
majority of these branches are from the anterior and posterior
communicating arteries. The network of sympathetic nerves
comprising the carotid plexus is continuous along the three main
branches which result from its trifureation. The distribution,
however, is very uneven. A few fibers‘continue along the ante-
rior and middle cerebral arteries for a short distance but the great
majority are found on the two communicating arteries which
supply the hypophysis; the posterior communicating artery is
particulary well supplied. From these extensions of the carotid
plexus numerous filaments are given off and pass along the
blood vessels to the stalk of the hypophysis, from which they
delve into the substance of the anterior lobe and are lost to
view. Some arterial branches have as many as three or even
four small filaments, the majority, however, only one or two.
The course of the fibers is fairly direct and very few branches
are given off. These filaments frequently entwine the vessels
but no minute plexuses or anastomoses are visible after leaving
the plexus on the main trunks. No nerves have been observed
on the external surface of the anterior lobe. All nerves going
to the hypophysis are in contact with the sheaths of minute
blood vessels. On reaching the stalk it is of course impossible
to trace this relation further. Their distribution in the gland has
not been observed.

338 WALTER E. DANDY

NERVES OF THE PARS INTERMEDIA

Only by dissection of the hypophysis can the nerve supply of
the pars intermedia be traced. By gently separating and retract-
ing the posterior lobe from the clasping mitten-like anterior lobe,
it is often possible to trace a single nerve fiber with its branches
passing down the stalk and spreading out over the pars inter-
media which envelops the posterior lobe (fig. 3).

NERVES OF THE POSTERIOR LOBE

It has been shown that the posterior lobe is supplied by a
median artery which is formed by the confluence of two branches,
one from each carotid artery immediately after its entrance into
the cavernous sinus. In the canine this vessel enters the pos-
terior lobe at the only point of dural attachment. Vital nerve
staining is somewhat more difficult in this region on account of
the relatively thicker dural covering which excludes the action
of the air and necessitates a delicate dissection of this vessel.
For a long time we were unable to find any trace of a nerve
entering the posterior lobe. Several branches were always visi-
ble at the origin of the vessels from the carotid but the fibers
were lost in the dura before the posterior lobe was reached.

However, it was finally possible to demonstrate nerve fibers
actually entering the posterior lobe along the artery. Certainly
the disparity between the nerve supply to the posterior and
anterior lobes is most striking—in the anterior lobe almost super-
abundant, in the posterior lobe very few. This contrast may
in some measure be due to the difficulties mentioned above; we
are however disinclined to lay much emphasis on them.

A most striking color contrast is demonstrated upon removing
the hypophysis after vital staining. The anterior lobe is a yel-
lowish white, the posterior a deep indigo blue, possibly due to
the (autogenic?) nervous character of the posterior lobe. The
blue is of a homogeneous character, no nerve fibers being differ-
entiable under the higher magnifications of the binocular micro-
scope. The intensity of the blue is even much more marked
than that of the adjacent, deeply staining oculomotor nerve.

NERVE SUPPLY TO PITUITARY GLAND 339

Fig. 1 Semi-diagrammatie representation of one side of the cavernous sym-
pathetic system of a canine, showing the nerves passing to the posterior lobe
along its artery. Other branches to the dura and a cluster (No) to the N. oculo-
motorius. The hypophyseal region is viewed from below with dura intact.

NERVES OF THE PARAHYPOPHYSIS

This little ‘nubbin’ resting in a small depression in the floor
of the sella, usually enclosed in dura, is present in over 80 per
cent of canines, and is evidently a remnant of the embryonic
Rathke’s pouch. In some adults it may be traced to the pars

340 . WALTER E. DANDY

Fig. 2 Semi-diagrammatic reconstruction of sympathetic nerves passing along’
the arterioles to the stalk of the hypophysis to supply the anterior lobe and
pars intermedia. Note relative dwindling of nerves away from the hypophyseal
region. ‘The view is from below with dura, hypophysis and carotid artery re-
moved.

intermedia; it varies greatly in size and histological character.
It has an individual blood supply, a small artery given off by
each posterior lobe artery. Frequently it has been possible to
trace a nerve some distance along this vessel toward this ‘‘body”’
but never have we been able to observe a definite nerve con-
nection.

NERVE SUPPLY TO PITUITARY GLAND 341

Fig. 3 Drawing to show the nerve passing from the plexus surrounding the
posterior communicating artery, down the stalk of the hypophysis to the anterior
lobe and the pars intermedia which covers the posterior lobe. The anterior
lobe has been dissected from the posterior lobe and gently retracted to permit
this view.

OTHER BRANCHES OF THE CAROTID PLEXUS

During observations on the hypophyseal nerve supply natu-
rally the distribution of the sympathetic filaments were noted
in the immediate vicinity. The dura of the sella region is excep-
tionally well supplied with filaments from the carotid plexus.
Several branches run from the carotid plexus direct to the oculo-
motor nerve. A couple of twigs were also observed entering the

342 WALTER E. DANDY

optic nerve; these branches were from the nerves in the adventitia
of the anterior cerebral artery. There is thus afforded a direct
nervous autonomic path between the optic and oculomotor nerves
and between these and the sympathetic trunk.

SUMMARY

The nerve supply to the pituitary body is from the carotid
plexus of the sympathetic system. Numerous branches radiate
to the stalk along the hypophyseal vessels and are immediately
lost to view in the substance of the anterior lobe.

The posterior lobe nerve supply is very scant, in marked con-
trast to the extensive innervation of the anterior lobe.

The pars intermedia receives its nerves from the stalk.

There is connection between the carotid sympathetic system
and the oculomotor and optic nerves.

The absolute differentiation between secretory and vasomotor
nerves is of course a matter of much dispute and is impossible.
The impression, however, from the character and course of the
nerve fibers their greatly increased number in the region of the
hypophysis, and their disappearance at a distance from the hy-
pophysis, the differences between the supply of the anterior and
posterior lobes, the connections established with the other cra-
nial nerves, leads us to regard them as secretory, in contradis-
tinction to vasomotor, the existence of which in the cranial
chamber has not been observed.

It is a pleasure to express my gratitude to Dr. Harvey Cushing
for his suggestions during the progress of this problem.

BIBLIOGRAPHY

(1) Pautesco, N. C. 1908 L’hypophyse du cerveau. Paris, Vigot Fréres.

(2) Herrine, P.T. 1908 The histological appearance of the mammalian pitu-
itary body. Quart. Jour. Exper. Physiol., 1, 121-159.

(3) Howrui, W. H. 1898 The physiological effects of extracts of hypophysis
cerebri and infundibular body. Jour. Exper. Med., 3, 245-258.

(4) Rerorp, L. L. anp Cusuina, H. 1909 Is the pituitary gland essential to
the maintenance of life? Johns Hopkins Hospital Bull., 20, 105-107.

(5) Gortscn, E., Cusnine, H. anp Jacosson, C. 1911 Carbohydrate toler-
ance and the posterior lobe of the hypophysis cerebri. Johns Hopkins
Hospital Bull., 22, 165-190.

NERVE SUPPLY TO PITUITARY GLAND 343

(6) Weep, L. H., Cusuinc, H. anp Jacosson, C. 1913 Further studies on
the réle of the hypophysis in the metabolism of carbohydrates. The
automatic control of the pituitary gland. Johns Hopkins Hospital
Bulle 245 33:

(7) Marie, P. 1886 Sur deux cas d’acromégalie; hypertrophie singuliére non
congénitale des extrémités supérieures, inférieures et céphalique. Rev.
de Méd., vi, 297-338.

(8) Cusnine, Harvey 1912 The pituitary body and its disorders. Phila-
delphia.

(9) Hente 1879 Anatomie des Menschen, 3,

(10) Berxury, Henry, J. 1894 The finer anatomy of the infundibular region
of the cerebrum including the pituitary gland. Brain, 17, 575.

(11) Danpy, Watter E. anp GorerscuH, Emin 1911 The blood supply of the
pituitary body. Amer. Jour. Anat., 9, 137.

(12) Witson, J.Gorpon 1910 Intravitam staining with methylene blue. Ana-
tomical Record, 4, 267.

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THE MORPHOGENESIS OF THE MAMMALIAN OVARY:
FELIS DOMESTICA

B. F. KINGSBURY
Department of Histology and Embryology, Cornell University

THIRTY-TWO FIGURES

It is a striking peculiarity in the development of the reproduc-
tive system in man and mammals (not to consider other forms of
the Vertebrata) that the duct system (modified in the female) is
laid down double, the duct system of the male disappearing in the
female or persisting as vestigial structures and vice versa, the
organs of the female duct system and the mammary gland being
vestigial in the male. The embryo in the course of development
thus passes through an indifferent period during which the sex can-
not be ascertained, followed by the period of sexual differentiation.
In the double appearance of the reproductive fundaments devel-
opment is protandrous—that is, the ‘male’ system appears earlier
than the female—this rule applying also to the differential devel-
opment of the ovary and the testis as well as the duct system.
No fully satisfactory explanation has been given for this double
development in the internal reproductive system. ‘Three or
four general explanations have been offered for this fundamental
law. (1) The primitive vertebrate was hermaphroditic and that
even in the higher forms the hermaphroditic tendency persists
showing itself in the development of a double system, male and
female, even causing the development in the male of arudimentary
mammary gland, in the female of a rudimentary prostate gland—
organs which did not extend back in the line of vertebrate de-
scent anywhere near the hypothetical hermaphroditic ancestral
form. ‘The occurrence of hermaphroditism among the ascidians
and lower vertebrates (that-is, Myxine) and the sporadic appear-
ance of a true or false, complete or incomplete, hermaphroditism

345

346 B. F. KINGSBURY

among the higher forms, up to and including man, is regarded as
supporting this contention. On this interpretation the indifferent
period would be a potentially bisexual or hermaphroditic period
in development. This interpretation is not necessarily opposed
to the view that the sex-determining factors are already present
in the egg at the time of its fertilization. (2) As a modification
of the above view might be mentioned the interpretation that the
early mammalian embryo during the indifferent period is truly
bisexual, containing the potentialities of either sex whose subse-
quent determination leads to the arrest, atrophy and more or
less complete disappearance of the organs of the opposite sex.
These two views are related and also bear on the problem of sex
determination whose cytological side is subsequently mentioned
and briefly discussed. (3) It has been argued that the reason
for the development of the Miillerian duct in the male is due to
the fact that it was the original reproductive duct and that the
utilization of the excretory duct system in the male as a repro-
ductive duct was a secondary acquirement. This interpretation,
supported by Waldeyer and Lenhossek, hardly suffices. The
theory would fail to explain the appearance of the rudimentary
mammary gland in the male, or the prostate and rete ovarii in
the female. (4) The converse of this view might be suggested,
namely, that the pronephric duct system (the male duct system)
represented the ancestral system in the nephridia in pre-chordate
forms, serving not only the nephric system but the reproductive
system as well, and conveying both the male and female repro-
ductive cells to the exterior. The shedding of the female repro-
ductive cells into the coelomic cavity from the surface of the
gonad and the development of a Miillerian duct in association
therewith would thus be a secondary adaption.' (5) By no
means exclusive of the preceding suggestions as to the meaning
of the double character of the reproductive organs, since they
deal with a distinct aspect of the problem, should be mentioned

1 Cf. Felix, W.; Theoretische Betrachtungen iiber das Genitalsystem der Verte-
braten. pp. 821-834, Handbuch der Entwicklungsgeschichte der Wirbeltiere, Vol.
3, 1, 1906.

MORPHOGENESIS OF THE MAMMALIAN OVARY 347

the attempts to analyse the mechanism of sex inheritance and
development. Upon the cytological side we have the inter-
pretations correlated with the large amount of work being done
at the present time upon the determination of sex and the chromo-
some pattern of the germ cells. This is not the place in which to
enter into a discussion of the complicated problem of the cytologi-
cal basis in the determination of sex. The careful and detailed
work on a number of forms, mainly invertebrate and chiefly
insects has, as is of course well known, established the existence
of an extra chromatin mass (heterochromosome) or chromosome
complex whose presence is correlated with the development of
the female sex.

Upon the basis of this fact theoretical considerations have led
generally to the conclusion that the heterochromosome is the
conveyor of ‘femaleness’, being an embodiment of ‘determiners’
of the female sexual characters, and that the female is homozygous
as regards sex characters, the male being heterozygous. The
presence of the hetercchromosome in the higher vertebrates and
its significance in the determination of sex are in a far from satis-
factory condition at the present time. In the case of man, Guyer,
Guthertz and Winiwarter have described heterochromosomes,
but their descriptions do not mutually support one another and
hence there is considerable uncertainty as to what the facts may
be. Their observations, however, together with those of Jordan
on the opossum and bat, Newman and Patterson on the armadillo,
Stevens on the guinea pig, Vejowsky on the cat, indicate the pres-
ence in the male gametogenesisof an extra chromatin mass (hetero-
chromosome). Jordan, however, states that it is absent in the
spermatogenesis of mongoose, cat, squirrel, rabbit and pig. Wini-
warter and Sainmont have observed in the oogenesis of the cat a
body that they suggest may be a heterochromosome. The mor-
phological basis of fact is therefore still very scant. The state-
ment may be vouchsafed, furthermore, that the correlation of
this individual chromatin mass with the development of female
sexual characters or even with the determination of the female
sex 1s unproven.

348 B. F. KINGSBURY

The assumption—to speak in Mendelian terms—that the female
is homozygous (homogametic, Wilson; ¢ ¢) as to sex, the male
being heterozygous (digametic, Wilson «), which appears to be
the more generally accepted view, does not seem to afford a satis-
factory hypothetical basis for the explanation of the double
development of the reproductive duct system of higher verte-
brates, the prostate and rete ovarii in the female, the mammary
gland in the male. Nor does the converse furnish an interpreta-
tion much more satisfactory—that the male is homozygous as to
sex ( 7 o), the female being heterozygous (9 #). If itis believed
to be of advantage to express sex in these terms, in so far as the
explanation of the development of the primary sexual characters
of vertebrates is concerned, it would seem necessary to consider
both male and female heterozygous as to sex, the male characters
being or becoming dominant in the male, the female characters
recessive, and vice versa, in accordance with the early sugges-
tion of Castle.

The only generalization that can be safely drawn, however,
from the cytological conditions that have been described would
appear to be that in the development of the female sex a greater
amount of ‘formative material’ is required. This is in general
accordance with the suggestions of Boveri, Goldschmidt and
Wilson, and is supported by the experimental work of R. Hertwig
and Kuschakewitsch, Miss King, and Riddle, indicating what has
been termed a ‘quantitative’ rather than a qualitative factor in
the determination of sex. It would seem as though on the cyto-
logical side little assistance were to be gained from the work upon
the ‘sex’ chromosomes as carriers or determiners of sexual char-
acters. In most of the work upon the ‘x,’ ‘sex’ or heterochro-
mosomes, the point of departure has been the correlation of
chromosomes or parts of chromosomes with the appearance in
development of definite morphological characters. The problem
of the early development in vertebrates of the reproductive organs
appears to be closely linked with hermaphroditism in the pattern,
which I believe—admittedly by Wilson—presents difficulties from
the standpoint of chromosomal sex determination. The problem
is, I believe, broader and there is involved the choice between what

MORPHOGENESIS OF THE MAMMALIAN OVARY - 349

I have termed a process interpretation and an ultimate particle
interpretation of structure. Furthermore there are two aspects
that may well be quite distinct so that the problem would be
essentially doubled—the determination of sex on the one hand,
and the origin and development of the sexual organs on the other.

From whatever point of view the development of the verte-
brate reproductive system may be considered, interest must center
in the essential organs—the ovary and testis—and the question as to
whether or not they, as well as the duct system, exhibit the sexual
dimorphism is particularly pertinent. Waldeyer (’70) suggested
that the potentialities for the development of both ovary and
testis existed in the same individual and that the male and fe-
male gonads developed from different portions of the germinal
ridge. Van Beneden (’80) from the conditions in the adult bat
advanced the hypothesis that ovary and testis were homologous
or at least analogous in their morphology, comparing the med-
ullary cords (cordons pleins) to the tubuli contorti (seminifer-
entes) the medullary tubes (cordons tubulaires) to the tubuli
recti, the reticular body (corps reticule; that is, rete ovarii) to the
rete testis (Halleri). Subsequent work on the development of
the ovary by Mihalkowies (’85), Janosik (’85) (’88), Coert (790),
Allen (’04), and Winiwarter (’00) particularly have seemed to jus-
tify the suggested homologization. It is the concensus of opinion
of these later writers that the medullary cords are developed
from the germinal epithelium, and are hence directly of meso-
thelial origin, they being early formed and succeeded later by the
growths that furnish the functional germ cells of the ovary, the
frequently described Pfliiger’s egg tubes or cords. It should be
stated, however, that the older interpretation of the origin of the
medullary cords from the Wolffian body, as held by Waldeyer,
Balfour, and Braun, is still adhered to by O. Hertwig (’11) and
Tourneux (’09), the latter regarding the tubuli contorti of the
testis as developed from the same source, while the former de-
rives them from the germinal epithelium (mesothelium).

The development of the rete ovarii (and the rete testis) seems
unquestionably to demand further investigation to determine its
mode of origin. Coert (’90), Mihalkowicz (’85), Janosik (’85),

r)

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 3

350 B. F. KINGSBURY

Allen (’04), and Felix (’11) have derived it from the mesothelium,
while Sainmont (’05) in his detailed study supports the older view
of its origin from the Malpighian corpuscles of the mesonephros.

The most recent study of the development of the mammalian
ovary, that of man, by Felix in the Keibel-Mall ‘‘ Handbook of
human embryology” leaves undiscussed (apparently purposely)
the question of the comparability of the medullary portion of the
ovary with the testis, the presentation in the second volume con-
trasting in this respect with the brief statement in the first volume
illustrated by means of an elaborate diagram supporting the testic-
ular homology of the ovarian medulla. Felix, in fact, distinctly
rejects this homologization, inferentially at least, by the form
in which the development of the gonads in man is described.
Briefly stated, in the indifferent stage, the gonad consists of an
inner epithelial mass, separated from the covering epithelium
by the (primitive) tunica albuginea. In the male this becomes
the permanent T. albuginea and the inner epithelial mass speedily
resolves itself into the spermatogenic tubules. In the case of
the ovary however, the inner epithelial mass becomes again
intimately associated with the mesothelial covering of theorgan
and a new peripheral zone closely blended with and probably
derived from the inner epithelial mass becomes developed, termed
by Felix the ‘neogenic zone.’ This is destined to form the defini-
tive cortex of the mature ovary while the central portion of the
inner epithelial nucleus or mass undergoes a progressive degenera-
tion toward the periphery. The inner epithelial nucleus there-
fore furnishes the material for the functional structures in either
ovary or testis, differentiation of the former proceeding peripher-
ally, and more slowly whereas in the testis the differentiation is
central and early. Any homolog of the seminal tubules of the
testis is lacking in the human ovary, according to Felix.

It will be seen that the exposition of the development of the
human ovary given by Felix is not in itself contrary to the accep-
tance of the ovarian medulla-testis homology,—previously men-
tioned—since the portion of the epithelial nucleus in the indifferent
organ that forms the spermatogenic tubules of the male takes no
part in the formation destined to furnish the ova and follicle cells

MORPHOGENESIS OF THE MAMMALIAN OVARY ab

of the fully developed ovary. The question of the double char-
acter of the ovary clearly hinges on the interpretation of the
neogenic zone as well as that of the portion underlying it. It is
evident however, that in any event the morphological comparison
of the medulla of the ovary and the testis is not very close in man.

For the converse of the ovarian-testis homology—the represen-
tation of the ovary in the testis—but little has been said. There
is nothing in the normal adult testis that can be construed as char-
acteristically ovarian, as this would of necessity he outside the
tunica albuginea. Janosik (85) has described a late formation
of large cells in the surface epithelium of the testis in the human
fetus as an attempt to form follicles. Biihler (’06) mentions the
occurrence of germ cells in the surface epithelium of fetal testes,
as apparently does Coert (90). It is clear that any proliferative
activity in the covering epithelium of the testis after the forma-
tion of the germinal cords is on any interpretation very slight.

The present investigation was undertaken to determine the
origin of the interstitial cells? of the ovary, so abundant in that
organ in the cat. While the monograph upon the development
of the ovary of the cat by von Winiwarter and Sainmont (’08)
has rendered unnecessary a detailed presentation of many aspects
of ovarian morphogenesis in this animal, a somewhat differing
interpretation and different point of view appear to Justify a brief
consideration by me.

The study in so far as presented is a purely morphological one
based upon 60 series of sections of ovaries from relatively late
fetal to adult life, the periods particularly considered being there-
fore those of the fetal, post natal, and pre-sexual development.
While several methods of fixation and staining were employed,
as indicated in the foot-note,*? depending upon what it was de-
sired to bring out, the technique already described (Kingsbury

2 Published as a separate article.

’ The ovaries upon which the study is based are as given below. Only a por-
tion of the animals were reared in the laboratory, the greater number were pro-
cured from householders and hence the data as to age is not as exact as it would
otherwise have been. My observations confirm those of Sainmont and v. Wini-
warter that there is a large variation in ovaries of the same age. Age is therefore

302

B. F. KINGSBURY

11) was found particularly useful because of the easy differen-
tiation of the cells containing ‘lipoid’ and so-called ‘mitochondria’

(representing undoubtedly lipoid in masked form).

The mor-

a poor indicator of stage of development, and the seriation may be easily deter-
mined from the internal structure of the ovary itself.

NO.

CO SIO) Or Ww

| fetus

STAGE AND LENGTH

75 mm.
75 mm.
80 mm.
80 mm.
95 mm.
95 mm.
95 mm.
95 mm.
112 mm.

embryo
embryo
fetus
fetus
fetus
fetus
fetus

fetus

3 to 4 day P.P.
3 to 4 day P.P.
6 days P.P.
Ca. 10 days P.P.
8 days P.P.

10 days P.P.
10 days P.P.
12 days P.P.
14 days P.P.
16 days P.P.
Ca. 14 days

21 days P.P.
Ca. 4 wk.

Ca. 4 wk.

33 days P.P.

Ca. 5 wk.

| 10 wk. P.P.

Ca. 10 wk.

| Ca. 3 mo.

Ca. 3 mo. (eystic)
Ca. 4 mo.
half grown

| approaching sexual maturity

young adult; virgin
young adult; virgin
young adult; virgin

young adult; virgin
adult (young?) corp. lut.
adult (young) corp. lut.
adult

adult (injected)

adult preg. (#132 a)

| adult preg. (#* E-1)

adult preg. (#150 a)
adult preg. (fetus *73)

| adult preg. (fetus)
' adult (preg. near term)

| adult nursing

adult (corp. lut.)
adult old (17 yr.)
(1 ovary pathol.)

PLANE

Trans.
Trans.

FIXER

| Flemmings FI.
| Flemmings FI.
Flemmings FI.

| Flemmings FI.

| Flemmings Fl.

| Flemmings FI.
Flemmings El.

| Zenker’s Fl.

| Zenker’s FI.

| Zenker’s Fl.

=|
.| Flemmings FI.

| Zenker’s FI.
Z(?)
Z. an Mullers

re

+++
Sse

NNNSSNNNI

Flemmings Fl.
Flemmings F1.

Cu. dichr, etc.

N
ae
Se
=

Hermann’s FI.
Z. + M.; Benda’s FI.
Z. + M.

Picro-acetic

Ale.
Z. and M.
Z.

Z. + Pot. dichr.

Fore! s+ Mullers; Z. +

STAIN

iron hematox.
iron hematox.
iron hematox.
iron hematox.
iron hematox.
iron hematox.
iron hematox.
I. H. and Safr.
I. H.; Bi. and E.
H

Be SS Or ests © rn
BREESE S

Q
(=
a

bt
bafta bts? s
eae
bs

(Ol ral sales elles!

No stain; Safr.

Cu.H.; Benda’s

Cu.H.: Visits

H. and orange

islet

H. and E.; H. and Pf;

isk

Culbie- iene

LA Culbi Eo andebt

H. and E.; I.H.; Cu.H.

H. and E.

He Sair

I.H.; H. and Pf.; H. and
E

H. and E.; H.Pf.; I.H.;
Elastin St.

| I.H.; Safr.

Safr.; I.H.; no stain

MORPHOGENESIS OF THE MAMMALIAN OVARY oe

phological differentiation obtained by this technique is particu-
larly serviceable. The dark blue interstitial cells and the epi-
thelial structures a lighter blue contrasting strikingly with the
golden brown of the stromal tissue and thus permitting them to
be readily distinguished without high power examination, while
at the same time a good fixation of cytoplasmic and nuclear de-
tail was obtained. °

The study began with a fetus of 75 mm. total length, which cor-
responds closely with the forty-day fetus of Sainmont. At this
stage the morphology of the testis is fully established, as is also
the general morphology of the ovary, which consists of primitive
medulla and primitive cortex both composed of cordlike groups of
epithelial or epitheloid cells as the parenchyma, with strands of
stroma cells interspersed. The parenchyma of the primitive
cortex is in broad connection with the surface epithelium and
consists largely of genitoid cells with but few indifferent cells dis-
cernible. The strands of stroma cells are small and relatively
insignificant. The parenchyma of the primitive medulla is made
up-of genitoid cells and indifferent cells the latter present in much
greater amount as compared with the cortex. ° The genitoid cells
resemble those of the primitive cortex markedly. While some of
them lie free in the stroma, the majority are surrounded by indif-
ferent cells, some few possessing an evident follicular epithelium.
Certain of these cells have taken on the character of ‘egg cells.’

The parenchyma of the primitive medulla and primitive cortex
are broadly confluent with each other, the separation of the two
being largely indicated by a more marked accumulation of stromal
tissue in the intermediate zone, in the form of a trabecular mesh-
work of strands of spindle-shaped stroma cells, and blood vessels
which have a general longitudinal direction in this region. These
blood-vessels form convenient landmarks. The primitive cor-
tex (as well as the definitive cortex later) is strikingly free from
larger vessels.

At the outset it may be said that it is obvious that in the mor-
phogenesis of such an organ as the ovary, its increase in size must
be kept constantly in mind, in the analysis. The ovary of the
75-mm. embryo or fetus in which the structural components—

354. B. F. KINGSBURY

so-called medullary cords, egg cords, rete ovarii, and stroma—
have already appeared, can nearly be contained in a single large
Graafian follicle of the adult organ. An understanding of the
morphological transformations that lead to the establishment of
the adult organ is to be found only in a correct analysis of its
growth. In the published descriptions of the development of
the ovary the marked and rapid increase in size, it is felt by the
writer, is frequently not adequately expressed or given due con-
sideration. Thus, the so-called egg tubes of Pfluger are spoken
of as ‘down-growths’ of the surface (germinal) epithelium,
whereas they more exactly represent cell trails left behind in the
advancement of the surface as the organ increases in size. This
does not apply apparently to the earliest proliferative growth of
the germinal epithelium in the indifferent period, in which appar-
ently there is an actual displacement of the underlying tissue
(Relixe 192

The medullary cords in similar manner represent the earliest
proliferations of the mesothelium whose immediate activity ap-
parently largely determines the early growth. Whether or not
the mesothelium loses its connection with these early formed
masses, the stroma growing between as a tunica albuginea primi-
tiva as described by Sainmont, I cannot of course consider, since
the stages studied do not include the early development. In
the 75-mm. fetus a sharp separation into primitive medulla and
cortex by a distinct stromal layer does not exist.

In the further prenatal growth of the ovary, as illustrated in
the stages examined, the ovary more than doubles its diameters.
This size increase is due to the double activity of the epithelial
elements (ova and indifferent or follicle cells) and of the stroma.
The surface mesothelium retains throughout its connection with
the epithelial masses to which the term egg cords may be applied
and which early assumed a more elongated form due to their
greater separation by strands of stroma and the increase in size
of the ovary due to their growth and that of the stroma as well.
The seat of proliferation lies mainly peripherally in the primitive
cortex and the growing zone becomes more superficial as develop-
ment proceeds. Cell divisions also occur frequently in the sur-

MORPHOGENESIS OF THE MAMMALIAN OVARY S55.

face mesothelium, being apparently more abundant in the earlier
stages in which the- egg cords are relatively more broadly con-
nected with it. It but shares with the underlying egg cords a
common growth activity and in no sense can it be regarded as the
sole direct center of proliferation from which the latter are formed.
Indeed, at birth the mitoses are more abundant in the outer por-
tions of the egg cords than in the surface epithelium with which
they are connected. The synizesis figure of the germ cells forms
a convenient landmark which serves to mark the centrifugal
march of differentiation within the egg cords. Mitoses continue
to occur within the peripheral portion of the egg cords until about
two weeks after birth.

von Winiwarter and Sainmont have described the divisions of
the oogonia as practically ceasing at the time of birth, marked
multiplication appearing again in the twenty-first-day ovary and
becoming permanently arrested soon thereafter. I have seen no
evidence of such a periodicity in the oogonial divisions in the
material employed by me, which, it must be confessed, was not
as abundant as that studied by these authors. The advance of
the ‘wave of synizesis’ appeared a fairly steady centrifugal pro-
gression. In the thirty-three-day ovary the superficially located
germ cells of the cords were in the synizesis stage, all the more
deeply placed cells being postsynizetic. In all subsequent stages
the ova were definite oocytes and in the seven-weeks’ ovary the
definitive cortex with its primary or resisting follicles was differ-
entiating out of the primitive cortex.

In its development the ovary of the cat, as far as concerns
the growth and differentiation of the cells that—according to the
writer’s interpretation—are to become the definitive ova of the
period of sexual maturity, conforms to the plan described by Felix
for the human ovary, the egg cords being less distinct in the human
ovary and the neogenic zone described by Felix less developed
as a distinct zone in the cat. The distinctness of the neogenic
zone destined to furnish the ova of the mature period would appear
to be likewise evident earlier than in the cat. The stroma by
its growth evidently plays an important part in the breaking up
of the egg cords and the formation of the primary follicles of the

356 B. F. KINGSBURY

definitive cortex. In the deeper portions of the ovary marked
growth activity of the stroma introduces a complexity not en-
countered in the typical zone of the primitive cortex. In the
ovary before birth and for the first few days after birth the egg
cords project deeply into the ovary, occupying a position which
subsequently will become medulla. The egg cords are typically
branched, presenting the characteristic appearance well known
from previous published descriptions by several workers (figs. 8-9).
In the deeper portions of the egg cords the ova are the most ad-
vanced in development and both types of cells—egg cell and
follicle cell—may be recognized in this portion of the egg cord.
The surrounding stroma is relatively denser in this middle zone
of the ovary and the growth activity of the stroma appears par-
ticularly marked here. The strands of stroma cells run irregu-
larly, some having a more radial, others a more tangential direc-
tion.

Because of the presence of egg cords in this zone, it is usually
grouped as part of the primitive cortex. It might with equal
propriety be regarded as part of the medulla, since medullary
cords are also contained therein—that is, groupings of mainly
indifferent cells with only an occasional definite ovum. This
zone is in fact an intermediate zone and its peripheral portion
persists as a boundary zone during the period of postnatal growth
of the ovary.

In their deeper portions the egg cords become separated into
cell groups containing one or more ova and in this way numerous ~
follicles are formed. Their number appears to be added to by
continued ‘cutting off’ of egg cells from the egg cords in the inter-
mediate zone. In this zone of the postnatal ovary great com-
plexity exists. It seems to represent the main line of advancing
stromal growth following upon the peripheral (and superficial)
zone of epithelial proliferation, perhaps associated with the be-
ginning follicular differentiation.

In illustration of the more general features of the morphogen-
esis, figures 8 to 15 may be consulted. The distinctness of the
cortex becomes progressively more marked until it assumes its
definitive structure. This applies as well to the other zones of

MORPHOGENESIS OF THE MAMMALIAN OVARY oT

the adult ovary. What has been spoken of as the intermediate
zona is most distinct in the growing ovary in the postpartum
period (fig. 10), losing its identity in the adolescent and adult
period (figs. 14 and 15) as part of the zona parenchymatosa. The
zone vascularis, on the contrary, can hardly be said to exist as
such in the fetal ovary, and becomes more distinct as maturity
is approached. Marginal growth, along the line where ovary
and ovarian ligament join (represented by the white line of the
adult human ovary) appears to play an important part in the
assumption of the adult morphology. In the marginal zone
growth continues apparently as long as the increase in size con-
tinues in the presexual period (approximately four months), the
growth including the stroma as well as the parenchyma (elements
of epithelial origin). |

The zones of the ovary are but the expression of the mode of
growth and type of blood supply, and hence possess no intrinsic
or genetic significance such as the cortex and medulla of the
suprarenal organ, for example, possess; but even so, the termin-
ology of the regions of the mammalian ovary is not altogether
satisfactory. The older terms, medulla and cortex, as well as the
B.N.A. terms, zona vascularis and zona parenchymatosa are both
employed and are both applied to the mature organ. The latter
terms may be very satisfactorily employed in the description of
the adult. ovary but they are not so applicable in the developing
_ structure due to the mode of growth. The terms introduced by
Sainmont (’05) in his study of the development of the cat’s ovary
and subsequently adopted in the monograph by von Winiwarter
and Sainmont (’08) are quite serviceable and are those which
will be used here, with two or three modifications which render
them more serviceable as expressions of the present writer’s con-
ception of the morphogenesis. The primitive cortex becomes the
definitive cortex during development. This is a somewhat more
restricted use of the term than that usually employed. It repre-
sents therefore only the outer portion of the definitive cortex in the
more usual use, or the zona parenchymatosa. It is, however,
as employed by His (’65) in the eat’s ovary. The outer portion
of the nucleus epithelio-stromalis centralis (of Sainmont) is the

358 B. F. KINGSBURY

intermediate zone described above. Into the composition of the
zona parenchymatosa of the adult there goes, therefore, the defini-
tive cortex, the intermediate zone and a portion of the epithelio-
stromal nucleus. The zona vascularis is developed out of the
basal nucleus (connective tissue) of the growing ovary extended
at the expense of the epithelio-stromal nucleus, while the mar-
ginal growth of the ovary plays a part in establishing it. The
primitive medulla would include the epithelio-stromal nucleus
and the basal nucleus.

The developmental changes that take place in the deeper por-
tions of the growing ovary, in the primitive medulla, are complex,
and this is intensified by the desirability of determining, for theo-
retical reasons, the exact mode of growth and the morphological
value at the different stages of the so-called medullary cords
which have so frequently been regarded as the equivalents of
the seminal tubules of the testis. The rete ovaril occupies a posi-
tion in the cephalic portion of the medulla (fig. 9). Its tubules
are, in the youngest embryo studied (75 mm.) easily distinguished,
of definite cuboidal-columnar epithelium, with a distinct lumen.
Subsequently, in older fetuses, the epithelium becomes more
distinctly flattened and the rete character more accentuated
(fig. 8). Whether the cell cords that form the structure are derived
from the mesothelium at the cephalic end of the ovary, or are
‘ingrowths’ from the mesonephros, or are of double origin and
nature, has not been made an object of investigation by me, and |
hence of course cannot be adequately considered at this time. The
structure as observed in the growing organ suggests the last view.
The rete furthermore undergoes a progressive change after birth
and remains as a persistent structure in the adult ovary. It
requires no very extensive study of the ovary of older fetal and
new-born animals to determine that at least some of the medul-
lary cords are connected with the rete ovari, as has been described
by others (von Winiwarter and Sainmont ’08) and this requires
therefore no extended description or comment at this point. Two
other features of the medullary cords in the older embryos, the
presence of fat-granules in the cells, and the occurrence of large
cells within the cords, require brief discussion. Droplets of a

MORPHOGENESIS OF THE MAMMALIAN OVARY 359

fatty (‘lipeid’) nature were found in the medullary cords of the
95 mm. and 112 mm. fetus, three to four-day kitten, and still
demonstrable in the six-day kitten. They may have been pres-
ent in the earlier stages examined, but the technique was not of
a nature to demonstrate their presence easily. von Winiwarter
and Sainmont found the fat globules appearing about forty-five
days p.c. (i.e., 70 mm. length, Sainmont), and no longer present
three days p.p. They reject the interpretation of Allen (’04) in
the pig, that the presence of fat is indicative of degeneration of
the medullary cords. They emphasize on the contrary the pres-
ence of fat as evidence of profound metabolic change taking place
in the medullary cords at this time. This, perhaps, could hardly
be questioned. It might be added, however, that one can hardly
speak of a “disappearance of lipoid from the medullary cords,” since
the application of what I may term a ‘mitochondrial technique’
adduces evidence of the presence of fat in abundance in masked
form in all the epithelial cells within the ovary—so-called medul-
lary cords, follicle cells and ova—at all stages of their later
growth, at least, appearing as free lipoid globules in the last, in the
process of their vitellogenesis. Hence the question resolves itself
into the reason for the existence of droplets of free lipoid in the
epithelial cells (medullary cords) in the deeper portions of the
ovaries. Inasmuch as I find evidence of dwindling and disap-
pearance of these cell cords in the deeper portions of the ovary
at about this time (after birth) I incline to the interpretation
offered by Allen.

The ‘large cells’ present in the medullary cords and the surface
epithelium have not been specially studied by me. They were
encountered in the two youngest embryos and in the surface
epithelium, particularly in the zone bordering the hilum, well
along in postpartum stages. I have seen no reason for drawing
a sharp line between these cells and obvious ‘germ cells,’ as I
believe that all intergradations between them and the latter may
be found. On the other hand, most of them, at least in the ante-
partum ovary, do not give rise to the definitive germ cells of
the adult ovary, and hence are not primordial germ cells in the
original sense (‘Ureier,’ of Waldeyer). It is unnecessary to

360 B. F. KINGSBURY

introduce a discussion of their interpretation and significance.
Reference is simply made to the excellent discussion of von Wini-
warter and Sainmont (p. 67). Those investigators who accept a
definite cell-lineage for the germ cells, strongly suggested by the
observations of Allen (’11), Rubaschkin (’09, ’11) Wood, Dodds,
and others, may interpret them as unproductive side lines of the
germ-track or as a more or less temporary hypertrophy, for un-
known reasons of ‘indifferent cells’ cells, as accepted by von
Winiwarter and Sainmont, who believe that they subsequently
return to normal size. Upon a purely physiologic or process
interpretation, however, these cells might still be grouped with
the germ cells as an expression of the oogenetic processes at work
in the developing ovary.

The period of the postpartum growth leading up to the appear-
ance of medullary follicles and their growth is a critical one in
the theoretical interpretation of the morphology. The primi-
tive cortex and medulla become accentuated and extended. The
ovary in the first five weeks quite doubles its diameter. The
stroma ovarii continues its obvious growth activity. The in-
crease in the epithelioid cords and their morphological trans-
formations furnish the characteristic feature of this period. They
increase in bulk and while very irregular in contour often assume
a more tubular form. ‘Their cells increase in size and by their
arrangement assume a form more characteristically epithelial,
as about a potential lumen. Their nuclei lie more peripherally
and the inner ends of the cells are more elongated, vacuolar and *
reticulate. Abundant ‘mitochondrial’ substance is present here.
The structural appearance in such cases forces a comparison with
the tubules of the testis, and the comparison becomes enhanced
by the relation of the cell cords within the medulla of the ovary
to the rete. The resemblance to the tubules of the undifferen-
tiated embryonic testis, or more closely to the tubules of the cryp-
torchid is particularly striking. von Winiwarter and Sainmont
(’08) have called attention to this resemblance of the medullary
cords to the tubules of the testis, as the former (?00) had pre-
viously done in his paper on the development of the rabbit’s ovary,
and as several have done in a comparison of the ovarian medulla

MORPHOGENESIS OF THE MAMMALIAN OVARY 361

with the testis. Such a comparison, to which the writer at first
inclined, seems to him upon maturer consideration a superficial
one. The elongated form, simulating a tubule is not the uni-
versal form of growth of these cell masses nor do they possess,
when elongated, the morphology of the seminal tubules. The
form assumed is quite irregular and is obviously a factor of the
growth of the epithelial cords and stromal strands in their mutual
relation to one another in the growth of the ovary during this
period.

The source of the cells that compose the cell cords and masses
so prominent during the period of expansion is also important in
this connection. Two modes of origin present themselves as
possible: (1) A development by centrifugal growth of the primary
medullary cords apparent in the embryo so that from them come
all the epithelial cords inside the zone of the primitive cortex.
This mode of growth would make the medullary structures a unit
and strengthen the ovary-testis comparison. (2) That the cords
and masses of epithelial cells within the medulla, while in many
instances connected with the primary medullary cords and quite
possibly grown out from these cell groups, are nevertheless de-
rived in part from the indifferent cells contained in the egg cords
of the embryo. The differences of the epithelial cells would be
purely a matter of position and relation and not intrinsic or mor-
phological.

It is this last view that seems to me undoubtedly the correct
one. Small groups of epithelial cells are to be found at all stages
of the postpartum growth in the central nucleus, the interme-
diate zone and, especially later, in the inner portion of the primi-
tive cortex. They are formed by the breaking up of the inner
portions of the egg cords into primary follicles. Many of these
primary follicles and epithelial cell groups without an ovum en-
closed come to lie within the medulla, in the epithelio-stromal
nucleus where they subsequently grow and play a part in the de-
velopment of the medullary follicles, presently to be described.

4 Bremer, J. L. Amer. Jour. Anat., vol. 11, no. 4, May, 1911, pp. 393-416. Huber,
G. Carl, and Curtis G. M. Anat. Rec., vol. 7, no. 6, June, 1913, pp. 207-220.

362 B. F. KINGSBURY

In connection with the comparison of the structures within the
ovary to the testis tubules just considered, it may be said at this
point that the cells are as strikingly follicle cells at this stage and
place as subsequently in the development of the follicles of the ado-
lescent and adult periods. This is particularly clearly shown in a
comparison of such cells when a mitochondrial technique has been
employed. The disposition of the mitochondrial granules in the
inner ends of the cells in each instance serves to make the agree-
ment more striking. The fact that these so-called medullary
cords develop into follicles should leave no doubt of their ovarian
character.

The medullary follicles. The development of the follicles with-
in the medulla of the ovary of the kitten has been studied in de-
tail by von Winiwarter and Sainmont, who apply the name of
‘medullary follicles’ and regard their appearance as marking a
third stage in the development of the ovary. Their description,
briefly stated, is as follows: At eight days postpartum the med-
ullary cords are markedly elongated; at sixteen days they have
increased in volume and the cells have hypertrophied and be-
come more columnar in shape. This stage is the last one in which
a connection of the medullary cords with Pfliiger’s egg cords
exists. Ovules exist within the medullary cords, generally smaller
than those of the primitive cortex. Small medullary cords are
described in the zone bounding the primitive cortex and contain-
ing ovules which belong to Pfliiger’s egg cords. Such they inter- |
pret as ovules of Pfliiger’s egg cords which have become isolated
with a medullary cord. At twenty-three days p.p., primordial
medullary follicles, both uni- and pluri-ovular, are beginning
their development. At thirty-five days, the next described
stage, the medullary follicles have become voluminous struc-
tures. These they group as follicles in process of growth and folli-
cles fully formed, the latter possessing an antrum comparable to
the antrum of the definitive Graafian follicles of the adult period.
The medullary follicles now undergo a peculiar degeneration dur-
ing the next two or three weeks, so that, at sixty to sixty-five
days p.p., all remains of the medullary follicles have completely
disappeared, this degeneration being accomplished or accompa-

MORPHOGENESIS OF THE MAMMALIAN OVARY 363

nied by an enucleation of the follicles by the ovarian stroma.
The naked ova, so ‘shelled out,’ undergo a degeneration in the
midst of the stroma. Not all ova degenerate through the destruc-
tive agency of the stroma, but undergo progressive degeneration
within the medullary follicle. With the degeneration of the med-
ullary follicles the indifferent cells and ova derived from the first
proliferation of the germinal epithelium of the ovary entirely
disappear.

An identical fate (i.e., degeneration) awaits the Graafian folli-
cles derived from the egg tubes of Pfliiger, or the second prolif-
eration of the germinal epithelium.’ Beginning about sixty to
sixty-five days, Graafian follicles develop from the deeper portions
of the cortical zone, the first ones being nearly always pluri-ovular,
containing two or three or more ova. Very large follicles are thus
formed, so that, about three and one-half or four months’ post-
partum, the ovary attains a large size, becoming subsequently
reduced in size with the degeneration of these follicles and their
absorption. At this same time, the remaining resting or primary
follicles formed from the second proliferation have practically
disappeared. A third proliferation’ then furnishes the ova and
follicle cells destined to develop into the Graafian follicles of the
period of sexual maturity.

5 yon Winiwarter and Sainmont 1908, p. 85: ‘‘Nous nous proposons d’exposer
dans le présent chapitre qu’un sort identique est reservé & tous les ovules et folli-
cules de de Graaf qui dérivent des tubes de Pfliiger ou cordons corticaux (seconde
proliferation). Cette déchéance n’atteint pas tous les ovules au méme degré de
développement. Les uns, et ils sont majorité, ne dépassent pas le stade de fol-
licule primordial. Les autres se transforment en folliclues de de Graaf plus ou
moins volumineux. Neanmoins le résultat est le méme: leur ensemble est voué
i la mort. Deplus, la dégénérescence des follicules de de Graaf suit une marche
trés particuliére, etablissant une transition manifeste et graduelle entre l’atrésie
des follicules médullaires que nous avons décrite et l’atrésie typique, telle qu’elle
a été étudiée dans l’ovaire adulte. C’est pourquoi nous faisons suivre la descrip-
tion des cordons médullaires par celles de l’évolution des cordons corticaux, afin
de faire ressortir combien le développement de l’ovaire est continu et progressif.”’

6 yon Winiwarter and Sainmont, 1908, p. 89: ‘“‘Il arrive un moment (vers 33
i 4 mois p. part.) ot, pratiquement, tous les follicules primordiaux ont disparu.
On ne voit plus alors sous l’épithélium de revétement qu’une serie de cordons
épithéliaux, représentant tout au moins en partie les anciennes cellules follicu-

leuses. Nous disons en partie, car, ainsi que nous le verrons ultérieurement,
tandis que ces phénoménes régressifs se déroulent, l’épithélium de revétement

364 B. F. KINGSBURY

I have sketched the general results of von Winiwarter and
Sainmont because of the monographic character of the study made
of the development of the ovary of the cat and the uniqueness
of some of the results and interpretations offered.. It is therefore
with some hesitancy that I venture, from a study of material in-
ferior in amount to theirs, to offer interpretations that differ in
certain fundamental respects.

In as far as concerns the medullary follicles, the difference of
interpretation has three aspects. In the first place, as has been
fully indicated in the discussion of the medullary cords in the:
foregoing paragraphs, the writer is unable to accept the sharp
distinction between the products of a ‘‘first and second prolifera-
tion,’”’ a distinction that plays so important a part in the interpre-
tations of von Winiwarter and Sainmont. The distinction of
medullary cord and sex cord seems to the writer more a matter of
differentiation, location, and convenience for descriptive purposes
rather than a sharp distinction of materials of morphologically
different values. The follicles that begin their development dur-
ing this period are thus but the earliest follicles which from posi-
tion may appropriately be termed medullary follicles, it is true,
but only for descriptive and topographical reasons. Their devel-
opment, peculiarities and fate are closely involved in-the growth
processes taking place in the ovary at this time.

The second point of different interpretation involves the source
of the ova and the early transformation of the inner portions of
the ‘egg cords.’ As has been already stated, it has seemed to the
writer clearly apparent from a comparison of the successive series
of ovaries of advancing development, that going hand in hand with
the peripheral growth of the egg cords there has been a central
ne reste pas inactif. Une nouvelle prolifération (la troisiéme), celle des invagi-
nations épithéliales, apparait et les colonnes cellulaires qui les composent, trav-
ersent l’albuginée et se mélent aux amas des cellules, folliculeuses. Comme ces
deux formations sont constituées de cellules épithéliales ordinaires, il est impos-
sible morphologiquement de distinguer ce qui revient aux unes et aux autres.
Toujours est-il que c’est 4 leurs dépens que se formeront la zone corticale défini-
tive et les oeufs définitifs de l’adulte.’’ (p. 260): ‘‘Ces invaginations, jointes
aux cellules folliculeuses de la zone corticale primitive, aboutissent 4la formation

de la zone corticale définitive de l’ovaire, 4 laquelle, seule, sera réservée la pro-
duction des oeufs définitifs. Son histoire appartient 4 un chapitre ultérieur.”’

MORPHOGENESIS OF THE MAMMALIAN OVARY 365

disassociation or breaking up of the egg cords in which the growth
of the stroma has been largely instrumental. This leads to the
isolation of the ova or groups of ova, and from these have come
the ova which occur in the medullary follicles that begin their
development in the third week after birth. In illustration of
this interpretation there are submitted as text figures 1 to 6,
six photographs of sections taken entirely at random and there-
fore not of illustrative value otherwise. In these the centrifugal
oogenetic wave is indicated by the synizesis stage apparent in all
save the first and last in which the ova are in the pre- and post-
synizetic stages, respectively. From a comparison of these it
will become apparent, I believe—if the growth of the ovary be
kept in mind—that the large ova in the deeper portion which in
the last two figures are in developing medullary follicles are derived
from the progressive breaking up of the inner portions of the so-
called egg-cords.

The third difference of interpretation of the medullary follicles
concerns the meaning of the peculiarities of their form. The view
of von Winiwarter and Sainmont, that the great irregularity and
peculiar form of the follicles encountered in the ovary of the cat
at this stage is due to the destructive activity of the ovarian stroma
by penetrating and enucleating the follicle, has been noted above.
The striking peculiarity of the follicles developing during this
period was observed by myself before I had become aware of the
work of von Winiwarter and Sainmont and they had been inter-
preted in quite the reverse direction, namely, as progressive rather
than regressive pictures. Further examination and considera-
ation has but confirmed me in the interpretation. The medul-
lary follicles began their progressive development early in the third
week after birth, although a close limitation of the time of their
appearance cannot be given. The numerous ova in the ovary
within the zone of the primitive cortex possess well-marked fol-
licular epithelium. Some of them are definitely within so-called
medullary cords of elongated form, others within more irregular
masses. A number lie free in the stroma, surrounded only by a
single layer of follicle cells which may vary from flattened to col-
umnarinshape. Such ova are particularly found in the peripheral

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 3

366 B. F. KINGSBURY

Fig. | 8O mm. fetus. * 120. Fig. 4 Twenty-one day kitten. < 60.

Fig. 2 95 mm. fetus. X 110. Fig. 5 Thirty-three day kitten. 60.
Fig. 3 Three to four day kitten. & 60. Fig. 6 Five to six weeks kitten. 60.

MORPHOGENESIS OF THE MAMMALIAN OVARY S67

portion of the medulla and in the intermediate zone. Interspersed
with these are cords or clusters of follicle cells whose connection
with egg cells is not apparent. Some of these appear to come from
the indifferent cells of the egg cord in its breaking up, or to have
survived the degeneration of an egg cell which they once envel-
oped (fig. 16). Such epithelial structures with the surrounding
stroma and the interstitial cells compose the epithelio-stromal
nucleus of the two-week ovary. Marked growth of the masses
and cords of follicular cells and of the stroma produces the ovary
of two months. Very great irregularity is shown in the growth
of the cords of follicle cells, and their relation to the ova is par-
ticularly interesting. A typical follicle formation of this period
is shown in figures 24 and 25.

It is structures of this kind that have been interpreted by von
Winiwarter and Sainmont as follicles in degeneration attended
by penetration of the stroma and enucleation. Figures 16 to
25 are introduced in illustration of the reverse view. From
the morphological relations there shown it would appear that
the eggs are becoming enveloped by the growing follicle masses.
Figure 16 shows two small egg cells of smaller size and close to
each a cluster of follicle cells, doubtless derived from adjacent
indifferent cells of the original egg cord. Each egg is invested by
a layer of follicle cells of its own. By growth of the follicular
cluster the ovum, in its epithelium, becomes partially surrounded
(fig. 17, 18) and finally completely invested (figs. 19, 20) by the
growing cell mass; and with the appearance of an antrum—indi-
cated from the beginning—a Graafian follicle more or less irreg-
ular, is developed. Several ova may thus become included in a
single follicle which may be of rather tubular form (fig. 23). The
layer of follicle cells belonging to the ovum and immediately
surrounding it may be traced well into the complete investment.
The investing mass fuses with it and its identity is finally lost.
It is indicated in figures 17 to 21.

Whether the investing follicular mass at the beginning is al-
ways or even usually distinct from the sheathing follicle cells
is not easy to determine. In many instances, at least,-it would
appear that the investing mass is developed from one side of the

368 B. F. KINGSBURY

primary follicle or medullary cord and in the process of growth
simply becomes wrapped around,enclosing ovum and its follicular
epithelium of the opposite pole, as well as some of the stroma.

In support of the above interpretation and as opposed to the
reverse one of regressive change by a process of enucleation, may
be advanced: (1) the fact that in general the smaller egg cells are
less enclosed; (2) that they lie generally more peripherally; (3)
that the line of fusion of the investing epithelium may often be
detected (figs. 19, 20, 22); (4) that the free egg cells possess in
most instances at least a sheathing simple follicular epithelium;
(5) that degenerating egg cells lying free in the stroma are but
seldom encountered.

Accepting the interpretation above set forth, it can hardly be
doubted that these follicles owe their irregular form relations to
a compounding of the growth of the stroma and that of the follicu-
lar elements. It may be suggested that their peculiarities are to
some extent due to an attempt at follicle formation in an ovary
during rapid growth; or, put differently, the growth of follicular
elements and ovarian stroma in lines at variance with each other
prevents the free expansion and uniform growth so characteristic
of the adult ovary, so that the growing follicular cords must
grow around the ovum and hence come to invest it (figs..24 and
25). The reverse, from the same point of view, might easily be
conceived to oceur and the follicular cells be stripped off as a re-
sult of the pressures and tensions of the stromal growth, and thus
the ovum be left naked in the midst of the stroma, producing thus
the effect of enucleation without attributing to the ovarian stroma
a specific activity in the process. Indeed, from the figures of
‘ von Winiwarter and Sainmont, as well as my own observations,
this appears frequently to be the case. What chemical corre-
lations may likewise be involved cannot be estimated. -

The follicular structures in the ovary at this period are of very
varied form. In order to obtain a three-dimensional view of the
relations, a model was made of a portion of an ovary (no. 26) in
which follicular cords, follicles and ova inside the primitive’ cor-
tex are illustrated. The ova shown are in all cases immediately
surrounded by their simple follicular epithelium. Of two of the

MORPHOGENESIS OF THE MAMMALIAN OVARY 369

follicles so shown, enlarged drawings are given in figures 29 and
30; figure 29 may be compared to the sectional view shown in fig-
ure 23 and figure 30 may be similarly compared with figure 18.
Within the larger masses an ovum was contained completely en-
closed. The picture that is afforded by the reconstruction sup-
ports, I believe, the evidence which the histology furnishes, name-
ly, that in most instances the process illustrated by these peculiar
follicle formations is one of inclusion rather than of exclusion of
the ovum, in most instances at least.

Observations of these formations by earlier workers than von
Winiwarter and Sainmont in the cat’s ovary, or similar structures
in the ovaries of other animals, appear to be lacking. von K6lli-
ker, it is true, and Biihler in his earlier work, regarded the follic-
ular epithelium as coming from the medullary cords and these
‘in turn from the mesonephros, investing the egg cells (whieh were
derived from the germinal epithelium) by growing around them,
so that relations similar to those met with in the kitten’s ovary
may have been influential in forming their interpretations. But
neither their description nor figures give us definite evidence.

The fate of these peculiar follicles is one of considerable impor-
tance in their interpretation. While many of these follicles evi-
dently degenerate, the majority I believe remain as the large irreg-
ular, usually pluri-ovular follicles which are found in the ovaries
of kittens before the onset of sexual maturity. Such follicles are
shown in figure 13 and were figured by Sainmont (fig. 15) in his
earlier paper, wherein they were not specifically discussed, al-
though they were obviously thought of as derived from the ‘‘egg
tubes of Pfliiger.”’ In the later and larger work with von Wini-
warter, so often referred to in this paper, these follicles were de-
scribed and regarded as developed out of the material of the
second proliferation (Pfliiger’s egg tubes), succeeding as a second
set the ‘medullary follicles’ formed from the medullary cords of
the first proliferation which entirely degenerated. This inter-
pretation is a necessary corollary to the interpretation given by
them of the peculiar follicle formation with which we have just
been dealing. Figures showing the irregularity and pluri-ovular
character of these follicles are given (11 text figures, p. 90) butnone

370 B. F. KINGSBURY

showing their development, so that it is somewhat difficult to
judge of the evidence—aside from the interpretation of the pecu-
liar medullary follicles of younger ovaries—that lead them to
regard these as quite distinct from the follicles of the earlier
period. Sainmont’s figures (4 to 9) appear to me far from con-
vincing. In their description their resemblance to the medullary
follicles is pointed out and the similarity in their mode of ‘degen-
eration.’ They differ in the presence of a better developed theca
and the relation and character of the interstitial cells.

As in their formative period—as I interpret it—these follicles
are characterised by their great irregularity and the numerous
ova usually contained. This appears in figure 13 of a single sec-
tion, and more so in a model made from a section of the same
ovary. In between these follicles occur numerous cords of cells
(which-were reproduced in part) and strands of interstitial cells-
(not shown in the model). The stroma forms a well defined
theca for the follicle and it is there that the interstitial cells chief-
ly occur. Two of the irregular pluri-ovular follicles are drawn
separately (figs.31 and 32). Their irregularity is clearly apparent
from these. The cords of cells are apparently vanishing struc-
tures—small groups of follicle cells derived from the medullary
cords or of cortical origin. In these follicles several ova are
contained.

The approach of sexual maturity is attended by a profound
degeneration of the Graafian follicles formed during the pre-sexual
period. In ovaries of this stage nearly all the larger Graafian
follicles are found to be in some stage of aresia, as illustrated in
one of the figures in my paper on the interstitial cells. Eight
ovaries of this period were examined. The conclusion of von
Winiwarter and Sainmont that the degenerations of this period
involve also the primary follicles as well and that the ova of the
sexual maturity are derived from a third ‘down-growth’ from the
surface epithelium, renders this period one of marked importance.
As far as the material studied by me goes, it has afforded no-evi-
dence of such a new formation of ova; in nearly all cases an abun-
dance of resting or primary follicles in the cortex was found to be
present, and it is believed that the primary follicles found in the

MORPHOGENESIS OF THE MAMMALIAN OVARY 371

ovary during the adult period are formed in the differentiation of
the cortical zone in the pre-sexual period. But one ovary (no.
39) was secured, clearly belonging to this period, in which the
primary follicles had nearly completely disappeared, giving the
picture described and figured by von Winiwarter and Sainmont.
This was, however, regarded as a case of exceptionally profound
degeneration, and showed no evidence of a renewed prolifera-
tion of oogonia from the surface epithelium. The margin of
the ovary bordering the hilum long (up to three months, at least)
remains a seat of proliferation and growth during the enlargement
of the ovary in the pre-sexual period, and it is quite possible that
in the second growth period (that immediately preceding sexual
maturity) following the period of profound degeneration and cor-
responding diminution in volume, the marginal surface epithe-
lium particularly resumes its proliferative activity with a result-
ing increase in the number of primary follicles. It is also quite
possible that the profound degeneration and consequent third
proliferation are related, and not constant but variable; only an
extreme morphological interpretation would reject the possibility.
The material, however, is insufficient for the settlement of these
points. Neither the preliminary paper of von Winiwarter and
Sainmont, nor the statements in their subsequent papers present
the evidence in a form that seems to me fully convincing, and the
promised chapter’? upon the development of the third prolifera-
tion will be looked forward to with interest.

If it isa matter of deep-seated importance and not a mere
expression of growth, a proliferation of ova just before sexual
maturity will be found to occur in other mammals. It may be
questioned, however, whether Rubaschkin (712) is justified in
regarding what he terms a ‘third proliferation’ from the surface
epithelium of the guinea pig ovary, occurring before birth, as
homologous with a (third) proliferation of ova just preceding
sexual maturity, such as von Winiwarter and Sainmont describe
for the cat.

As a result of the profound degenerations before the onset of
sexual maturity there is left an ovary smaller but richer in stroma

7 See quotation, from p. 260, in footnote 6, p. 363.

372 B. F. KINGSBURY

(fig. 14). The Graafian follicles that develop during the period
of sexual maturity are almost always uni-ovular in the cat, are
more regular and uniform in shape and the ova are characterized
by a markedly different structural appearance as compared with
the ova of the pre-sexual Graafian follicles as noted by von Wini-
warter and Sainmont.

A very frequent accompaniment of the degeneration of the
Graafian follicles of the pre-sexual period in kittens approaching
sexual maturity, may be incidentally mentioned, namely, the
occurrence of polar spindles, polar body formation and a form of
degeneration of the ovum by fragmentation, simulating a par-
thenogenetic cleavage. It is unnecessary to consider here the
question of their parthenogenetic nature. Similar conditions have
been described in several forms. Bonnet has considered and
rejected their parthenogenetic character. In the ‘‘ Hertwig Hand-
buch,’’ Waldeyer has discussed them and pronounced against
their parthenogenetic significance, while Hertwig regards them
as essentially a beginning parthenogenesis. In the cat they
occur most abundantly in old kittens (ca. four months) in the
large Graafian follicles that have begun degeneration as atresia
folliculi. While no particular search for them has been made,
four polar spindles and several fragmenting ova (fig. 28) were
encountered in a single ovary and were found to occur in four
ovaries of this period. In illustration of the typical and ‘normal’
appearance, figures 26 and 27 are submitted, showing a first
polar body and second polar spindle (?) in the same ovum. That
there is not a factor peculiar to the period of adolescence which
determines the introduction of maturation, is evinced by the oc-
currence of the polar spindle formation in a degenerating folli-
cle of an adult ovary. That the phenomenon is not simply an
indication that the growth period had been completed and the
follicle essentially mature when it enters upon atresia, is also
shown by the occurrence of a polar spindle in an obviously young
Graafian follicle. In most instances, however, the follicles in
which polar spindle formation occurs are of large size. The con-
stant correlation is that with degeneration of the follicle. There
appears to be, therefore, some factor connected with atresia

MORPHOGENESIS OF THE MAMMALIAN OVARY 373

folliculi that can, under certain unknown conditions, induce
maturation.

In the period of sexual maturity the development of the
Graafian follicles is quite variable and the morphology of the
ovary fluctuates correspondingly. Figure 15 shows a typical sec-
tion of the zones of the adult ovary, the zona parenchymatosa
consisting of cortex and zone of Graafian follicles (epithelio-
stromal zone), and the zona vascularis.

GENERAL CONCLUSIONS

The results of this study of 60 series of cat ovaries may be
summed up briefly at this point. From the stage with which the
study began, when the ovarian character of the organ is clearly es-
tablished (75 mm. fetus) to sexual maturity, the ovary increases
in diameter approximately five times. The growth zone appears
to be mostly peripheral, in and beneath the mesothelial covering
epithelium that is the source of the ova and follicle cells. The
greatest activity appears in the primitive cortex beneath the sur-
face epithelium rather than in the latter, and growth continues
here well into the postpartum period, as von Winiwarter and
Sainmont have shown. Up to approximately the third week
postpartum the surface epithelium and the underlying cell cords
of mesothelial origin form a common mass, the latter being con-
nected with the former. Subsequently (third week) they are
separated by the appearance of a tunica albuginea.

At the margins of the ovary bordering the hilum the growth
activity continues much longer (up to approximately three to
four months) and along this bordering zone the surface epithelium
- retains its connection with the underlying egg and follicle cell
groups.

The deeper masses of cells of mesothelial origin are the older
and differentiation follows a centrifugal course save at the mar-
gin where younger stages in the oogenesis are to be encountered
long after they have disappeared from the remainder of the ovary.
In this growth the stroma plays an important part. The growth
of the ovary may be said therefore to be mainly peripheral and
marginal, and the differentiation centrifugal and marginal. .

374 B. F. KINGSBURY

Accompanying the centrifugal wave of differentiation and
erowth there is a progressive advance in the state of development
attained. Early in development (antepartum) large genitoid
cells appear in the central portion mainly in the medullary cords.
These disappear. The earliest follicles are central but do not
attain large size of advanced development but degenerate. After
birth (third week), come the irregular medullary follicles which,
however, degenerate. An irregular centrifugal wave of degen-
eration might therefore be said to follow after the wave of
differentiation.

The wave of differentiation and growth does not run through
to completion but in the periphery proceeds much more slowly,
leading, therefore, to the establishment of a cortex in which the
follicles remain long in a resting stage, as the well-known primary
follicles.

Degenerations affecting follicles of all stages of development
occur at all periods of the life history as well. The small and.
immature ovary of the anti- and postpartum periods is obviously
‘unable’ to provide adequate blood supply for the follicles be-
ginning development during these periods and hence the sugges-
tion at once arises that therein is to be found the reason for the
profound degenerations occurring in the pre-sexual period and
after sexual maturity; that the processes, which in the adult ovary
lead to the formation of the mature Graafian follicle, are opera-
tive from the beginning but continually fail, due to the absence
of the necessary conditions (nutritive or otherwise) reaching pro-
gressively more advanced stages as development and growth pro-
ceed. The lack of proper vascular and nutritive conditions is
doubtless the cause of many of the degenerations® (atresia folliculi; .
degenerations of primary follicles) particularly in the adult period.
Inasmuch as it is extremely doubtful if the developmental
factors are all intrinsic—that is, the growth and differentia-
tion within the ovary independent of the rest of the organism—
it is equally improbable that all the degenerations of the growth

8’ Compare the conclusions of Clark, from the study of injected human ovaries,

immature and adult. J.G.Clark. Johns Hopkins Hospital Reports, vol.9, 1901:
pp. 593-676.

MORPHOGENESIS OF THE MAMMALIAN OVARY 370

period can be explained as due simply to the intrinsic conditions
of growth.

The relations of the zones that mark the morphogenesis of the
cat’s ovary may be illustrated by a series of schemata (text fig-
ure 7) wherein schema A indicates approximately the conditions
in the 75 mm. fetus, schema D, the zones of the adult ovary.
These schemata may also be directly compared with the photo-
graphs reproduced as figures 8 to 15. The nomenclature of the
ovary has been briefly discussed (p. 357).

Fig. 7 Schema to illustrate the appearance of the zones in the morphogenesis
of the cat’s ovary. C.P., primitive cortex; C., cortex (definitive) ; V.E., epithelio-
stromal nucleus; V.B., nucleus basalis; Z.J., intermediate zone; Z.P., zona par-
enchymatosa; Z.V., zona vascularis.

In an attempted analysis of the morphogenesis of the mamma-
lian ovary it would be necessary to compare the development in
small, medium-sized and large animals in detail, in order thereby
to eliminate the effects of size, and arrive if possible at the intrin-
sic aspects of the organogenesis. Such an analytical comparison
of mammalian ovaries has hardly been done. The recent account
of Felix of the development of the human ovary, as already
sketched, indicates possibilities, and to it, along general lines, the
development of the ovary of the cat appears to conform. The
existence of ‘medullary cords’ and Pfliiger’s “egg tubes’ must be
regarded as of secondary significance as but variations in the
mode of growth. The neogenic zone of Felix obviously compares
with the primitive cortex of the ovary of the cat. It is to be
regretted that the description did not include the important
period of development during childhood.

376 B. F. KINGSBURY

The development of the cat’s ovary, according to my interpre-
tation, affords no support for the view that the ovary proper is
superimposed as a distinct growth on a vestigial testis represented
by the rete ovaril and the medullary cords. The latter, while
they present a superficial resemblance, histologically, particularly
to the tubules of a cryptorchid testis, are nevertheless obvious-
ly ovarian. Their apparent testicular character is due to their
form of growth. They contain ova and form follicles, while their
cells clearly agree with the follicle cells in structure, and this is
also clearly due to the processes occurring within them, which are
themselves in part a function of their relations. There remains
the fact of the existence of a rete ovaru, the undoubted homolog
of the rete testis, particularly well developed in some animals,
such as the eat, and connected with some of the so-called medul-
lary cords which thus would appear clearly homologous with the
tubuli contorti of the testis. The apparent dual structure of the
ovary is but a part of the larger problem of the double develop-
ment of the internal organs of reproduction in typical vertebrates,
referred to at the beginning of the paper. The development of
a rete and its connection with the parenchyma of the gonad in
the female is but part of the tendency to develop the duct system
of the male. The conclusions to which one is almost inevitable
compelled is that of a deep-seated hermaphroditic tendency in
the development of vertebrates, which finds expression in the
double character of certain definite organs and structures. This but
describes the morphogenetic pattern with a suggestion of its phy-
logenetic origin. Upon the analytical side, it might be affirmed
that processes determining both male and female duct systems
were present in development of each sex. If it were believed to
put the problem on a better basis, it might be said that each sex
is-heterozygous in this respect, the factors determining the devel-
opment of the male duct system becoming dominant in the course
of development of the male and vice versa. The development
of the ovary indicates strongly that the development and double
character of the reproductive system must be clearly differen-
tiated from that of sex itself. The development of the reproduc-
tive system is double—has two aspects: the establishment of the

MORPHOGENESIS OF THE MAMMALIAN OVARY 3H

fundamental plan of the system, and the underlying mode (meta-
bolic attitude) that determines the direction taken—the ‘‘deter-
mination of sex.’’ In the development of the human ovary and
testis, for example, Felix has shown that in both sexes there is
the same fundamental material which is worked over, so to speak,
along one or the other lines of differentiation.

It was the suggestion, of Wilson I believe, that sex and the sex-
ual characters might be differently determined, the latter by
the heterochromosome. Suggesting in turn that the hetero-
chromosome stands for the former; instead, it might be stated in
closing as at the beginning of this paper, that the evidence indi-
cates a quantitative rather than a qualitative sex difference, a
different metabolic habit, degree or tendency that determines the
result.

SUMMARY

1. In the development of the ovary of the cat, growth is mainly
peripheral and marginal.

2. Differentiation therefore follows centrifugally.

3. The epithelial elements (parenchyma) occur in the form of
cords.

4. Medullary cords and egg cords are not to be sharply
distinguished.

5. The growth determines the appearance of fairly definite
zones: (a) cortical, (b) intermediate, (c) epithelial stromal.

6. Degenerations occur throughout the period of growth and
in the adult period.

7. In general, the degenerations follow a centrifugal course.

8. The stroma obviously plays an active and important part
In ovarian growth.

9. The primitive cortex is interpreted as directly forming the
definitive cortex containing the primary follicles.

10. No evidence was found of a new formation of ova just prior
to sexual maturity. .
11. Profound degeneration of the early formed Graafian
follicles occurs, being most marked before the advent of sexual

maturity.

378 B. F. KINGSBURY

12. Polar spindles, polar body formation and fragmentation
(abnormal cleavage?) occurs particularly in the atresia folliculi
preceding sexual maturity.

13. The Graafian follicles of the adult period are of a somewhat
different type as compared with those of the growth period (pre-
sexual). Intergradation is, however, obvious.

BIBLIOGRAPHY

An extensive list of literature references is not given. The ovary of the cat
has been a favorite object of investigation: Pfliiger (’63), His (’65), Rouget (79),
Schulin (’81), Janosik (’85, ’88), Giacomini (’87), Coert (90), H. Rabl (98), Ganfini
(07), Sainmont (’05), von Winiwarter and Sainmont (’08), have considered its
structure and development. The last named, however, have so fully considered
the literature upon the development of the mammalian ovary and that of the
cat in particular, that a repetition here would be quite unnecessary. Only the
titles of papers upon the mammalian ovary, referred to in the foregoing brief dis-
cussion, are therefore here included.

ALLEN, B. M. 1904 The embryonic development of the ovary and testis of the
Mammalia. Amer. Jour. Anat., vol. 3, pp. 89-146.

1911 The origin of the sex-cells of Amia and Lepidoeteus. Jour.
Morph., vol. 22, pp. 1-55.

VAN BENEDEN, E. 1880 Contribution 4 la connaisance de l’ovaire des mammi-
féres. Arch. de Biol., vol. 1.

Biuxter, A. 1906 Geschlechtsdriise der Saugetiere, in Handbuch der Entwick-
lungsgeschichte der Wirbeltiere, herausgegeben von O. Hertwig. Vol.
3, 1. pp. 716-742. Jena. ;

Corrt, H. J. 1890 Over de Ontwikkeling en den Bouw van de Geschlachtasklier
bij de Zoogdieren meer in het bijzonder van den Hierstok. Diss. Inaug.
Leiden. 1898

Fretrx, W. 1911 Chapter TX, in Handbuch der Entwicklungsgeschichte des
Menschen, herausgegeben von F. Keibel und F. P. Mall, vol. 2, pp.
857-885. Leipzig.

Janosik, J. 1885 Histologisch-embryologische Untersuchungen tiber des Uro-
genitalsystem. Sitzungsber. d. Kais. Akad. f. Wiss. Wien. Bd. 91,
Abt, 3, pp. 97-199, Math-Naturw. K1.

1888 Zur Histologie des Ovariums. Sitzungsber. d. Kais. Akad. d. |
Wiss. Vol. 96. Abt. 3, pp. 172-192. Math-Naturw. Kl. (December
1887).

Krneassury, B. F. 1911 The histological demonstration of lipoids. Anat Rec.,
vol. 5, no. 6, June, pp. 314-318.

MORPHOGENESIS OF THE MAMMALIAN OVARY 379

Mrnarkovics, V. von 1885 Untersuchungen iiber die Entwickelung des Harn-
u. Geschlechtsapparates der Amnioten,—I. Die Excretionsapparate.—
II. Die Geschlechtsgange.—III. Die Geschlechtsdriisen. Internat.
Monatschr. f. Anat. u. Histol., Bd. 2, pp. 41-106.

Rupascukin, W. 1909 Uber die Urgeschlechtszellen bei Sa&iugetieren. Anat.
Hefte. Bd. 39, pp. 605-652.

1912 Zur Lehre von der Keimbahn bei Siugetieren. Ueber die Ent-
wickelung der Keimdrusen. Anat. Hefte, Bd. 46, pp. 345-411.

Satnmont, G. 1905 Recherches relatives 4 l’organogenése du testicule et de
l’ovaire chez le chat. Arch. de Biol., vol. 22, 1905-1907, pp. 71-162.

WatuperyerR, W. 1870 Eierstock und Ei. Leipzig.

von WintwarterR, H. 1900 Recherches sur l’ovogenése et l’organogenése de
Vovaire des mammiferes (lapin et homme). Arch. de Biol., vol. 17,
pp. 33-199.

von Wintwarter, H., er Sarnmont, G. 1907 Uber die ausschliesslich post-
fotale Bildung der definitiven Eier bei der Katze. Anat. Anz., Bd. 32,
pp. 613-616.
1908 Nouvelles recherches sur l ovogenése et l’organogenésde l’ovaire
des mammiféres (chat). Arch. de Biol., vol. 24, 1908-1909. pp. 1-142;
165-276 ; 373-431 ; 628-650.

PLATE 1
EXPLANATION OF FIGURES

8 Transection of ovary, fetal kitten, 95 mm. (no. 5); rete ovarii, medullary
cords (occupying central portion), egg cords, are shown. The ‘synizetic wave’
occupies approximately the middle of the zone of egg cords. Photograph. XX 35.

9 Longisection of ovary, kitten 3 to 4 days, P. P. (no. 11). The position of
the rete in the cephalic portion (at the right) is illustrated. There are shown
the branched egg cords occupying the primitive cortex and extending down into
the epithelio-stromal zone, the basal connective tissue nucleus. Medullary cords
(in the epithelio-stromal zone) are not shown at this magnification. Photograph.
x 15.

10 Transection of ovary, kitten 14 days, P. P. (no. 20). The zones of the
ovary appear more definite. Medullary follicles beginning. Photograph. X 20.

11 Transection of ovary, kitten, Ca. 4 weeks (no. 22). The medullary fol-
licles developing. In the primitive cortex, the primary follicles are forming.
Photograph. X 20.

12 Transection of ovary, kitten, 5 to 6 weeks (no. 24). The peculiar medul-
lary follicles whose development is illustrated in figures 16 to 25 are here shown
developing. The cortical egg cords are now dissolved into primary follicles,
the primitive cortex becoming the definitive cortex. Photograph. X 15.

13 Transection of ovary, kitten, 3 months (no. 35). The irregular, pluri-
ovular medullary follicles are now large. Their irregularity and pluri-ovular
character are indicated. Compare figures 31 and 32. The cortex is occupied by
primary follicles. Two sections of rete ovarii are shown in the center. Photo-
graph. X 12.

380

MORPHOGENESIS OF MAMMALIAN OVARY PLATE 1

B. F. KINGSBURY

o8l

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 3

PLATE 2
EXPLANATION OF FIGURES

14 Transection of ovary, young virgin adult cat (no. 45). The Graafian folli-
cles of the presexual period have practically disappeared and those of the adult
period are developing. The zona parenchymatosa is well indicated by the ova-
rian stroma. Photograph. X 15.

15 Section of ovary, adult cat (no.51). The surface of a corpus luteum is cut.
The zona parenchymatosa is occupied by the Graafian follicles in varying stages
of development. The zone of primary follicles (cortex) is best indicated at the
right and left. Photograph. X 12.

16 Two primary follicles with adjacent nests of follicle cells. From the inter-
mediate zone of ovary no. 20 (14 day), showing ‘stage I’ in the development of the
‘irregular medullary follicles. Photograph. % 300.

17 Developing medullary follicles, ‘stage 2.’ Ovary no. 29. Photograph. X 250.

18 Developing medullary follicle, ‘stage 3.’ Ovary no. 29. Photograph. X 260.
A nodule of stroma is included between the investing and sheathing follicle
cells. Two groups of interstitial cells are seen at the right, between them a small
cluster of follicle cells.

19 Medullary follicle (young), ‘stage 4.’ The line of junction of the invest-
ing follicle mass is shown at the lower right hand corner (arrow). Photograph.
* 250.

20 Similar to figure 19. The line of junction may be seen (7). Note also a
nodule of included stroma (s). Photograph. 250.

382

MORPHOGENESIS OF MAMMALIAN OVARY PLATE 2

B. F. KINGSBURY

383

PLATE 3
EXPLANATION OF FIGURES

21 Medullary follicle, illustrating the investment by the follicle mass. A group
of stroma cells in the form of interstitial cells are being included (7). Photograph.
<250!

22 Irregular cumulus oophorus from a medullary follicle. The line of junction
is still indicated (7). A small group of included stroma cells is also shown (s).
Groups of interstitial cells le external to the follicle (¢). Photograph. X 250.

23 The development of medullary follicles. The elongated form of some of
the follicle masses is illustrated. Four ova are related to the one shown, each with
its peculiar sheathing follicular epithelium, two of them being cut through the
nucleus. Photograph. »X 60.

24 Two developing medullary follicles. Note the stromal investment of each.
Groups of interstitial cells are shown interspersed. Photograph. X 100.

._ 25 Two developing medullary follicles. Nodules of stroma are included
between the investing and sheathing follicular epithelium. Photograph. 100.

26 Mitotic spindle (polar spindle?) in a degenerating ovum. Ovary no. 39.
Photograph. X 250. ;

27 Polar body. Degenerating ovum. From a section adjacent to that of fig-
ure 26. Photograph. X 250.

28 Ovum degenerating by fragmentation. A single nucleus is shown in the
larger fragment. Two nuclei appear in the fragment immediately to the left of
it. Photograph. X 250 (approx.).

MORPHOGENESIS OF MAMMALIAN OVARY PLATE 3

B, F, KINGSBURY

PLATE 4
EXPLANATION OF FIGURES

29 Drawing of a single follicle grouping from a model of a segment of ovary
no. 26. Two ova are shown each of which is within its own follicular epithelium.
The irregularity of the ‘investing’ follicle mass is shown.

30 An ovum partially invested. From the model as in figure 29. A cordlike
follicular mass lies adjacent to it.

31 Drawing ofa single irregular pluri-ovular follicle from a model of a segment
of ovary no. 35.

32 Asin figure 31. The marked irregularity and cordlike extensions are shown.

386

MORPHOGENESIS OF MAMMALIAN OVARY PLATE 4
B. F. KINGSBURY

387

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THE DEVELOPMENT OF THE CYTOPLASMIC CON-
STITUENTS OF THE NERVE CELLS OF THE CHICK
I. MITOCHONDRIA AND NEUROFIBRILS

E. V. COWDRY
Associate in Anatomy, Johns Hopkins University

From the Anatomical Laboratory of the University of Chicago

FIVE PLATES

CONTENTS
Introduction. ©... 22.42... Werte See. SNE Re ae Te 389
WATE CEA GUEC Gece 3.) s cet) 2) 5 i ee Ee 03g tale a a ae wtela tie See 391
Motteria nancy MmebhOUs..y..<: 5.054 sae ee eR ELA yei- cos cies se ele 33g eielet megane 396
Mitochondria in early stages before the differentiation of neurofibrils: em-
bryos of 0:0, 14 somibes.i75. o: ee i ee ele kee ics le ieee 400
Mitochondria and neurofibrils in later stages: embryos of 15 to 33 somites 402
1. The stage of development at which the first neurofibrils are formed... . 402
2. Type of cell in which neurofibrils first develop.......................-- 403
3. Region of cytoplasm in which neurofibrils originate..............-..... 404
4. Comparison of mitochondria and neurofibrils in embryological series:
embry oso 19; torsll SOMES a ane eee see ole esi. ce a eter 405
| DT ESYEyUIS|S) CCS 1 Wea IM eI Ae Oa TA Oi. o/s. 6 Seco a eS 408
1. Foundation of the theory that neurofibrils are developed from
TEATGARC TON EVAN «1s, asco eeiras eae 5 al hea ag RCA se es 5 ie fen She OR 408
2. Criticism) of the theory. 2) aan. BUaid-/A.0- 5-00' GOR ORO One aNEI ae Raa ERC 0 409
Syabhesonigin of meurohlnils s.r erent ecto ss oe ere cA gle 414
SO LLRNANI NE TEV We sor etd. tay es aula Se Recreate a ok REINA Se tes ova See ac: Sesh 416
IBY!) oO Gyot2} 0) 0 eee EE Ae nk chee kT NG A Cer G5. 4.6.9 dio O's 0.6.0 2 GIR OIRO CREE tor 417
INTRODUCTION

The attention which has been focussed, during the last four
or five years, upon mitochondria as fundamental components of
the cytoplasm of all cells has stimulated investigators in their
attempts to determine the relation which these bodies bear to.
the activities of the cell and to the genesis of the cytoplasmic
constituents. Thus we find that Meves (’07, p. 403) was the

; 389

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 4
JANUARY, 1914

390 E. V. COWDRY

first to claim that they are transformed into neurofibrils. He
brings forward the following generalization (’08, p. 845): that,
with the specialization of the embryo into different organs and
tissues, primitively similar cells assume special functions which
find expression in characteristic structures or differentiations.
All these products, no matter how heterogeneous they may be,
arise through the metamorphosis of one and the same elemen-
tary plasma-constituent, the chondriosomes (mitochondria). Thus
the different filamentous formations, such as the various fibrillar
structures of epithelial cells, the protoplasmic filaments of epi-
dermal cells, the fibrils of smooth and striated muscle, the neuro-
fibrils, neuroglia threads and connective tissue fibrillae are, accord-
ing to his conception, to be classified as products of chondriosomes.

Dr. Bensley pointed out to me certain facts which are incon-
sistent with this hypothesis of the transformation of mitochon-
dria into neurofibrils. The far-reaching biological significance of
this generalization of Meves, relating, as it does, to the funda-
mental properties of protoplasm; and the pressing need for further
light upon the nature of the cytoplasmic constituents of ani-
mal cells, from the standpoint of both ontogenesis and phylo-
genesis, in order that investigations dealing with changes in cells
under different functional conditions may be based on a secure
foundation, have induced me to undertake this investigation.

I therefore decided to follow up my previous paper (712 a) in
which I determined the morphological independence of the mito-
chondria, chromidial substance, canalicular apparatus and neu-
rofibrils in adult spinal ganglion cells of the pigeon, by further
investigations the object of which would be (1) to determine as
far as possible, the origin of the neurofibrils, chromidial sub-
stance and canalicular apparatus; (2) to study the morpholog-
ical relations of these structures to each other in the course of
development; and (3) to elucidate their functional significance.
I have attempted throughout to give a full and accurate descrip-
tion of my findings, to state concisely their bearing upon the
problems involved and to avoid transgressions from the realm of
fact into the domain of theory, because we are at present ham-
pered by a dearth of facts and a multitude of theories.

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 391

I am much indebted to Prof. R. R. Bensley and to Prof. C.
Judson Herrick for their kind advice and invaluable criticism
in connection with this work.

LITERATURE

In this connection we shall consider briefly the literature deal-
ing with the mitochondria in early stages of development, the
theory relating to the transformation of mitochondria into neu-
rofibrils, the regional differentiation of neurofibrils, and finally
certain general points of criticism which apply to the work upon
the development of the cytoplasmic constituents of the nerve
cells of the chick as a whole.

I know of no observations dealing with mitochondria in the
ectodermal structures of chick embryos of less than fifteen hours
incubation (Meves ’08); but since the early stages have not been
characterized by any data except the length of the period of in-
cubation it is difficult to say how early observations have act-
ually been made. Rubaschkin (’10) has described mitochondria
in guinea pig embryos as early as the four cell stage. Moreover
it has been shown that the mitochondria are distributed approx-
imately equally on cell division to the two daughter cells, and
that they are transmitted from one generation to another through
the medium of the egg and in all probability of the sperm also.
On account of considerations such as these the presumption is
warranted that the mitochondria occurring in the nervous sys-
tem of chick embryos are derived from those of the parental sex
cells.

It has already been mentioned that Meves (07, p. 403) was
the first investigator to claim that mitochondria played a roéle
in the formation of neurofibrils. He states subsequently (’08, p.
838), in the description of one of his figures, which was drawn
from a preparation of the spinal cord of a chick embryo of three
days and nine hours incubation and which shows a chain-like
arrangement of the chondriokontes (mitochondria) in the cyto-
plasm of the neuroblasts, that he regards this illustration as evi-
dence that these chains of chondriokontes produce the primitive
neurofibrils, for he believes them to be the same threads which

392 E. V. COWDRY

are blackened by silver impregnation after Cajal. He adds that
the completed neurofibrils (in the later stages of ontogenesis)
cannot be stained by mitochondrial methods in the same way that
the forerunners of the neurofibrils in the cells of the medullary
tube, which stain by silver impregnation from the beginning of
the third day, may be so demonstrated. He believes that there
is a period in development when the neurofibrils may be stained
by mitochondrial methods and also through silver impregnation.

Duesberg (10, p. 612) refers to the researches of Meves and
Hoven regarding the development of neurofibrils from mito-
chondria and remarks that the number of chondriosomes di-
minishes with the increase in the age of the embryo until in the
adult nerve fiber none remain stainable by Benda’s method.
Meves, in a recent paper (’10 a, p. 655), apparently confirms this
observation for he concludes that no chondriosomes remain, as
such.in adult spinal ganglion cells.

Hoven (710) has furnished the most detailed observations in
support of the mitochondrial origin of neurofibrils. He studied
the formation of neurofibrils in the cells of the neural tube and
spinal ganglia of chick embryos by the application of the Benda
method and of Cajal’s silver impregnation method. He found
that the morphology and cytoplasmic arrangement of the chon-
driokontes, as demonstrated by Benda’s method, bears a very
striking resemblance to the appearance of the earliest neurofi-
brils demonstrable by silver impregnation in cells of the same stage
of development. The chondriosomes at this period-form a re-
ticulum of undulating filaments (p. 475). Furthermore, in later
stages the chondriokontes decrease in number as the neurofibrils
increase in amount, and he asks the question if the chondrio-
kontes do not give rise to the neurofibrils what becomes of them?
He believes that the few which do persist in the adult nerve cell
correspond to the internal reticular apparatus of Golgi, the Bin-
nennetz of Kopsch, etc. As corroborative evidence he draws a
close analogy between the formation of myofibrils, as indicated by
Duesberg (’09 and 710), the transformation of chondriosomes
into connective tissue fibrils (Meves 710) and+the formation of
neurofibrils from chondriosomes.

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 393

Marcora (’11, p. 952), so far as I have been able to determine,
was the first investigator to criticise this conclusion and to em-
phasize the following differences between the chondriosomes and
the primitive neurofibrils: (1) that the chondriokontes are short,
thick, wavy filaments, never being continuous with the few chon-
driosomes in the axis cylinder; whereas the neurofibrils are fine,
long, straight threads arranged near the greater process of the
neuroblast and distinctly continuous with the numerous neuro-
fibrils in the axis cylinder; and (2) that it is not possible to stain
distinctively the neurofibrils with the mitochondrial dyes.

Furthermore, Duesberg (’12, p. 745) returns the verdict ‘non
proven’ in the case of the neurofibrils, but maintains that he
has incontestably demonstrated that myofibrils arise in like
fashion from mitochondria.

The regional differentiation of neurofibrils in the chick will
be considered under three headings:

(a) The stage of development at which the first neurofibrils
appear. Besta (’04, quoted from Collin ’06, p. 238) gives the
degree of differentiation as that of sixty-five hours incubation;
Cajal (07, p. 178) fifty-six to sixty hours; Gerini (’08, p. 182)
forty hours, and Hoven (’10, p. 441) 44 somites and seventy-six
hours incubation. Other investigators working prior to these
with more imperfect methods give the date even later.

(b) Type of cell in which neurofibrils first develop. Besta (04,
quoted from Collin ’06, p. 238) and Gerini (’08, p. 182) conclude
that the neurofibrils in the chick are formed in the bipolar cells
of the outer layer of the neural tube; Cajal (’07, p. 178) in the
apolar cells bordering the ventricle (i.e., in preneuroblastic cells) ;
and Meves (’07, p. 403) and Hoven (10, p. 478) assert that they
are first differentiated in the cytoplasm of the neuroblasts.

(c) Region of the cytoplasm in which neurofibrils originate.
Investigators are apparently agreed that the neurofibrils in the
chick are first formed in a definite, restricted zone of the cyto-
plasm; for Besta (04, quoted from Collin ’06) claims that they
are formed in the protoplasmic substance about the nuclei;
Cajal (07, p. 178) that they are differentiated in a network
which arises in a fibrillogenous area in the distal portion of the

394 E. V. COWDRY

cell; Meves (’07, p. 838) that the chains of chondriokontes in the
ax's cylinder process and in the adjacent portion of the cytoplasm
are converted into neurofibrils; Gerini (’08, p. 182) that they are
formed at either pole of the nucleus from minute granules which
stain deeply by Cajal’s method; and, finally, Hoven (10. p. 475)
asserts that the neurofibrils are generated from a reticulum of
mitochondrial filaments in the cytoplasm of the neuroblasts and
ganglion cells.

Most of the work dealing with the development of the cyto-
plasmic constituents of the nerve cells of the chick seems to me
to be open to criticism on the basis of the following considera-
tions:

(1) Point of view. Goldschmidt’s hypothesis (’09, p. 107) that
mitochondria belong :to the category of chromidial apparatus
(Fauré-Frémiet ’10, p. 483); Smirnow’s conception (’07), am-
plified by Hoven (’10, p. 479), of the similarity of the mitochon-
dria and the reticular apparatus; and, lastly, Meves generaliza-
tion (08, p. 845) that the fibrils in various types of cells are
developed from mitochondria, forwarded as they were before
definite proof was forthcoming, rendered the dissociation of the
cytoplasmic constituents more difficult for investigators working
on morphogenesis.

(2) The confusion which has arisen by the application of many
ill-chosen terms to a definite and concrete class of cell granulations
called mitochondria by Benda in 1899. Among these may be men-
tioned ‘Chondriosomen’ (granules), ‘Chondriokonten’ (thread-
like granules), ‘Chondriomiten’ (granules distributed in rows) and
‘Chondriom’ (the cellular content of chrondiosomes). Meves has
devised yet another series, ‘Plastosomen,’ ‘Plastochondrien’ and
‘Plastoconten.’ When one considers in addition the vast array
of names applied in the older literature to mitochondria, such
as for instance cytomicrosomes, interstitial granules, Flemming’s
fila, Altmann’s bioblasts, fuchsinophile granules; although at the
present day we recognize that each of them comprises also many
granules which are not mitochondria, so that none of them may
be regarded as synonyms for mitochondria, and also the fact
that each term is modified more or less depending upon the na-

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 395

tionality of the investigator using it (ef. Chondriokonten, chon-
driocontes, condrioconti) it becomes at once evident that any
additional terms only complicate the situation, especially when
they have been devised to proclaim some functional interpretation.
Moreover, we have no assurance that the slight differences im-
plied by these terms will be appreciated by the majority of the
investigators of the future as well as those of the present. It
would be much safer and simpler to make use of the word ‘mi-
tochondria,’ recognising that similar granules may assume differ-
ent shapes under varying functional conditions, such as rate of
multiplication, surface tension, and so forth, because by so doing
we need not subscribe to any theoretical considerations respect-
ing their significance.

(3) The inadequacy of the technique employed. Meves’ gen-
eralization was apparently based, in the first instance, on the
application of a slightly modified iron hematoxylin method of
technique alone. The fact that Michaelis demonstrated in 1899
that mitochondria could be stained specifically by janus green,
and that Bensley recognized the importance of this discovery,
introduced it into histological technique and repeatedly called
attention to it in his publications (1910 and again in 1911) has
been completely ignored. It is true that we have not yet got
a satisfactory dye for the neurofibrils and the canalicular appa-
ratus, and that we cannot in fixed tissues even, demonstrate, in a
constant fashion, the neurofibrils and the mitochondria specifically
stained in the same cell. Thus we are frequently forced to piece
together in our mind’s eye the relations of the different cytoplas-
mic constituents to each other although we have to study them
singly in separate preparations. Pending the discovery of vital
dyes and synthetic methods all the available methods of tech-
nique must be employed so that the popular pitfall of supposing
that one or two methods alone will tell the whole story may be
avoided. Even when these precautions have been taken, the
biologist should stop to consider what a multitude of factors
associated with the structure of cells the microscope still fails
to reveal to us.

396 E. V. COWDRY

(4) The absence of controls. The different stages in the em-
bryological series have not as a rule been sufficienty accurately
characterized. In some contributions the only criterion has been
the number of hours of incubation, in others the length of the
embryos alone, so that it is frequently difficult to tell what stage
the investigator in question is describing. Furthermore, it has
been overlooked that synthetic comparisons between the struc-
tures of cells which do not occur in the same parts of the nervous
system of embryos of the same degree of differentiation are not
valid, since all regions of the nervous system are never uniformly
developed.

MATERIAL AND METHODS

The material consists solely of chick embryos and is in part
summarised in table 1. The degree of specialization of the em-
bryos was determined by a consideration of: (1) the number of
mesodermic somites, (2) the period of incubation at 39°C., (3)
the length (measured before removal from the egg) and (4) a
comparison of their external morphology with the different stages
figured and described in Duval’s Atlas d’embrylogie and in Kei-
bel and Abraham’s Normentafel zur Entwicklungsgeschichte
des Huhnes (Gallus domesticus). Reference was frequently made
to Lillie’s text-book on the development of the chick, and to
Keibel’s Normentafel zur Entwicklungsgeschichte des Kiebitzes.

The methods include the observation of living tissue, with
and without the use of vital dyes; and of the impregnation and
staining of fixed material, imbedded in paraffin and in celloidin.
Each embryo was fixed, dehydrated and imbedded individually.
The technical methods employed are given in table 2.

In the study of living tissue the portion of the nervous system
under investigation was isolated in warm salt solut'on under the
binocular microscope and immersed in the staining fluid. The
stains were generally used in a concentration of 1 : 10,000 of 0.85
per cent sodium chloride solution. Preparations were examined
from time to time until the desired intensity of coloration had
been attained.

397

NERVE CELLS

OF

CYTOPLASMIC CONSTITUENTS

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398 E. V. COWDRY
TABLE 2
Methods
FOR METHOD FIXATION
intravitam

mitochondria (1) Michaelis-Bens-
ley |

(2) Meves’ 1908, p.|
832

| (3) Benda’s Hoven,

1910, p. 430

STAIN

janus green 1:10,000

Flemming’s fluid
(modified)

Flemming’s fluid
(modified)

(4) Bensley’s anilin
fuchsin methyl
green 1911, p. 309 |

acetic osmic bichro-
mate mixture

iron hematoxylin
with or without
erythrosin

alizarin crystal vio-
let

anilin fuchsin with
methyl green, tolu-
idin blue, or meth-
yleneblue eosinate
or methylene blue

erythrosinate

(5) Bensley’s copper
chrome hematox-
ylin, 1911, p. 310 |

acetic osmic bichro-
mate mixture

neurofibrils (1) Paton’s modifica- |

hematoxylin

| silver nitrate
tion of Bielschow- |
sky’s method, 1907,
p. 576 |
== | ="
!
(2) Cajal’s formula | silver nitrate none
I, 1907a, p. 215 |
chromidial | (1) Carnoy, ete. toluidin blue and
substance | erythrosin
canalicular | (1) Kopsch, 1902 _ osmic acid | none
apparatus | | |
controls (1) formalin, neutral for-| toluidin blue ery-

malin, formalin
acetic, chrome sub-
limate, sublimate
acetic, sublimate

formalin acetic,
picro-sulphuric,
osmic acid, nitric

throsin; hematoxy-
lin; mitochondrial
dyes, etc.

acid, etc.

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 399

Embryos imbedded in paraffin were cut in serial sections by
means of a Minot rotatory microtome. The great majority of
the series were cut transversely; but others were made in coronal
and sagittal planes so that the one-sided conceptions so likely to
arise from the perusal of sections cut in a single direction, have
been eliminated. All the material imbedded in paraffin was cut
into sections 4 u thick.

The celloidin method of imbedding was employed, in the case
of several embryos, on account of the objections raised by cer-
tain authors (Cajal ’07, p. 172, et al.) to paraffin. Complete
series were made about 7 u» thick by the technique advocated by
Maximow (’09, p. 184).

This investigation has been controlled as follows:

(1) The entire nervous system has been studied; but detailed
comparisons have only been made between the results of differ-
ent methods in the neural tube and the neural crest on the right
side in the region of the sixth somite of embryos of the same
degree of differentiation.

(2) The commoner artefacts due to fixation are shrinkage, dis-
tortion of form relations, and destruction, solution or change in
the nucleus and cytoplasmic constituents. Shrinkage is de-
pendent, to some degree, upon the water content, and since this
probably varies in different regions we must look for unequal con-
traction. The diminution in the longitudinal axis was measured
in the case of the mitochondrial and neurofibrillar fixatives; in
the former it amounted to 23 per cent, in the latter to 19 per ~
cent; (compare the size of the nuclei represented on plates 3
and 4). The finer form relations, as seen after fixations in vogue
for mitochondria and neurofibrils, were controlled by comparison
with the results obtained after a large number of fixations, es-
pecially adapted for their accurate delineation, and with living
tissue teased out in warm physiological salt solution. It ap-
pears that the shape of the cells is but little altered by the fixa-
tions, and I am inclined to believe that, in the chick, the difficulty
encountered in the observation of cell boundaries is in some meas-
ure due to shrinkage. Moreover, Harrison (’10, p. 793) states
that the cells in the neural tube of frog embryos are perfectly

400 E. V. COWDRY

distinct, and ascribes the difficulty experienced by investigators
in the demonstration of cell boundaries to inadequate technique.
I have already (’12 a, p. 486) mentioned the precautions neces-
sary in the fixation of the cytoplasmic components of adult nerve
cells and the same apply without reservation to the embryo.

(3) The inevitable contraction, resulting from dehydration has
been reduced by decreasing the period of immersion in the grades
of aleohol to a few minutes only, and by the use of bergamot oil
for clearing, which renders the use of absolute alcohol unnec-
essary.

(4) It is difficult to dissociate the effects of fixation and imbed-
ding. The shrinkage due to imbedding in paraffin and celloidin
was slight. I fail to see any very great or constant differences in
the appearance of .tissues treated by these two methods. In my
experience the paraffin method does not produce any more arte-
facts than the celloidin method.

(5) Sectioning, if carefully carried out, does not produce se-
rious artefacts (Cowdry ’12 b, p. 491).

(6) The methods of impregnation and staining control each
other, for the neurofibrils appear to be essentially similar in spec-
imens prepared by Cajal’s and Paton’s technique, and the mito-
chondria in preparations made by the methods enumerated in
table 2.

MITOCHONDRIA IN EARLY STAGES BEFORE THE DIFFERENTIATION
OF NEUROFIBRILS: EMBRYOS OF 0 TO 14 SOMITES

Both filamentous and granular mitochondria undoubtedly ex-
ist in the nerve cells of the chick in these early stages, but the
filamentous form is much more numerous. The illustrations (figs.
1 to 5) tend to minimize the uniformity in the morphology of the
mitochondria on account of the fact that the long axes of the
mitochondrial filaments are directed in all planes relative to the
drawing and that they are cut in pieces of varying size at all
angles during sectioning. Most of the mitochondria are of neces-
sity oriented parallel to the long axis of the cells in which they
occur. The true filamentous nature of the mitochondria is there-
fore obscured in sectioned tissues, particularly in sagittal series

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 401

where the sections are cut in most instances transversely to the
length of the cells with the result that the mitochondria appear
either uniformly granular or as short rods depending upon the
thickness of the section in question. The preponderance of the
filamentous form of mitochondria may be conclusively demon-
strated in specimens stained with janus green intravitam.

The morphology and cytoplasmic arrangement of mitochondria
are represented in figures 2, 3, and 1, which illustrate progres-
sive stages in development. ‘The specimens from which these
figures were drawn were prepared by Meves’ modification of the
iron hematoxylin method so that the mitochondria appear a
dark blue-black color and may be readily distinguished from the
dark browinsh black yolk globules. The first (fig. 2) shows epi-
thelial cells from near the middle line of the medullary plate of
an embryo of twenty-four hours incubation at 39°C. in which
the primitive groove fold and pit alone were differentiated. The
mitochondria are seen in the form of filaments and granules dis-
tributed evenly throughout the cytoplasm, whereas the yolk
spherules show a tendency to become grouped together in the
distal, (i.e., remote from the membrana limitans interna) por-
tions of the cells. The second (fig. 3) illustrates the appearance
of epithelial and germinative cells from the neural fold in the
region of the head process of an embryo of 3 somites and twenty-
four hours incubation. The amount of yolk material is reduced
and the mitochondria are far more abundant in the distal than
they are in the proximal parts of the cells. I have not, however,
been able to distinguish a similar heaping up of mitochondria
in cells stained by janus green intravitam (fig. 23). The third
drawing (fig. 1) is from the ventral half of the neural tube in the
region of the tenth somite of an embryo of 12 somites and thirty-
four hours incubation. The mitochondria are here distributed
evenly in the cytoplasm and the yolk gloubules tend to be accu-
mulated in the distal portions of the cells, just as in figure 2.

There are two points of considerable significance from the
point of view of the theory of the transformation of mitochondria
into neurofibrils. The first is that there is little, if any, variation
in the morphology or staining reactions of mitochondria in these

402 E. V. COWDRY

early stages; and the second that these mitochondria in the cells
of the neural tube apparently do not differ in any way, capable
of detection by the methods of technique now in use, from the
filamentous and granular mitochondria occurring in the struc-
tures derived from the other two germ layers (i.e., mesoderm and
endoderm).

MITOCHONDRIA AND NEUROFIBRILS IN LATER STAGES: EMBRYOS
OF 15 TO 33 SOMITES

1. The stage of development at which the first neurofibrils are formed

I find that neurofibrils are first formed in the nervous system
of a chick embryo, the differentiation of which may be charac-
terized as follows: Somites 15, length 5.8 mm., incubation at
39°C + 40 hours (being slightly more advanced than Duval’s
embryo of 33 hours, fig. 268, p. 56). In such an embryo the neuro-
fibrils are most abundant in the marginal neuroblasts in the
hind-brain opposite the otic invagination (fig. 11). A few cells
containing them may be seen in the midbrain (fig. 12); but in the
forebrain the only indication of their presence is a more or less
continuous network, blackened with silver nitrate, in the distal
(remote from the membrana limitans interna) portions of the
cells (figs. 10 and 12). Traces of them occur further caudad in
the cells of the neural tube as far back as the fifth somite. There
is a large area comprising the posterior portion of the midbrain
and the anterior part of the hind-brain in which no neurofibrils are
differentiated, even in the stages as far advanced as 20 somites.

Since the neurofibrillar methods of technique employed by me
depend upon impregnation with silver nitrate, they are not suf-
ficiently accurate to permit of complete enumeration of the cells
in the different parts of the nervous system containing neurofi-
brils, and I have therefore been prevented from studying the
regional differentiation of neurofibrils on a percentage basis and
comparing my results with those obtained by Paton (’07) and
Coghill (09, ete.) who worked on fish and amphibian embryos:
respectively.

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 403

In general, the earliest neurofibrils appear to be formed in
chick embryos in three chief localities: (1) in the hind-brain oppo-
site the otic invagination (stages of 15 somites on); (2) in a center
on either side of the extreme anterior end of the midbrain
(stages of 18 somites and on); and (3) in the nuclei and root
fibers of the cranial nerves (from stages of about 15 somites on).
They are not differentiated until very much later (i.e., beyond
31 somites stages) in spinal ganglion cells.

2. Type of cell in which neurofibrils first develop

I have found neurofibrils in bipolar cells, in apolar cells, in
marginal neuroblasts and in certain cells which occur within the
cavity of the neural tube.

The neurofibrils in a typical bipolar cell are illustrated in fig-
ure 7. The cell is located on the right side of the hind-brain op-
posite the otic invagination of an embryo of 24 somites, length
7.9 mm., and sixty-five hours incubation, prepared by Cajal’s
silver nitrate method. The cytoplasm is stained a little bit
darker yellow in the distal portion of the cell and the neurofibrils
appear thicker and more continuous than in the proximal part.

The neurofibrils in apolar cells bordering the central canal
are best shown in figure 22. This specimen was prepared by
Paton’s modification of the Bielschowsky silver impregnation
method. The cells illustrated occur in the neural tube opposite
the posterior part of the third somite of an embryo of 33. so-
mites, length 8.3 mm., and seventy-four hoursincubation. At aa
continuous circumnuclear neurofibrillar network is seen in black
against a dull red granular background. The appearance of such
a neurofibrillar network in apolar cells is of distinctly rare oc-
currence. No networks of this nature were observed in embryos
of 15 somites, which is the stage of development at which the
neurofibrils first appear.

Marginal neuroblasts containing neurofibrils may be readily
found in embryos of all stages intermediate between 15 and 32
somites, and they probably occur in further developed embryos
also. Figure 11 shows the primitive neurofibrils in a marginal

404 E. V. COWDRY

neuroblast from the hind-brain on the right side opposite the otic
invagination of an embryo of 15 somites, of 5.8 mm. in length
and forty hours incubation prepared by Cajal’s silver nitrate
method. The neurofibrils occur throughout the cytoplasm which
has assumed a rather dark yellowish tint. Figure 8 shows the
neurofibrils in a cell of the same type in a more advanced embryo.
Here the neurofibrils seem to be of finer diameter and more con-
tinuous, while the ground substance of the cytoplasm is of a
distinctly lighter and more brilliant yellow tint.

Neurofibrils also occur in cells which seem to lie within the
lumen of the neural tube, and in the processes of cells which
extend along the ventricular border of the central canal. Fig-
ure 9 has been drawn from the left side of the hind-brain opposite
the otic invagination of an embryo of 24 somites, length 7.5 mm.
and sixty-five hours incubation treated by Cajal’s silver nitrate
method. The cell (a) is apparently within the cavity of the
neural tube, and the process (b) of the cell (c) extends toward the
membrana limitans interna, all three of which contain well dif-
ferentiated neurofibrils. Figure 6, which represents a cell from
the same region of the same embryo, shows a similar condition.
Appearances such as these are distinctly rare, seem to be
restricted to the region of the hind-brain and do not obtain in
embryos during the early stages of neurofibrillar formation.

3. Region of the cytoplasm in which neurofibrils originate

Figures 10, 12 and 14 are from an embryo of 15 somites, length
5.8 mm. and ineubation forty hours at 39°C., which represents
the stage of development at which neurofibrils may first be
distinguished. All of these three figures show that the neuro-
fibrils are first formed in the immediate neighborhood of the
nuclei. The fact that these primitive fibrils are in the form of a
network is best illustrated at a figure 14. In the cells containing
neurofibrils the distal region of the cytoplasm (i.e., that near the
membrana limitans externa) stains a darker yellowish brown
color than does the more proximal part, on the opposite side
of the nuclei, whereas in cells without neurofibrils the cytoplasm

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 405

stains evenly throughout. It is in this distal, highly colored, por-
tion of the cytoplasm that the neurofibrils are later first completely
differentiated. It should be noted, in passing, that certain
portions of the cells of the adjacent myoblasts likewise stain a
similar dark yellowish brown color with silver nitrate.

4. Comparison of mitochondria and neurofibrils in embryological
series: embryos of 19 to 31 somites

It has already been mentioned that we have not at present
a technique at our disposal by means of which the mitochondria
and neurofibrils may be stained differentially in the same cell in
a constant and trustworthy fashion. For this reason a synthesis
must be resorted to. With this in mind I have endeavored to
build up a synthetic picture of mitochondria and neurofibrils as
they appear in cells of increasing degrees of differentiation. Two
series of embryos extending from stages of 19 to 31 somites, each
member of a series differing from the preceding by the acquisi-
tion of a single mesodermic somite, were employed, one being
prepared by Meves’ iron hematoxylin method for mitochondria
(figs. 15 a, etc.) the other by Cajal’s silver nitrate method for
neurofibrils (figs. 15 b, ete.). Four additional methods of tech-
nique were used for control, namely, janus green intravitam, Bens-
ley’s anilin fuchsin methyl green method, the Benda method
and Paton’s modification of the Bielschowsky silver impregna-
tion technique. All embryos were cut in complete serial trans-
verse sections. In making the comparisons morphological equiv-
alents (of approximately the same area and thickness, 4 ») alone
were used, or, in other words, cells occurring on the right side
of the neural tube opposite the sixth somite in embryos of the
same degree of differentiation. The two chief parallel series,
which are in part represented in plates 3 and 4, afford a fairly
secure basis for the comparison of the form, distribution, stain-
ing reactions and relative amount of mitochondria and neurofibrils
in developing nerve cells.

a. Form. The shape of mitochondria is, to some extent, de-
termined by the configuration of the cells containing them. Thus

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 4

406 E. V. COWDRY

in the rounded neuroblasts, in apolar cells, and in the irregular
shaped cells within the lumen of the neural tube they are either
in the form of granules or of straight or slightly curved rods with
rounded ends; whereas, in the bipolar cells they are longer and
straighter. I have never observed any network formation or
fusion of mitochondria in fixed preparations or in specimens
stained with janus green intravitam, although I have searched
carefully for it in the region of the axone and axone hillock. It
should again be emphasized that the illustrations tend to mini-
mize the uniformity in the morphology of the mitochondria on
account of the fact that their long axes are directed in all planes
relative to the drawing and that in sectioning they have been
cut at all angles.

The mitochondria show no variation in their morphology of
which abundant examples may not be seen in neighboring struc-
tures derived from mesoderm and endoderm. Neither do they
differ in shape from the mitochondria occurring in the neural
tube before the formation of any neurofibrils.

The appearance of neurofibrils is fairly constant in strictly
homologous cells of these four types. They are apparently
continuous, the mitochondria discontinuous: they seem to form
a reticulum, whereas the mitochondria never lost their individ-
uality; and finally the neurofibrils are of very fine diameter and
irregular outlines, in spite of the fact that the tendency of im-
pregnation methods is by precipitation, and in other ways, to
add to the size of the structures demonstrated.

b. Distribution. The mitochondria, like the neurofibrils, are
generally arranged parallel to the long axis of the cell, as in the’
case of the neuroblasts, the bipolar and epithelial cells. Occa-
sionally the neurofibrils are arranged in a whorl-like concentric
fashion about the nucleus, but the mitochondria are not simi-
larly oriented. Both neurofibrils and mitochondria occur in all
parts of the cytoplasm. In some specimens, stained with iron
hematoxylin, the mitochondria gather more thickly in the pe-
ripheral parts of the cell, although such an accumulation is not
apparent in cells stained with janus green intravitam (figs. 23 and
24),

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 407

c. Staining reactions. No variations or change could be
detected in the results of fixation or in the staining reactions of
mitochondria coincident with the development of neurofibrils.
Their degree of solubility in acetic acid is apparently constant,
and throughout the series of preparations (which of course is
much more complete than it was possible to represent on the plates)
they appear a dark blue black color, which is the same in figure
15 a, at a stage when no neurofibrils are differentiated, as it is
in figure 20a after a large nunber of neurofibrils have been
formed. Moreover, the staining reactions of mitochondria in the
neural tube of this series of preparations, running parallel as they
do to the differentiation of neurofibrils, do not differ, in any ap-
preciable way, from those of mitochondria in other neighboring
tissues of the same embryos. They are likewise similar to those
of mitochondria occurring in the neural tube before the differen-
tiation of any neurofibrils. The only variation in the staining
reactions of the neural tube in this series seems to be in the case
of the background and to be dependent upon the length of dif-
ferentiation in iron alum and the duration of the subsequent
washing in tap water.

It is very difficult for me to determine whether a change in
the reaction of neurofibrils to silver nitrate during the course of
development actually exists in my preparations. The varia-
tions in color which I have observed may be due to a host of
possible factors among which the duration and temperature of
impregnation, the presence or absence of light, and so forth, may
be mentioned. I have absolutely no reliable evidence, therefore,
of any change in the chemical composition of neurofibrils from
the time that they are first laid down to the most highly special-
ized embryos in my series.

d. Relative amount of mitochondria and neurofibrils... As I have
already stated, the figures on plates 3 and 4 constitute the essential
features of a comparison of the results yielded by Meves’ iron
hematoxylin method and by Cajal’s silver nitrate technique

‘The relatively smaller size of the nuclei in figures 15a to 20a is indicative

of the fact, already mentioned (p. 399), that the shrinkage is greater in the mito-
chondrial preparations than it is in the neurofibrillar ones.

408 E. V. COWDRY

respectively. In both cases, drawings have only been made from
sections of the neural tube on the right side opposite the sixth
somite, and the degree of specialization of the embryos from
which the figures on plate 3 were drawn corresponds severally
with that of those represented on plate 4, figure 15a to figure
15b, and so on. The preparations show that in progressive
stages in the differentiation of the cells of the neural tube there
is no decrease in the amount of mitochondria parallel to the
increase in neurofibrillar material.

DISCUSSION

1. Foundation of the theory that the neurofibrils are developed from
mitochondria

This hypothesis is based to some extent upon the following
statements, which have been advanced, in whole or in part, by
the investigators named.

a. That the neurofibrils increase in amount as the mitochon-
dria decrease until finally the adult condition is attained in which
the neurofibrils are completely differentiated and the mitochon-
dria absent. (Duesberg ’10, p. 512; Hoven ’10, p. 478; and
Meves 710 a, p. 655).

b. That microchemical transitions exist between mitochondria
and neurofibrils, since the primitive neurofibrils may first be
stained by mitochondrial methods, then by both mitochondrial
and neurofibrillar methods and, finally, by the various neurofi-
brillar methods of technique alone (Meves ’08, p. 838; Hoven
10, p. 478, etc.).

e. That morphological transitions also exist between mito-
chondria and neurofibrils: according to Meves (708, p. 838)
chains of mitochondria are transformed into neurofibrils; ac-
cording to Hoven (710, p. 475) the mitochondria form a reticulum
from which the neurofibrils are differentiated.

d. That the development of the myofibrils, connective tissue
fibrils and the fibrils in epithelial cells support this theory since
they, in a similar fashion, are developed from mitochondria.
This constitutes the argument from analogy (Benda ’99; Meves
07: Duesberg 710; Meves ’10; Firket ’11 and Duesberg ’12).

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 409
2. Criticism of the theory

The material and methods upon which the statements are based.
In many cases the embryological series have been made up of
embryos of different types of animals. It is a well-known fact
that similar portions of the nervous system of different animals
are often not differentiated at the same rate, so that comparisons
made in a heterogeneous series are not valid. I know of no in-
vestigator who has clearly and concisely described either the ex-
tent or the composition of his series of embryos. The criteria
used for the characterization of the different developmental
stages have been lamentably deficient. Every biologist knows
that embryos of apparently the same period of incubation may
differ widely in their degree of differentiation. This is particu-
larly true in the case of eggs which have been bought from
dealers and about which we consequently know but little.

Complete series, cut in transverse, sagittal and coronal planes,
have not, as a rule been studied; and care has not been taken,
in forming a synthetic picture of the cytoplasmic constituents,
to draw material only from strictly homologous cells (Herrick ’09)
of the same degree of differentiation. Few methods of technique
have as arule been taken advantage of, and, in some instances,
conclusions have been deduced solely from the application of a
single method. In the majority of investigations no reference is
made to the use of subsidiary methods designed for the control
of the finer form relations, and resort has never been made to
vital dyes.

The statements. (1) My own observations are utterly at vari-
ance with the first argument, for I find that there is no decrease
. in mitochondria running parallel to the formation of neurofibrils.
Moreover the statement that mitochondria are absent in the
adult condition is wholly unwarranted in view of the fact that
several investigators have unquestionably demonstrated mito-
chondria in adult nerve cells. Altmann, as far back as 1890,
described and figured them in spinal ganglion cells of the frog
(fig. 2), and in the Purkinje cells of the cerebellum of the cat (fig.
3) as well as in the brain wall and in the medullary tube of cat

410 E. V. COWDRY

embryos (figs. 1 and 2). Held also has demonstrated mitochon-
dria by both the Altmann method (97, figs. 1 and 2) and the
iron hematoxylin technique (’97 a, fig. 10) in adult nerve cells.
In addition I, myself, have shown (1912 a and 1912 b) that mito-
chondria are present in large numbers in adult spinal ganglion
cells of the pigeon.

(2) The second statement postulates the existence of three
distinct phases in the developing neurofibril, each of which is
characterized by certain microchemical properties. In the
first stage the primitive neurofibrils may, it is said, be stained by
mitochondrial methods, in the second by both mitochondrial
and neurofibrillar methods and in the third by the various
neurofibrillar methods of technique alone.

I find that neurofibrils in chick embryos may be stained by
such mitochondrial methods as the iron hematoxylin method of
Meves (fig. 4), Benda’s method, the anilin fuchsin methylene
blue erythrosinate (fig. 27) and the anilin fuchsin toluidin blue
(figs. 21, 26 and 25) methods, of Bensley; but the staining is not
specific and seems to depend upon the degree of differentiation.
A comparison of figures 21, 26 and 25 will show that this is the
case with respect to the last mentioned method. These three
figures have been drawn from neighboring sections of the same
embryo, mounted on the same slide and prepared by Bensley’s
- anilin fuchsin toluidin blue method. In the first (fig. 21) the
differentiation is practically nil, the mitochondria staining ex-
actly the same color as the neurofibrils; in the second (fig. 26)
it has been carried a little further with the result that the neuro-
fibrils have lost their bright crimson color and have assumed a
dull red shade; while in the last (fig. 25) the decolorization has
been carried to an extreme so that the neurofibrils have lost all
of the acid fuchsin and have become stained with the differentia-
tor, toluidin blue. It is to be noted that, in these progressive
stages of differentiation, the initial affinity of the neurofibrils for
an acid dye (acid fuchsin) in which they resembled mitochondria,
is gradually changed to an affinity for a basic dye (toluidin blue),
while the intensity of the coloration of the mitochondria with
the acid fuchsin remains unaltered. Furthermore, the fact that

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS All

this coloration of the neurofibrils by mitochondrial dyes is marked
in adult cells (Cowdry 712 b, figs. 1, 2, 5, 7 and 13) should be taken
into consideration before regarding it to be indicative of transi-
tions between mitochondria and the primitive neurofibrils.

Let us now consider the statement that the primitive neuro-
fibrils may be stained by both the mitochondrial and the neuro-
fibrillar methods (i.e., the second phase). The completeness of
the demonstration of mitochondria by the iron hematoxylin
method depends upon the presence in the fixative of chromic acid,
osmic acid and acetic acid, in suitable amounts, and on the mor-
danting action of iron alum; while their complete absence in the
neurofibrillar preparations is due to the unmodified action of sil-
ver nitrate. The neurofibrils seem to have a specific affinity for
silver nitrate, upon which all silver impregnation methods de-
pend. So that it is extremely unlikely, especially in the absence
of direct evidence, that so widely divergent methods stain the
same thing, namely, the primitive neurofibrils, for if the primi-
tive neurofibrils may be stained by both methods, they must of
necessity partake of the microchemical properties of both mito-
chondria and neurofibrils which are, to some extent, mutually
incompatible.

Finally, the neurofibrils are said to enter on a third phase in
their history characterized by the loss of their affinities for mito-
chondrial dyes. I have.nevertheless failed to find any conclusive
evidence that the neurofibrils change their chemical composition
after their first formation. My failure may be due to the un-
standardized condition of the neurofibrillar methods of tech- |
nique which still prevails. In any case the burden of supplying
the evidence rests with those who make the statement.

If neurofibrils are formed by a chemical transformation of
mitochondrial substance into neurofibrillar material, one would
expect to find variations in the effects of fixation and in the
staining properties of mitochondria during the formation of
neurofibrils. I have shown that the exact converse obtains. Both
the solubility of mitochondria in acetic acid and their staining
reactions in the cells of the neural tube in which neyrofibrils are
being formed are remarkably uniform and constant. More-

412 E. V. COWDRY

over these properties apparently differ in no wise from those of
mitochondria in the neural tube before the formation of neuro-
fibrils or from the mitochondria in other embryonic cells.

It is evident therefore that the facts do not justify the state-
ment that microchemical transitions exist between mitochondria
and neurofibrils.

(3) With respect to the evidence for morphological transitions
between mitochondria and neurofibrils I would state that I have
failed to confirm Meves’ contention that chains of mitochondria
are transformed into neurofibrils. Mitochondria are sometimes
oriented end to end and one may often observe very long fila-
mentous mitochondria, like those represented in figure 4, for
instance. It is however a very far cry from either a linear ar-
rangement of mitochondria or from long filamentous mitochon-
dria to neurofibrils. This is manifested, among other things, by
the fact, already mentioned, that there is nothing peculiarly dis-
tinctive about the morphology or the arrangement of mitochon-
dr'a in the cells of the neural tube during neurofibrillar forma-
tion: they are alike indistinguishable, on the basis of their
morphology and distribution, from the mitochondria in the neural
tube in stages prior to the differentiation of neurofibrils, and
from the mitochondria occurring in other embryonic tissues both
before and contemporaneous with the development: of neuro-
fibrils. So that on the ground of morphology and cytoplasmic
arrangement of mitochondria there is just as much evidence for
the formation of neurofibrils in structures derived from meso-
derm and endoderm as there is in the case of the neural tube.

Since Hoven’s own figures do not show a reticulum, but rather
an interlacing of independent mitochondrial filaments, and since
I have been unable to discover a reticulum composed of mito-
chondria in any of my preparations, the presumption is war-
ranted that the mitochondria do not lose their individuality by
coalescing to form a network.

The conclusion is likewise justified that the facts, so far as
we know them do not support the statement that morphological
transitions occur between mitochondria and neurofibrils.

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 413

(4) The argument from analogy is based on the development
of myofibrils, connective tissue fibrils and epidermal fibrils from
mitochondria according to the followig observers.

Benda (’99) and Meves (’07) were among the first to come to
the conclusion that mitochondria become changed into myofi-
brils. Duesberg wrote an excellent paper in 1910 in whichhe
supported their conclusion. In a subsequent contribution (712,
p. 745) he states that while the two above named works (those
of Meves and Hoven) render the plastochondrial (mitochondrial)
origin of collagenous fibrils and of nerve fibrils only probable,
the study of the fate of plastosomes (mitochondria) in myoblasts
permits their réle in the formation of myofibrils to be shown un-
questionably. I do not, however, feel ready to accept Duesberg’s
evidence until I have satisfied myself that the similarity which
he found in the staining reactions of mitochondria and myofibrils
by the Benda method is indicative of like chemical composition.
It is necessary for us to bear in mind that all structures which
stain alike by a single method are not necessarily of the same
nature. This similarity in staining is the only evidence which
he brings forward to prove the existence of microchemical tran-
sitions between mitochondria and myofibrils. It remains to be
proved whether it is sufficient or not.

Meves (’10) has studied the origin of connective tissue fibrils
in the tendons of chick embryos. The technique employed con-
sisted of fixation in his modification of Flemming’s fluid, stain-
ing with iron hematoxylin for mitochondria and of counter-
staining the fibrils with acid fuchsin. He found that the fibrils
first appear in the peripheral portions of the cells where the mito-
chondria are abundant and have become elongated, and claimed
to have established a quantitative relation between the increase
in the number of fibrils and the decrease in mitochondria. He
concluded that the connective tissue fibrils are formed by a mod-
ification of mitochondria. Since he has failed to show a grada-
tion between the black stained mitochondria and the brilliant red
colored fibrils, he assumes (p. 164) that there is a time when the
mitochondria change their chemical character so that they can-
not be stained by either iron hematoxylin or acid fuchsin, that

414 E. V. COWDRY

it is during this stage that they become oriented end to end and
fuse to form neurofibrils, and finally, that the fibrils change for
a second time their chemical composition so that they acquire a
marked affinity for collagen staining dyes.

The work of Firket (11) on the formation of epidermal fibrils
in the cells of the beak and feathers of chick embryos, and that
of Arnold (712) on the origin of fibrils in malignant tumors is
likewise open to criticism owing to their failure to demonstrate
transitions between mitochondria and the fibrils, and on ac-
count of their tacit supposition that structures which stain by
the same method of technique are of similar chemical composi-
tion (Bensley 710).

3. The origin of neurofibrils

In attacking the problem of the origin of neurofibrils it is
necessary to bear in mind all the possible factors, which, by any
stretch of the imagination, may be involved. These should be
carefully studied and eliminated one by one from consideration;
but in so doing, it is absolutely essential to remember that the
cell maintains its existence by virtue of the interaction of these
same factors, and that any attempt to dissociate and analyse
them is artificial. The nucleus, chromidial substance, canalicular
apparatus and the ground substance ought therefore to be con-
sidered in addition to mitochondria.

I have already criticised the theory of the mitochondrial origin
of neurofibrils and this discussion has been carried sufficiently
far to show that the mitochondria should not be selected as being
the sole agents in the formation of neurofibrils. It is a very
different thing to conclude that neurofibrils are differentiated by
an actual transformation of mitochondria than it is to enter-
tain the possibility that mitochondria may, in some way, be
associated with the histogenesis of neurofibrils; for I am not a
great believer in the feasibility of attempts to dissociate cyto-—
plasmic activities.

Changes in the nucleus prior to and contemporaneous with the
development of neurofibrils have been described by Gerini (’08,

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 415

p. 182). He finds that the nucleoli in the peripheral cells of
the ventrolateral portion of the cord of chick embryos assume a
bipolar arrangement; that many minute granules, staining darkly
by Cajal’s method, appear at either pole of the nucleus; and that
the first neurofibrils arise about the periphery of these granules.
I, also, have found that the first indications of neurofibrils appear
in a part of the cytoplasm which stains especially darkly by
Cajal’s method. :

The Nissl substance may apparently be excluded from the dis-
cussion, since, according to Marcora (711, p. 946), it first appears
in the spina! ganglion cells of chick embryos of about six days’
incubation. Collin fixes the time even later for he states (’06,
p. 259) that it begins to manifest itself after about ten days’
incubation. Moreover, I have failed to demonstrate this material
in the nervous system of chick embryos of from 15 to 20 somites,
which is the stage during which neurofibrillation begins.

The canalicular apparatus may, likewise, be eliminated. Mar-
eora (711, fig. 22) has demonstrated it in the cells of the neural
tube of a duck embryo of three days’ incubation. Repeated
efforts, on my part, have not given any indication of its presence
in the earliest stages of the formation of neurofibrils, although
indications of it possibly occur in figures 21, 25 and 26, where a -
system of clear, unstained canals may be seen in the cytoplasm.

It is my opinion that further investigation will show that both
the canalicular apparatus and the chromidial substance appear
much earlier in development than has hitherto been supposed,
for they and the neurofibrils are, to some extent at least, indica-
tive of the same thing, namely, the functional maturity ofthe
cell. Furthermore, the tendency in the past has been for inves-
tigators to trace back these constituents of the cytoplasm into
earlier and earlier stages of development with each advance in
technique, and our present technique is by no means perfect.

It is impossible to rule out, in a similar fashion, the ground
substance from consideration because it is, like the mitochondria,
inseparably connected with all stages in the life of the cell.
The neurofibrils are therefore, in all probability, developed from
the ground substance, or from formed elements within it as yet un-

416 E. V. COWDRY

known, in response to physiological demands incident at the time
of their differentiation. The darker staining of the perinuclear
cytoplasm with silver nitrate (figs. 10, 12 and 14) in early neuro-
fibrillar stages strongly indicates the possibility that the nucleus
either contributes substance or participates in some way in the
formation of neurofibrils.

SUMMARY

1. The neurofibrils are first formed in developing chick embryos
as a differentiation of the ground substance (in the majority
of cases of the peripheral neuroblasts) at a stage of develop-
ment characterized by possessing 15 somites, being about 5.8 mm.
in length and having been incubated for forty hours at 39°C. =
(p. 402).

2. In the early phases of the development of the chick the
earliest neurofibrils are formed in three chief localities: (1) in the
hind-brain opposite the otic invaginations (stages of 15 somites
on); (2) in the nuclei and root fibers of the cranial nerves (from
stages of about 15 somites on); and (3) in a center on either
side of the extreme anterior end of the midbrain (stages of 18
somites on) (p. 403).

3. There is no evidence that mitochondria are transformed into
neurofibrils. The facts are these: (1) that there is no decrease
in the amount of mitochondria in development parallel to the
increase in the neurofibrils; (2) that the mitochondria do not
show, either by a variation in their morphology, staining reac-
tions, or in any other fashion, capable of detection by our pres-
ent methods of technique, indications of being transformed into
material of different chemical composition; and (3) that during
the first part, at least (embryos of somites 15 to 32) of the period
of formation of neurofibrils, the mitochondria in the neural tube
retain the similarity in their morphology and staining reactions
to the mitochondria occurring in the structures derived from the
other two germ layers (i.e.,;mesoderm and endoderm) which
they possessed before any neurofibrils became differentiated
(pp. 405-408).

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS 417

4. Mitochondria are present in the early stages in the differ-
entiation of the nerve cells of the chick (embryos somites 0 to
32) and there is ample evidence that they occur throughout cy-
tomorphosis. The neurofibrils, on the other hand, are only pres-
ent in later stages (embryos of 15 somites on) in evident adap-
tation to functional demands. Mitochondria may therefore be
regarded as cytoplasmic elements of a generalized nature, not
participating in so specialized a cell function as the develop-
ment of neurofibrils; while the neurofibrils are to be looked upon
as indicative of the differentiation of the cells in which they are
found (p. 403).

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des EHies von Ascaris megalochephala. Arch. f. mikr. Anat., Bd. 76,
pp. 683-713.

Mrcwaetis, L. 1899 Die vitale Farbung eine Darstellungsmethode der Zell-
granula. Arch. f. mikr. Anat, Bd. 55, pp. 558-575.

Paton, S. 1907 The reactions of the vertebrate embryo to stimulation and
the associated changes in the nervous system. Mitth. aus der Zool.
Stat. zu Neapel. Bd. 18, pp. 535-581.

RusascHKIn, W. 1910 Chondriosomen und Differenzierungsprozesse bei Siu-
getierembryonen. Anat. Hefte, Bd. 41, pp. 399-481.

Smirrnow, A. E. von 1906 Uber die Mitochondrien und den Golgischen Bil-
dungen Analoge Strukturen in einigen Zellen von Hyacinthus orientalis.
Anat. Hefte, Bd. 32, pp. 146-153.

EXPLANATION OF PLATES

All the illustrations have been drawn by Mr. A. B. Streedain, artist to the De-
partment of Anatomy of the University of Chicago, to whom I wish to acknowledge
my thanks. Preparations of nerve cells of developing chick embryos alone have
been used. Camera lucida, Zeiss apochromatic objective 1.5 mm. and compen-
sating ocular 6 were employed for all except figures 21 and 23-27, of which figures
21 and 25-27 were drawn with objective 1.5 mm. and ocular 8, figure 23 with ob-
jective 2 mm. and ocular 4 and figure 24 with objective 2mm. and ocular 6. They
have not been reduced in reproduction so that their magnification as they now
appear on the plates is as follows: for figures 21 and 25-27, 2200 diameters; for
figure 23, 820 diameters and 1050 diameters for figure 24, for all the others 1500 di-
ameters. All sections were cut 44 in thickness. Only specimens from transverse
sections have been figured. The colors have invariably been put in by daylight
so that the uncertainty and variability involved in artificial illumination has been
avoided.

ABBREVIATIONS

m.l.int., membrana limitans interna g.sp., ganglion spinale
m.l.ext., membrana limitans externa s., mesodermic somite
r.ant., radix anterior

PLATE 1
EXPLANTION OF FIGURES

This plate illustrates, among other things, the behavior of mitochondria in
early stages of development. All the specimens figured have been prepared by
Meves’ modification of the iron hematoxylin method so that the mitochondria
appear as black, filamentous or granular structures against a gray background.

1 Epithelial cells from the ventral half of the neural tube in the region of the
tenth somite in an embryo of 12 somites, thirty-four hours incubation (p. 401).

2 Epithelial cells from near the middle line of the medullary plate of an em-
bryo of twenty-four hours incubation at 39°C., in which the primitive groove, fold
and pit alone were differentiated. The mitochondria appear as granules and
straight or wavy filaments of a black color. They occur in all parts of the cyto-
plasm and may readily be distinguished from the black spherical yolk granules
(p. 401).

3 Epithelial and germinative cells from the neural fold in the region of the
head process of an embryo of 3 somites, twenty-four hours incubation. The
mitochondria are distributed throughout the cytoplasm but show a tendency to
become accumulated in the distal parts of the cell. Only a few spherical, jet-
black yolk granules may be distinguished at this stage (p. 401).

4 Radix anterior (7.ant.) with the neighboring portion of the neural tube,
which have become slightly separated from each other during the preparation.
Taken from opposite the tenth somite on the right side of an embryo of 31 somites,
seventy hours incubation. In the radix anterior mitochondria and neurofibrils
may be made out together; the former, rods or wavy filaments of different lengths,
the latter, threads of finer diameter and of a lighter color. The mitochondria in
the cytoplasm of the neuroblast (a) are shorter, curved more closely packed to-
gether and do not fuse together to form a network (p. 410).

5 From the neural tube opposite the tenth somite on the right side of an em-
bryo of 26 somites, and fifty-eight hours incubation. The cell outlines are indis-
tinguishable. The chromosomes in a dividing germinative cell are shown at (a)
and may be differentiated from the filamentous mitochondria by their character-
istic arrangement. The mitochondria are somewhat larger than in the preceding
figures (p. 401).

420

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS

AB Streedain del.

E. V. COWDRY

egy, ™.|.inf.

421

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 4

PLATE 1

1]

PLATE 2
EXPLANATION OF FIGURES.

All the preparations illustrated on this plate have been drawn from speci-
mens prepared by Cajal’s silver nitrate method (formula I). Figures 6 to 9 show
the different types of cells in which neurofibrils are formed: figures 10, 11, 12 and
14 the appearance of the earliest neurofibrils to become differentiated (i.e., in an
embryo of 15 somites) and figure 13 the neurofibrils passing out from the neural
tube among the cells of the myoblast in a later stage. Unfortunately the
preparations could not be illustrated in their original colors on account of the
cost of reproduction.

6 From the right side of the hind-brain, opposite the otic invagination, of an
embryo of 24 somites, length 7.5 mm. and sixty-five hours incubation. This
figure shows a cell sending out a process, containing neurofibrils, which pierces
the membrana limitans interna (m./.int.) and extends for some distance within the
cavity of the neural tube (p. 404).

7 Bipolar cell from the same region of the same embryo. The neurofibrils
are black. The membrana limitans interna (m.l.int.) and externa (m. l. ext.) are
shown for orientation (p. 403).

8 Marginal neuroblast from the same region of the same embryo. The neu-
rofibrils seem to form a more or less continuous network (p. 403).

9 From the same region of the same embryo but on the left side. The cell
(a) is apparently within the cavity of the neural tube and the process (b) of the
cell (c) extends toward the neural tube. All three contain neurofibrils (p. 404).

10 Cells from the right side of the forebrain of an embryo of 15 somites,
length 5.8 mm., incubation forty hours at 39°C, being shghtly further advanced
than Duval’s embryo of thirty-three hours, (fig. 268, p.56). The darkly staining
portion of the middle cell affixed to the distal pole of the nucleus contains
within it a blackened network which probably represents the first stage in the
histogenesis of the neurofibrils (p. 402).

11 More completely formed neurofibrils occurring in a marginal neuroblast
in the hind-brain on the right side, opposite the otic invagination of the same
embryo (p. 402).

12 Cells from the right side of the mid-brain of the same embryo showing a
stage in the formation of neurofibrils similar to that represented in figure LO (p.
402).

13. Neural tube with the adjacent myoblast in the region of the sixth somite
on the right side of an embryo of 29 somites, length 8.1 mm. and incubation sixty-
three hours. The neurofibrils are seen winding in and out among the mesoblastie
cells (p. 411).

14 Cells from the same region of the same embryo as figure 10 showing, rather
more satisfactorily than figures 10 and 12, the darkly staining distal part of the
eytoplasm in which the neurofibrillar network is formed (p. 404).

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS PLATE 2
E. V. COWDRY

—m.|.ext.

A.B. Streedain del.

PLATE 3
EXPLANATION OF FIGURES

Plates 3 and 4 constitute the essential features of a comparison of the results
yielded by Meves’ iron hematoxylin method and by Cajal’s silver nitrate tech-
nique respectively. In both cases drawings have only been made from sections
of the neural tube on the right side opposite the sixth somite and the degree of
differentiation of the embryos from which the figures of plate 3 were drawn cor-
responds severally with that of those represented on plate 4 (fig. 15 a to fig. 15 b
and so on). The drawings have been arranged so that the membrana limitans
externa is to the left hand side of the observer. A collation is therefore justified
since morphologic equivalents of approximately the same area and thickness
(4) are only being used. This comparison of homologous equivalents in increas-
ing stages of development prepared by these two methods, the one showing mi-
tochondria, the other neurofibrils, indicates: (1) That there is no decrease in the
amount of mitochondria parallel to the increase in neurofibrils. (2) That the
mitochondria do not show, either by a variation in their morphology, staining
reactions, or in any other way, capable of being detected by our present-day
methods, indications of being transformed into material of radically different
chemical composition (pp. 407 and 416).

15a From the right side of the neural tube in the region of the sixth somite
of an embryo of 17 somites, length 5.5 mm. and incubation forty-five hours. The
mitochondria are filamentous, their irregular shape, as seen in the drawings, de-
pends upon the fact that their long axes are directed in all planes relative to the
drawing and that they are cut in pieces of many sizes, at all angles during sec-
tioning. There is a remarkable variation in the color of the back-ground, for in
two cells, (a) and (b), the cytoplasm remains practically unstained, although
there is apparently no other difference between them and the neighboring cells
(p. 408).

16a Same region, embryo of 20 somites, sixty-two hours incubation. There
is no change in the mitochondria.

17a Same region, embryo of 22 somites, fifty-two hours incubation. The
section has been cut rather obliquely. There is no change in the mitochondria.

18a Same region, embryo of 24 somites, length 7.0 mm. and sixty hours in-
cubation. The section is even more oblique. The mitochondria show no change.

19a Same region, embryo of 28 somites, sixty-four hours incubation. No
change in mitochondria.

20a Same region, embryo of 31 somites, seventy hours incubation. There is
still no change in the mitochondria.

424

PLATE ~

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS

r. V. COWDRY

E

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2a bd
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16a

A.B.STreedain del.

Now

PLATE 4
EXPLANATION OF FIGURES

This plate logically belongs to the preceding (plate 3) in connection with which
it has been described. All the figures are from preparations made by Cajal’s
silver nitrate method.

15 b A portion of the neural tube from opposite the sixth somite on the right
side of an embryo of 17 somites, 44 hours and 15 minutes incubation. No neuro-
fibrils are formed, but the cytoplasm in the distal portions of the cells (a), (b)
and (c) is stained especially darkly with the silver nitrate.

16b Same region, embryo of 20 somites, length 7.4 mm., 88 hours, and 20
minutes incubation. The neurofibrils have appeared, in a single cell, and are
distinctly stained.

17b Same region, embryo of 22 somites. Considerable increase in neurofi-
brillar material.

18 b Same region, embryo of 24 somites, length 7.5 mm., 65 hours incubation.
Shows some neurofibrils passing through the membrana limitans externa into the
myoblast.

19b Same region, embryo of 28 somites, length 6.8 mm., 63 hours incubation.
Neurofibrillar material increased.

20 b Same region, embryo of 31 somites, length 7.3 mm., 62 hours incubation.
Shows a great increase in the number of neurofibrils, many of which now run par-
allel to the long axis of the neural tube.

PLATE 5
EXPLANATION OF FIGURES

Figures 21, 26 and 25 represent progressive stages of differentiation in Bens-
ley’s anilin fuchsin toluidin blue method. These figures have been drawn from
neighboring sections of the same embryo, mounted on the same slide. In the
first (fig. 21) the differentiation is practically nil, the mitochondria staining ex-
actly the same color as the neurofibrils; in the second (fig. 26) it has been carried
a little further with the result that the neurofibrils have lost their bright crimson
color and have assumed a dull red shade; while in the last (fig. 25) the decolori-
zation has been carried to an extreme so that the neurofibrils have lost all of the
acid fuchsin and have become stained with the differentiator, toluidin blue. It
is to be noted that in these progressive stages of differentiation the initial affinity
of the neurofibrils for an acid dye (acid fuchsin), in which they resembled mito-
chondria, is gradually changed to an affinity for a basic dye (toluidin blue), while
the intensity of the coloration of the mitochondria remains unaltered. Con-
clusions based upon the apparent similarity in the staining reactions of mito-
chondria and the primitive neurofibrils should therefore be received with caution
(p. 410 and 416).

21 Cell and accumulation of neurofibrils from the ventral portion of the neu-
ral tube of an embryo, length from cervical flexure to tail flexure 8.4 mm., 100
hours incubation, corresponding closely in degree of differentiation to Duval’s
embryo of 96 hours. Stained by Bensley’s anilin fuchsin toluidin blue method

426

PLATE 4

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS

E. V. COWDRY

{
(
a

tt

AB Streedain del.

17b

EXPLANATION OF PLATE 5 (CONTINUED) ae

with but slight differentiation, the neurofibrils and the mitochondria staining
exactly the same color. The clear, uncolored spaces within the cell may consti-
tute the canalicular apparatus, because it is demonstrated, in a similar manner,
by this technique applied to adult spinal ganglion cells (Cowdry, ’12 a, fig. 1).

22 Apolar cells bordering the central canal of the neural tube opposite the
posterior part of the third somite from an embryo of 33 somites, length 8.3 mm.
and 74 hours incubation, prepared by Paton’s modification of the Bielschowsky
method. The circumnuclear neurofibrillar network are shown in black (p. 403);
compare this with Cajal 1907, figures 1 and 2.

23 Pear-shaped neuroblast isolated from the neural tube opposite the tenth
somite on the right side of an embryo of 25 somites, length 7.45 mm., stained in-
travitam in a 1:10,000 solution of janus green in 0.85 per cent sodium chloride so-
lution. The magnification is less, so that the granular mitochondria, shown in
green, seem smaller than in the other illustrations (p. 401).

24 Cells from the same region of the same embryo stained in the same way, the
sole difference being the substitution of ocular 6 in place of compensating ocular
4 in making the drawing. Mitochondria bluish green. The cell walls are quite
distinet (p. 401).

25 Taken from the dorsal portion of the neural tube of the same embryo as
figure 21, prepared by the same method and mounted on the same slide. The
process from the cell (a) is closely applied to that from the cell (b), both piercing
the membrana limitans externa together. The differentiation has been carried
to an extreme so that the neurofibrils have completely lost all shade of acid fuch-
sin and have assumed the blue color of the differentiator, toluidin blue, whereas
the mitochondria still retain their original bright crimson coloration. The neu-
rofibrils and the mitochondria are seen side by side in the processes of both cells
differentially stained. The clear spaces in the cell (b) may, as in figure 21, rep-
resent the canalicular apparatus (p. 410).

26 Section from the same embryo, slide and method, which was also taken
from the dorsal part of the neural tube. The process from the cell (a) pierces
the membrana limitans externa and extends among the spinal ganglion cells (g. sp.).
The differentiation is here intermediate between that represented in figures 21
and 25. The neurofibrils have consequently lost their bright crimson coloration
(fig 21) and have become a brownish red color so that they may readily be distin-
guished from the mitochondria which are in the interfibrillar substance in the
cell process. A few clear, tortuous, uncolored spaces are seen in the spinal gang-
lion cell (g.sp.) which are possibly the canalicular apparatus (p. 410).

27 A portion of the radix anterior from an embryo, length from the cervical
flexure to the tail flexure 8.4 mm., period of incubation 100 hours, corresponding
closely in degree of differentiation to Duval’s embryo of 96 hours, which has been
stained by Bensley’s anilin fuchsin methylene blue erythrosinate method. The
sheath cells (a) are shown and the mitochondria withinthem. The neurofibrils
appear a yellowish brown color and a few bright crimson mitochondria are visible
in the interfibrillar substance (p. 410).

428

CYTOPLASMIC CONSTITUENTS OF NERVE CELLS PLATE 5
E.v. COWDRY

ean:

24

A.B.Streedain del Werner & Winter, Frankfort2M_

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO.4

429

FEEDING EXPERIMENTS ON TADPOLES

II. A FURTHER CONTRIBUTION TO THE KNOWLEDGE OF ORGANS
WITH INTERNAL SECRETION

J. F. GUDERNATSCH

Department of Anatomy, Cornell University Medical College, New York
(From the Department of Histology and Embryology, University
of Munich. Director: Prof. S. Mollier)

TWO DOUBLE PLATES

Certain mammalian glands with a so-called internal secretion,
when given as food, can enact a decided influence on the growth
and differentiation of amphibian embryos. This was shown by
experiments that were carried out during the summer of 1911 on
tadpoles of Rana temporaria and Rana esculenta.

During the spring of 1912 these experiments were repeated
and at the same time varied to such an extent that no doubt as
to their results remained. Although the experiments of the two
seasons revealed many precise data, there is still a great number
of obscure features regarding the definite action of internally
secreting glands when fed to the tadpoles. While the influence
on growth and differentiation resulting from feeding some of the
glands was striking, others exerted no marked effects. The ac-
tion of the latter, in part or entirely, may not be concerned with
those most important physiological processes in the embryo, name-
ly, growth and differentiation. They might play their chief réle,
then, in the household of the postembryonic organism. Or, since
taken from mammals, some of these internally secreting glands,
if at all connected with embryonic development, may fail to re-
veal this influence, when fed to amphibian embryos. Their ac-
tion must finally be studied in experiments on higher vertebrates.

The experiments in 1912 were performed on tadpoles of Rana
temporaria, Rana esculenta, Bufo vulgaris and Triton alpestris.

431

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 4

432 J. F. GUDERNATSCH

The eggs of these species were collected from ponds in the vicin-
ity of Munich and reared in the laboratory. They were kept
in large aquaria and after hatching were transferred to smaller
dishes, each dish containing about 100 individuals. After the
larvae had grown for some time and seemed to be crowded in the
dishes they were separated into smaller groups. To avoid pos-
sible errors, a great number of individuals was always used for
each kind of experiment. All in all, over 20,000 tadpoles were
treated.

The eggs from different localities were kept separate and, as
far as possible, eggs apparently coming from the same mother were
grouped in one set and used for a special experiment. When-
ever the eggs necessary for one experiment could not all be sup-
plied by a single female I endeavored to select eggs coming from
the same locality, and apparently in the same stages of develop-
ment. For example, set III probably contained eggs from three
mothers, but the time of hatching, April 7 and 8, and their
sizes, 10 to 11.5 mm at the beginning of the experiment, show
them to be in similar stages of development. Again, for other
experiments in which a large number of animals was required, as
in sets VIII to XI over 4000, several sets in apparently the same
stage of development were thoroughly mixed and then divided
into smaller groups. In these last sets the tadpoles hatched be-
tween April 10 and 13 and a month later, when the feeding
bégan, their sizes varied from 13 to 18 mm; this difference is not
greater than one would encounter in eggs from a single individual.

The water was changed at least once a day. While standing,
its temperature varied with the room temperature, a process that
corresponds to natural conditions. However, even in a room
with apparently uniform temperature, the water will not show
exactly the same temperature in all the dishes, as Barfurth has
pointed out. To overcome any unequal influence of light, air
and temperature resulting from the position of the dishes, they
were shifted several times a day so as to progress in a certain
order. With this precaution there were no detectable differences
of temperature in the dishes assigned to one set. There was
sometimes a slight difference of temperature between the dishes

FEEDING EXPERIMENTS ON TADPOLES 433

used for the different experiments, but this was of no importance,
so long as the tadpoles used for one experiment were kept under
uniform conditions.

As during the previous year, the food in small pieces was placed
in the water and there voraciously taken by the animals, with
the exception of pancreas which never seemed to excite their
appetite very greatly.

Last year the tadpoles had their natural food previous to the
experiments, this year in all but one experiment no other food was
received except the one chosen for the studies. In 1911 after
the feeding began the animals were kept exclusively on a one-
gland diet; in 1912 these experiments were repeated with part
of the animals while in addition a mixed, either two-animal tis-
sues or gland-plant diet, was introduced.

The following were used as foods: thyroid, adrenal, liver,
spleen, hypophysis, brain, pancreas and muscle from the horse,
testicle, ovary and thymus from cattle, and as vegetable food
Elodea canadensis and Ceratophyllum demersum.

It would not have been possible to perform such an extensive
series of experiments but for the courtesy and generosity of Prof.
S. Mollier, in whose laboratory in Munich the experimental part
of this paper was carried out. It is with pleasure that I express
my best thanks for the many kindnesses shown me during my
stay in his laboratory.

Experiment I

Rana temporaria, Set I. Figure 1, a to 2f. Hatched April 4 to 7,
1912. Feeding began April 13, 1912. Original size 10.2 to 12 mm.

The diary reads as follows:

April 4 The feeding begins.

April 22 Thyroid-fed have hind legs forming.

April 24 Thyroid-fed have fore legs forming.

April 25 Thyroid-fed begin to die off.

April 29 Last thyroid-fed animals dead. Thymus- liver- spleen- and
muscle-fed tadpoles are larger than the other groups.

April 30 Muscle, liver and spleen ones show hind leg buds.

May 4 Adrenal cortex, adrenal medulla and ovary have hind leg
buds faintly noticeable.

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434

May 14

May 24

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June 14

June 17
June 19
June 20

June 21

June 22
June 23

June 24

June 25
June 26

June 27
June 29
June 30
July 6
July 7

FEEDING EXPERIMENTS ON TADPOLES 435

To this time the controls have been starving, in order to test
the possible influence of hunger on development. They
show no signs of advancement, but are far behind in size.
From now on they are fed on Elodea canadensis.

A group originally intended for a hypophysis diet also starved
up to this day, and from now on is fed on brain.

The liver-fed ones have a greenish color, the thymus-fed ani-
mals are deep black.

The smallest individuals in all sets begin to die off. The
spleen-fed ones become very black, the bigger ones on ad-
renal cortex become very light, lighter than on adrenal
medulla.

The first liver-fed ones show hind legs; this is 49 days later
than the time at which the thyroid-fed ones attained the
same condition.

Hind legs appear in adrenal-cortex-fed tadpoles. Their body
assumes a peculiar triangular shape (fig. 7 b), very pointed
at the anterior end and broad at the posterior. They are
all becoming light, while all the adrenal-medulla-fed ones
are dark.

A number of each group, 70 to 80, which have no hind legs
except the small buds, are placed on thyroid diet.

The light color of the adrenal-thyroid tadpoles is disappear-

ing.

Those liver-fed ones that have put out fore legs since June
12 shorten their tails.

The liver-thyroid animals develop hind legs.

Adrenal medulla-thyroid and muscle-thyroid tadpoles de-
velop hind legs. The former are extremely active.

Liver-thyroid and muscle-thyroid ones assume a frog shape.

Spleen-thyroid and adrenal cortex-thyroid ones develop hind
legs. The first liver-fed frog leaves the water.

Spleen-thyroid and adrenal cortex-thyroid animals assume
frog shape. Brain-thyroid ones develop hind legs. Ad-
renal cortex tadpoles are extremely light greenish-yellow.

Brain-fed ones assume somewhat the triangular adrenal cor-
tex shape described on June 14.

Liver-thyroid and adrenal medulla-thyroid animals show fore
legs.

Spleen-thyroid-fed ones show fore legs.

Ovary-thyroid and brain-thyroid tadpoles assume frog shape,
the latter are very active.

Muscle-thyroid animals show fore legs.

Adrenal ¢ortex-thyroid and brain-thyroid ones show fore legs.

Ovary-thyroid ones show fore legs.

All the thyroid-fed ones are fixed, since many of them die.

Brain-fed tadpoles show hind leg buds.

436 J. F. GUDERNATSCH

July 11 Muscle-fed ones have the best developed hind legs, next the
ovary and adrenal-cortex ones which have short but very
strong legs, then adrenal-medulla, brain and spleen.

The color of the animals on the different diets is: control,
brown; muscle, dark brown; liver, dark greenish; spleen,
dark; adrenal cortex, very light brown; adrenal medulla,
dark brown; ovary, yellowish; thymus, dark; brain, brown.

July 19 Muscle-fed ones begin metamorphosing.

July 20 Ovary-fed ones begin metamorphosing.

Thymus-fed have no signs of legs, spleen-fed only small buds.

In this experiment the thyroid treatment proved to be as
effective as in the experiments performed in 1911. Tadpoles that
had been fed on no other food than thyroid grew hind legs 9 days
after the feeding began and fore legs only 2 days later. Normally
several weeks would have elapsed between the appearance of the
hind and the fore extremities. When these thyroid-fed tadpoles
put out their anterior limbs and began to shorten their tail, they
were 18 to 20 days old, calculating from the date of hatching.
Normally they would take from 10 to 12 weeks to complete their
metamorphosis. '

The tadpoles fed on ordinary meat, muscle (fig. 1d, 1),
showed distinct hind legs 44 days and fore legs 71 days later
than the thyroid-fed ones. The first individual in the muscle-
fed group to complete its metamorphosis was approximately 104
days old.

Liver-fed tadpoles (fig. 1 a, 12, 1 x) show the best growth, al-
though their growth is only slightly better than that of the spleen
and thymus sets. Next to the thyroid group the liver-fed tad-
poles also showed most rapid progress in differentiation. They
grew hind legs 40 days and fore legs 49 days later than the thy-
roid group. The first liver-fed specimen to complete its meta-
morphosis was approximately 78 days old.

The spleen- (fig. 1b, 11) and thymus- (fig. 1 c) fed tadpoles
show almost parallel courses in growth and differentiation. Dur-
ing the first weeks of the experiments the spleen group ran a
little ahead of the thymus specimens. The members of both
groups became extremely dark in color during the course of the
feedings. At the approximate age of 104 days, when the experi-

FEEDING EXPERIMENTS ON TADPOLES 437

ment was discontinued, none in either group had hind legs out,
although spleen-fed ones showed leg buds.

The three groups of tadpoles fed on adrenal cortex (fig. 1 f, 17),
adrenal medulla (fig. 1g, 1 ¢) and ovary (fig. 1 e, 1 p) were dur-
ing the first weeks of growth behind the muscle, liver, spleen and
thymus groups. Later, however, they grew more rapidly. Some
of those fed on ovary actually reached the average size of the
faster groups and completed their metamorphosis at the approx-
imate age of 105 days.

The adrenal cortex tadpoles were somewhat faster in develop-
ment and grew better than the adrenal medulla ones. The former
budded hind legs on the 71st and the latter on the 8lst day. At
the conclusion of the experiment the adrenal cortex ones bad
strong well developed legs, while the legs of the medulla-fed
individuals were still short and drawn close to the body.

In previous experiments, in which adrenal cortex and medulla
had not been given separately, it was seen that the adrenal-fed
tadpoles became extremely light in color after 3 or 4 weeks of
feeding. The pigment cells were found to be completely con-
tracted. The suggestion was made ‘‘that the extract from the
chromaffine cells of the medulla which dissolved in the water
caused the pigment cells to contract”? and ‘‘former experiments
with adrenalin would warrant such a suggestion.”’ In the present
experiments, however, separate sets of tadpoles were fed on cor-
tex and medulla respectively. After 5 weeks’ feeding those fed
on adrenal cortex became much lighter than those fed on adre-
nal medulla or any other food. ‘This difference in color became
more evident as the experiment proceeded, until the cortex-fed
tadpoles had an extremely light, greenish-yellow tint. Thus the
above suggestion is doubtless incorrect, at least in these experi-
ments, yet the true cause of the light pigmentation is still obscure.

These cortex-fed tadpoles also differed from the other groups
in still another feature, namely, the peculiar triangular shape
(fig. 7 b) of their bodies described above. <A few of the brain-fed
tadpoles somewhat approached this condition.

The chief purpose of feeding the set I on different foods was
to study the influence of the thyroid treatment, after the ani-

438 J. F. GUDERNATSCH

mals had fed for sometime on various other tissues. The tadpoles
were about 10 weeks old, when the thyroid was given. They
were fed only four times. This feeding caused the same accel-
eration in differentiation that was noticed in former experiments.
Within 3 to 7 days the reaction to the treatment became evident.

There were differences in the rapidity of this reaction among
the different sets of tadpoles, as may be seen from the diary, but
whether or not ‘these differences are significant as to the value
of the food given before the thyroid treatment, remains an open
question.

The liver-thyroid-fed tadpoles (fig. 1 k) were the first ones to
develop hind and fore legs. This fact might easily be explained
on the ground that the liver-fed group was the most advanced
in the entire series (May 8, June 1, June 12, and June 28, in
the diary). Yet why those fed, for instance, on spleen, the slow-
est lot in the series, having no legs even on July 19, and those fed
on adrenal medulla which were at the start of the thyroid treat-
ment smaller than the liver-fed ones, should react to the thyroid
stimulus as fast as the liver group, cannot be explained on the
above basis suggested for the liver-fed group.

On the other hand, the adrenal cortex group though of about
the same size are 10 days ahead of the adrenal medulla group
in differentiation. Yet after thyroid application they do not
react with as great speed as the latter, and fall behind in the series.

Throughout the experiment it was observed that muscle-liver-
and spleen-fed tadpoles belonged to one class as far as their
growth was concerned, while those fed on adrenal cortex, adrenal
medulla and ovary formed another class. As soon, however, as
the thyroid factor was introduced this classification was dis-
turbed. Thus the previous feeding may influence to some de-
gree the mode of reaction to the thyroid stimulus. No variety
of previous feeding can, however, completely prevent this reac-
tion for any length of time. After a shorter or longer interval
(in this experiment the difference was only from 3 to 7 days)
every tadpole, as far as macroscopic features are concerned, will
respond to the thyroid stimulus, no matter what kind of food had
been previously given.

FEEDING EXPERIMENTS ON TADPOLES 439

To show the different degrees of response to the thyroid food
in the various groups, table 2 may be added. While it does not
reveal, as said before, any important points as to the respective
value of the different foods given before the thyroid diet, it shows
some striking changes.in the subsequent positions in the series
of the various groups, especially those fed on adrenal medulla,
spleen and adrenal cortex. -
TABLE 2

AVERAGE SIZE AT THE

_ se Ee 7 DAYS LATER SUMINTENGTH | TIME O¥ REACTION
muscle | muscle | adr. med, 12.761 | liver (fig. 1 k)
liver | liver liver, 14.777 | adr. med. (fig. lu, 1d
spleen adrenal med. brain, 15.053 | muscle (fig. 1 0, 1 za)
adrenal cortex | brain muscle, 15.454 | spleen (fig. 1 m, 12)
adrenal medulla | ovary | ovary, 16.129 | adr. cor. (fig. 1s, 1 2c)
brain _ spleen adr. cor., 28.421 | brain (fig. 1 w, 1 ze)
ovary | adrenal cortex spleen, 32.692 | ovary (fig. 1q, 1 2b)

Experiment II

Rana temporaria, Set IV. Figures 4,a to p. The tadpoles hatched
on April 5 to 6, and the feedings began April 13, 1912. Original length
of the specimens was 12 to 12.5 mm.

The diary records of the experiment are given below:

April13 The feeding was started.

April 16 Those fed on thymus and spleen have become larger than
the others.

April17 Thyroid-fed tadpoles have become considerably smaller than
thymus-fed ones and the thymus ones are getting dark.

April 18 Thymus-fed tadpoles are much darker than the others, spleen-
fed specimens are also getting dark. Thyroid ones now show
frog-like bodies.

April19 Thymus-fed individuals are almost black, spleen-fed very
dark, muscle-fed much lghter, thyroid-fed darker than
muscle, but lighter than thymus and spleen. Thyroid ones
have distinct frog shape, their hind legs are beginning to
bud (14 days old).

One set of thymus-fed individuals is now changed to a
thyroid diet, thymus-thyroid I.

April 20 The resorption of the tail in thyroid-fed individuals is dis-
tinctly noticeable. The specimens all lie on the bottom of
the dish and appear frail, taking no more food, while the
others in the experiment swim actively.

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441

442
April 21
April 22

April 23
April 25
April 29
May 6
May 9
May 10
May 138
May 15

May 18
May 22

May 28
June 2
June 5
June 6
June 7
June 12

June 17

June 18

J. F. GUDERNATSCH

The thyroid individuals are budding fore legs, the left one
first ;} 16 days old.

Thyroid ones begin to die.

Only 3 days after the thymus-fed ones were changed to
thyroid the influence is noticeable. They have become
reduced in size, take on frog shape and bud hind legs.

Fore legs budding after a treatment of only four days. Thy-
mus-thyroid I do not seem as frail as animals fed on thy-
roid alone.

Thyroid-fed anjmals do not develop their limbs any further,
nor do they reduce their tails much further since last ob-
served; progress has ceased.

The last thyroid and thymus-thyroid I fed animals die.

A second group of thymus-fed animals is changed to thyroid,
thymus-thyroid IT.

Thymus-thyroid II have hind legs appearing and the frog
shape is noticeable.

Thymus-thyroid II have fore legs budding out, the left one
first.

A third group of thymus-fed animals is changed to thyroid,
thymus-thyroid ITT.

The control animals have not been fed so far. They do not
show any sign of differentiation and very little growth.
From now they are fed on Elodea canadensis.

All thymus-thyroid II have both fore legs.

Thymus-thyroid III have hind leg buds.

All thymus-thyroid IIT have their hind legs well developed.

Other thymus-fed animals changed to thyroid, thymus-thy-
roid IV.

Thymus-thyroid III have fore legs.

Thymus-thyroid IV have hind legs.

Thymus-thyroid IV have fore legs.

Thymus-thyroid II begin to die.

Some muscle-fed tadpoles have hind legs beginning.

Another group of thymus-fed animals put on a thyroid diet,
thymus-thyroid V.

Thymus-thyroid V have hind legs beginning and the frog
shape faintly noticeable.

Again a group of thymus-fed tadpoles changed to thyroid
diet, thymus-thyroid VI.

Part of muscle and spleen fed animals put on a thyroid diet,
muscle-thyroid and spleen-thyroid.

Last thymus-thyroid II dies.

The members of the thymus-thyroid V group have fore leg
buds.

1 Barfurth states that in 80% of the tadpoles the right fore leg appears first.
I have always observed the opposite.

FEEDING EXPERIMENTS ON TADPOLES 443

June 19 All thymus-thyroid V die, thyroid had been placed in the
water every day.

June 20 Thymus-thyroid VI have hind leg buds.

June 21 Muscle-thyroid have hind leg buds.

June 24 Spleen-thyroid have hind leg buds.

June 25. Thymus-thyroid VI and muscle-thyroid groups have fore leg
buds.

June 26 Spleen-fed tadpoles have hind legs.

June 27 Spleen-thyroid group have beginning fore legs.

June 8 Thymus group have hind leg buds.

June 29 Control tadpoles have hind leg buds:

July 3 Last animal of the thymus-thyroid III group dies.

July 6 Last individuals of the muscle-thyroid and spleen-thyroid
animals are preserved.

July 10 The remaining thymus-thyroid VI are preserved.

July 14 Last one of the thymus-thyroid IV group dies.

July 20 The experiment is discontinued.

The muscle-fed tadpoles are best differentiated.

Since Experiment III was performed along the same lines as
Experiment II, it may be well now to give the data of Experiment
III and discuss the two groups together.

Experiment III

Rana temporaria, Set III]. Figure 3, a to n. Contained apparently
three lots of eggs mixed; they hatched April 7 to 8 and the feedings
began April 13, 1912. The original lengths of the tadpoles were from
10 to 11.5 mm.

The experimental data are as follows:

April 13 Food is placed in the dishes for the first time.

April 15 Food is first taken by the animals.

April 18 Thymus-fed specimens become darker in color.

April 20 Some of the muscle- and thymus-fed tadpoles show hind leg
buds.

May 6 Part of the thymus-fed animals changed to thyroid diet,
thymus-thyroid I.

May 10 All individuals of the thymus-thyroid I group have hind legs
and the frog shape is noticeable.

May 13 Thymus-thyroid I group have fore legs budding, the left one
first. They lie on their backs and breathe rapidly.

A second group of thymus-fed animals is put on thyroid, thy-

mus-thyroid II.

May 15 The control has not yet been fed. It remaims far behind the
other groups in size and shows no signs of differentiation.
From now on the control group is fed on Elodea canadensis.

GUDERNATSCH

F.

J.

444

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May 18 The thymus-thyroid I animals are hardly able to swim; when
disturbed they move for a few seconds with convulsive
jerks, then drop again to the bottom of the dish.

May 19 The thymus-thyroid II group (fed thyroid three times only,
May 13, 14 and 17) have hind legs and the frog shape
becomes noticeable.

May 22 A third group of thymus-fed animals put on thyroid, thymus-
thyroid ITI.

May 27. Thymus-thyroid IT have fore leg buds.

May 28 Thymus-thyroid III (fed four times on thyroid, May 22, 23,
25 and 26) have hind leg buds.

June 2 Thymus-thyroid IIT have fore leg buds.

June 7 A fourth group of the thymus-fed tadpoles are put on thy-
roid, thymus-thyroid IV.

June 9 The last specimens of the thymus-thyroid I group are dying.

June 12 The thymus-thyroid IV animals have hind leg buds and the
frog-shape is noticeable.

June 17 A fifth group of the thymus fed animals is changed to a thy-
roid diet, thymus-thyroid V.

Also some of the muscle-fed ones are put on thyroid.

June 18 The thymus-thyroid IV animals have fore legs, but these
specimens are dying.

June 20 Last of the thymus-thyroid II are dying.

June 21 Thymus-thyroid V have hind legs.

June 25 Thymus-thyroid IV have fore legs.

June 26 The muscle-thyroid animals have hind legs.

June 30 The muscle-thyroid animals have fore legs.

July 1 A group of thymus-fed specimens put on a liver diet.

July 4 Some of the thymus-thyroid III group that have been kept on
vegetable food since June 5 seem to recover from the thy-
roid influence.

July 7 The last thymus-thyroid V are dying.

July 14 Muscle-fed tadpoles grow hind legs.

July 20. The experiment is discontinued, and only the muscle-fed ani-
mals have extremities.

Experiments II and III were performed for the purpose of de-
termining whether tadpoles of different ages would react to the
thyroid diet in similar or different ways. For this purpose a
great number of tadpoles were kept on a thymus diet and groups
of these were changed to a thyroid diet at various times. Table
5 shows the time of reaction to the thyroid stimulus in the differ-
ent groups. The number of days in the first columns indicates
the respective ages of the animals at the start of the thyroid feed-

FEEDING EXPERIMENTS ON TADPOLES 447

TABLE 5
SET 1V SET III
14 days hind 3 days
fore 4 days
30 days hind 4 days 28 days hind 4 days
fore 6 days fore 7 days
37 days hind 6 days 35 days hind 6 days
fore 15 days | fore 14 days
46 days hind 6 days | 44 days hind 6 days
fore 11 days | fore 10 days
62 days hind 5 days | 60 days hind 5 days
fore 11 days | fore 11 days
72 days hind 3 days | 70 days hind 4 days

fore 8 days fore 8 days

ings. The figures in the other columns give the number of days
required by each set in developing hind and fore legs.

The similarity of the results in the two series is striking. There
is a gradual decrease in the rapidity of action of the thyroid
influence up to the age of about 5 weeks followed by a steady
increase after this time. Whether or not this is a general rule
must be decided by further experiments. It is also difficult to
give a satisfactory explanation for this phenomenon, yet the fol-
lowing suggestion may be advanced. The young tadpoles were
very quickly affected by the thyroid feedings because the pre-
vious thymus diet had not acted long enough to delay or counter-
balance the thyroid stimulus. Older animals had enough thymus
agens accumulated to retard the thyroid action. Still older tad-
poles were more nearly approaching the normal time of meta-
morphosis and may thus have been ripe to respond to an acceler-
ating stimulus. The last argument, however, seems especially
weak, since it introduces a new factor in the action of thymus
diet; namely, that if it is prolonged, it loses the retarding influ-
ence and thus is less able to counteract the thyroid. This factor
has not yet been demonstrated; thus no conclusions as to the
rapidity of the thyroid influence on different age tadpoles can be
based upon it.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 4

448 J. F. GUDERNATSCH

It has been shown, however, in the experiments of 1911 that
thymus will affect older tadpoles less than younger ones. An-
other point may also be mentioned without attempting to explain
the situation. Table 6 shows that old thymus-fed tadpoles, when
put on a thyroid diet, respond more quickly to the thyroid stimu-
lus than do tadpoles of the same age which have been fed on
other substances. One should have expected just the contrary,
judging especially from Experiment VI in 1911, in which liver-
thyroid-fed tadpoles developed their fore legs 3 days earlier than
thymus-thyroid-fed ones.

TABLE 6
| SET IV: 72 DAYS OLD | SET 111: 70 DAYS OLD
| Thymus- | Muscle- Spleen- Thymus- Muscle-
| thyroid | thyroid thyroid thyroid thyroid
Hind legs in | Sdays | 4days 7 days 4 days 9 days
Fore legs in | 8 days | 8 days | 10 days | S8days 13 days

In Experiments IT and III those tadpoles (fig. 3 ctoh, 3k, 3m,
3n; fig. 4e toz, 41, 4 to p) that were transferred from thymus
to thyroid diets were fed on the latter gland only three or four
times. When they had developed their fore legs water-plants
were placed in the dishes. It had been noticed that thyroid-fed
tadpoles would die very soon after putting out fore legs. In these
experiments those that were placed in dishes containing plants,
although they were never seen to feed on the plants, could be
kept alive for some time, 20 to 53 days, while those specimens
remaining in water in which thyroid was placed longer than was
absolutely necessary for developing the extremities died within
10 to 12 days after the beginning of the thyroid diet. Those liv-
ing longer did not develop any further than those dying early,
but remained stationary except for a further reduction in size,
especially of the tail.

Table 7 gives the length of time that the different thymus-
thyroid-fed sets were kept alive in the two experiments. Tad-
poles in Experiment III were about 2 days younger than those
in Experiment IV, when the feeding began.

FEEDING EXPERIMENTS ON TADPOLES 449

TABLE 7
SET IV SET III
Age at (hors time Days he ing p after thyroid ae at the time thy roid Days living after
thy roid was given prcanmon Was given thyroid treatment
14 days 10 days (no olanea)
30 days | 43 days | 28 days 33 days
37 days | 51 days 35 days | 38 days .
46 days 53 days 44 days | 50 days
62 days 12 days (no plants) | 60 days 11 days (no plants)
72 days 23 days 70 days | 20 days

A glance at table 5 will show that the tadpoles of 37 and 46
(or 35 and 44) days showed the slowest reaction to the thyroid
treatment. The possible reasons for this behavior were dis-
cussed above.

Experiment IV

Rana temporaria, Set II. Figure 2, a to n. 960 tadpoles hatched
on April 6 to 7, 1912, and fed on muscle until June 3. The experiment
started on June 5.

This experiment confirmed the results of the two previous ones.
Tadpoles (fig. 2, a) which had lived for 50 days on an ordinary
meat diet were later affected by the thyroid stimulus as greatly
as animals fed only on a thyroid diet. The main object of this
experiment, however, was to ascertain how long the thyroid agens
had to be applied before results were noticeable, and furthermore,
whether or not it would be possible to counteract the thyroid in-
fluence after one or more days feeding. A great number of tad-
poles were reared on muscle and at the age of 50 days were di-
vided into groups of 80 individuals each. The different groups
were then fed on thyroid from 1 to 5 days and after this treat-
ment five of the groups were put on a vegetable diet (EKlodea
canadensis) (fig. 2, f to 7) and 5 others on muscle diet (fig. 2, c to e).

One group (fig. 2,b) was allowed to starve to test whether
hunger following a long period of feeding (over 6 weeks) would
bring about a quicker differentiation.

Still another group (fig. 2, 1) was kept in water in which small
pieces of thyroid gland were placed, but so arranged that the

GUDERNATSCH

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452 J. F. GUDERNATSCH

animals could not feed on it. The gland was placed in a small
glass vial closed with gauze and allowed to stand upright, the
open end being beneath the surface of the water. Thus no thy-
roid particles could get out into the water, merely an extract or
emulsion of thyroid constituents which diffused from the vial into
the dish. Animals kept in these dishes developed their hind
legs 8 days after the beginning of the experiment and the frog
shape became noticeable about the same time. Six days later
they all died without having grown fore legs, but their size on
that day, June 19, shows that they were completely under the
thyroid influence. Therefore the product of the thyroid which
caused the rapid differentiation in all the previous experiments
must be soluble in water.

The control animals which were kept starving after the 50 days
feeding did not grow hind legs until July 5, considerably later
than the thyroid treated ones. Thus the results after thyroid
application are different from those Barfurth reports in his ‘‘ Der
Hunger als foérderndes Prinzip in der Natur,” and cannot be
attributed to starvation of the tadpoles. Among the ten main
eroups of the experiment the thyroid influence manifested itself
in different ways. As a general result it may be stated that a
thyroid application of only 24 hours sufficed to show decided ef-
fects (fig. 2c, 2f), and that a treatment of more than 24 hours
(2 to 5 days) gave the typical results described in all previous
experiments.

In the five groups that received vegetable food following the
thyroid dose and in the five groups that were fed again on muscle,
a great number of individuals, 50 to 60 per cent, mainly the shorter
ones, died within 2 or 3 days after the first feeding. This ac-
cords with observations in other cases where thyroid was given
after previous feeding, but is in striking contrast with the hap-
penings in experiments where thyroid food was applied from the
start. In the latter experiments very few animals were lost dur-
ing the first days. It remains to be answered why the applica-
tion of thyroid should affect tadpoles that had not been previ-
ously fed on other diets less harmfully than those previously fed
on various tissues.

FEEDING EXPERIMENTS ON TADPOLES 453

The tadpoles which again received muscle after the thyroid
treatment reacted more strongly to the stimulus than those put
on a vegetable diet. They all grew hind legs within from 1 to
5 days, the frog shape became noticeable very soon, and they
died in great numbers, so that 7 days after the beginning of the
experiment only 20 per cent were alive. The animals treated
from 3 to 5 days grew fore legs on the 8th day, those treated 2
days were all dead on the 9th day; this group was affected most
strongly, which may be a mere coincidence. The groups fed only
one day grew fore legs on the 42d day, at this time only four
of this group were alive, only one of the 3 day group, arid none
of the 4 and 5 day groups, the last ones having died on the 29th
day of the experiment. That is, out of 400 individuals only 4
in the 1 day group and 1 in the 3 day group were able to survive
the thyroid shock. The table of growth on page 450 shows that.
these surviving individuals actually begin to grow again. These
4 animals of the 1 day group seem to have absorbed a very small
quantity of the thyroid agens. They stop their precocious dif-
ferentiation very soon and do not grow fore legs until July 17,
which is 34 days later than the 3 and 5 day groups.

The second set of tadpoles, those put on a vegetable diet after
a thyroid treatment, reacted in a similar fashion to that just de-
scribed, except perhaps as stated above, they were somewhat less
affected. This might suggest that a meat free diet may help to
counteract thyroid influences. The 3 and 5 day groups grow
hind legs on the 5th day of the experiment, the 2 day group on
the 6th day, and the 1 day group not until the 14th day. Fore
legs appear in the 3, 4 and 5 day groups on the 10th day, in the
2 day group on the 9th day, in the 1 day group none had ap-
peared on the 46th day, when the experiment was discontinued.
While many animals died during the first few days, some were
able to keep alive. On the 27th day the last ones of the 5 day
group died, and on the 33d day all of the 2 and 3 day groups
were dead. ‘There were five of the 1 day group and one of the
4 day group alive on the 42d day. These, as the table of
growth of page 450 shows, start again to grow.

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Experiment V

Rana temporaria, Set VIII to XI, voluntarily mixed. These hatched
April 10 to 13 and the experiment started May 8, 1912.

In addition to the organs used in the previous experiments,
pancreas and testicle were given as food in this experiment.
Each organ was given in two forms; with one group of tadpoles
a small piece of the tissue was placed in the water; to a second
group the food was given in a crushed form, a piece of the tissue
being squeezed between the fingers, until it was broken into
minute particles. This was done for two reasons. First to al-
low juices that might form an active part of the glands to ooze
out into the water as quickly as possible, second to give the
weaker individuals a chance to find food particles: In almost
every experiment it was seen that a number of the smallest in-
dividuals did not grow and after a time began to die. It seems,
however, that this is not due to an inability to obtain food, but
because they were less fit to live and were simply weeded out in
the early stages of development.

The tadpoles fed on the crushed glands developed in essentially
the same manner as the others, thus after several weeks both
sets were placed in one dish and fed alike.

One set of animals was fed on clotted blood. They did best
of all. A control set was fed on Elodea canadensis and another
was starved.

All of the tadpoles were about 4 weeks old at the beginning
of the experiment and had not been fed. When starved the ani-
mals eat the dead ones, if these are not removed, but their rate
of growth can by no means compare with that of tadpoles re-
ceiving plenty of food.

The thyroid-fed tadpoles showed the same rapid development
as observed in previous experiments. They could not be kept
alive longer than 23 days, although after 10 days they were put
in water through which a constant current of air was passed.
The groups fed on other tissues also showed the usual rate of
growth. Those individuals fed on spleen, liver and thymus ran
rather parallel courses and progressed somewhat faster during the

FEEDING EXPERIMENTS ON TADPOLES 457.

early days of the experiment than the tadpoles fed on testicle,
ovary and adrenal. The latter three groups also ran similar
courses. The set fed on plants showed a rather rapid growth,
about equal to that of the spleen, liver and thymus groups. The
tadpoles in these groups reached on the average a length of 30
to 31 mm at the time of metamorphosis, while the specimens
in the slower growing groups reached a length of 33 to 34 mm
before they metamorphosed. This fact might lead one to con-
clude that the materials fed to the faster groups, vegetable, blood,
spleen, liver and thymus, contain an agens which causes a more
rapid differentiation than the foods given to the second groups,
testicle, ovary, adrenal. This, however, is not the case, since in
the first group the spleen- and thymus-fed tadpoles exhibited no
signs of an approaching metamorphosis at a time when the tad-
poles of the second group had begun to leave-the water.

Attention should be called to the fact that in this experiment
as in the previous ones of 1912 the spleen- and thymus-fed groups
(spleen had not been fed in 1911) showed almost parallel courses
of development, neither reaching the stage of metamorphosis
when the other groups had. Whether or not this indicates an
influence of the spleen, or perhaps of all lymphatic organs, simi-
lar to that of the thymus in retarding differentiation I am at
present unable to decide.

The tadpoles intended for a pancreas diet did not feed freely
and most of them died early.

The starved animals did not differentiate beyond their original
stage and began to die about 4 weeks after the beginning of the
experiment.

The pigmentation exhibited by the animals was very much the
same as in previous experiments. The thymus-fed individuals,
however, did not become as dark as usual. Those fed on blood
and plants became very dark.

Experiment VI

Rana temporaria, Set Vto VII. Figure 5, a to e. Probably three
sets mixed. They hatched April 6 to 8 and were not fed until May
24, when the experiment began.

458 J. F. GUDERNATSCH

During the early part of the season hypophysis was not available,
so that no experiments with it could be carried out simultaneously with
the other. Therefore this late experiment of hypophysis feeding is not
of great significance. It is reported here to show the rates of growth
of tadpoles fed on glandular and others on the nervous parts of the
hypophysis.

TABLE 10
Measurements in millimeters, and diary
DATE Beets ot HYPOPHYSIS GLANDULAR | HYPOPHYSIS NERVOUS
|
19 .0-21 .0! 17 .0-26 .0 | 17 .0-26 .0
eo | 7.0- 8:0 6.0- 9.0 | 6.0- 9.0
ite Ta eiw sal) piel OSL SRO 10 .5-16 .0 | 10 .5-17 .0
3.5- 4.5 4.0- 6.0 | 4.0- 6.0
19 .0-22.0 17 .0-26 .5 | 17 .0-28 .0
Mage3ats as: 7.0- 8.5 6 .5-10.0 6 .0-10.0
11 .0-13 .5 10.5-18 .0 | 11 .0-18.5
4.0- 5.0 4.0- 6.0 | 4.0- 6.0
19 .0-22.5 17 .0-27 .5 1 17 .0-29 .0
| a ed 7.0- 8.5 6 .5-10.0 6 .0-10.0
11 .0-14.0 11 .0-18 .5 11.0-19.0
4.0- 5.0 4.0- 6.0 4.0- 6.0
23 .0-25 .0 17 .5-30.5 17 .0-30.5
Tarslon mceee. 8.0- 9.5 6 .5-11.5 6 .0-11 .0
15 .0-15.5 11 .0-19.0 11 .0-19 .5
5.0- 6.0 4.5- 7.0 4.0- 7.0
Hind legs

Ue eb sn Eee voluminous bodies.

( One individual grows
faster than the others
and develops hind
legs

JME SO; eee eee 39 0
11.0
21.0
7.0
( 25 .0-27 .0 19 .5-30.5 20 .0-32 .0, 34
9 .0-10.0 7.0-11.5 7 5-11 .0, 11
16 .0-17 .0 12.5-19 .0 12 .5-21 .5, 23
Talon. ee 5 .0- 6.0 4.5- 7.0 4.5- 6.5, 7
Short, but very strong} Only one, the biggest,
extremities. Beginto| has hind legs, but
die still drawn close to
the body

1 When four measurements are given, the first refers to the entire length, sec-
ond to length of body, third to length of tail, fourth to breadth of body.

FEEDING EXPERIMENTS ON TADPOLES 459

The difference between the rates of growth of the two sets is
not marked, there may be a little faster growth in the set fed on
neural hypophysis. The tadpoles fed on glandular hypophysis
(fig. 5 b, 5 d) show decidedly better differentiation and gradu-
ally become less pigmented than those fed on neural hypophysis
(fig. 5 e).

Experiment VII

Bufo vulgaris, Set I. Figure 8, a to d. Hatched April 10 to 20,
1912, and the feeding began May 6.

The experimental data are as follows:

May 5 The feeding began.

May 11 All the thyroid-fed specimens have hind legs and the frog
shape is noticeable. They were fed only four times.

May 18 Thyroid-fed animals have developed fore legs.

May 22 Algae were placed in with the thyroid-fed tadpoles.

June 18 Last thyroid-fed ones die.

June 20 Thymus-fed ones have hind leg buds.

June 24 Control animals have hind leg buds.

July 11 The muscle-fed tadpoles have the best differentiated hind legs.

July 14 Muscle-fed animals grow fore legs.

July 20 The first metamorphosis in muscle-fed group. The control
still have very small hind leg buds. Thymus-fed animals
have their hind legs still drawn close to the body.

This experiment introduced a new species into the study, Bufo
vulgaris, yet the results, as one might have expected, are essen-
tially the same. The thyroid-fed group (fig. 8, d) showed their
usual precocious differentiation, their decrease in size is not as
marked as in the Rana species. The thymus-fed group (fig.
8, c) did not grow much faster than the muscle-fed ones (fig. 8, )
and towards the end of the experiment they actually fell behind
in size, yet they showed little, if any, differentiation at the time
of the first metamorphosis of the muscle-fed tadpoles. A con-
siderable number of the thymus group died when they reached
a length of about 21 mm (status thymicus, thymus death).

TABLE 11

Measurements in millimeters

CONTROL
DATE

VEGETABLE | MUSCLE | THYMUS | THYROID
13-16 mm.!
Wavi6:e ee Cathet
7— 9 mm. ’
3— 5 mm.
14.0-16.0 15 .0-17.0 14.0-17.0 13.0-15.5
5.0- 7.0 6.0- 8.0 6.0- 8.0 5 .0= 7.0
May iSec.00 6 7.0- 9.0 8.0-10.0 8 .0-10.0 7.0- 9.0
3.0- 5.0 4.0- 5.0 AO=3520 3.0-'4.0
Legs 0.8
11.5-15.5
Micivall Speer: ee : :
3.0- 4.0
6.5-15.5
Maw S26) si4. 5
3.0- 4.0
| 11.0-15.5
| 4.5- 6.5
May 20... 65-90
3.0- 4.0
14.0-19.0 t6.0-19.0 14.0-20.0 11.0-15.5
ee 5.5- 8.0 7049.0 6.0- 9.5 4.5- 6.5
Bete ae ea 7.5-11.0 9 .0-11.0 8.0-11.5 5.0- 9.0
3.5- 5.0 4.0-5.0 4.0- 6.0 3.0- 4.0
14.0-20.0 16 .0-20.0 14.0-20.0 10.0-15.0
ren B50 7.0- 9.0 6.5- 9.5 4.5- 6.5
ee TES Tepa12.0. >) -OLOSNI05 8 .5-11.5 5.0- 9.0
#0=5:0..| (440= 5.0 4.0- 6.0 3.0- 4.0
14.0-20.0 16 .0-20.0 14.0-21.0 10.0-14.5
5.5- 8.0 7.0=19..0 7.0- 9.5 4.5- 6.0
June 6......... 7 .5-12.0 eerie Bes BOs 815
4.0- 5.0 4.0- 5.0 4.0- 6.0 3.0- 4.0
16 .0-20.0 16 .0-20.0 14.0-21.5 10.0-13.0
ae as 6.5- 8.5 7029.0 * 7 0='9:0 4.5- 6.0
sdesaes 23 8 .0-12.0 9.0-11.5 8 .5-12.0 4.0- 7.0
4.0- 5.5 4.0- 5.0 4.0- 6.0 3.0- 4.0
17 .0-20.5 16 .0-21.0 17 .5-22.0
ees 6.5- 8.5 7.0- 9.0 70-95
pa ace 9 .0-12.0 10.0-12.0 10.5-12.5
4.0- 5.5 4.0-5.5 4.0- 6.5
18 .0-21.0 18 .0-24.5 19 .0-22.5
July 11 6.5- 9.0 7.5-10.5 8.0- 9.5
ee a 10.012.0 | 10,5-13.0 11.0-13.0
4.5-6.0 | 4.0-6.0 4.5- 6.5

1 When four measurements are given the first refers to the entire length, second

to length of body, third to length of tail, fourth to breadth of body.

460

FEEDING EXPERIMENTS ON TADPOLES 461

The next three experiments to be described were performed
for the purpose of studying the influence of a mixed diet on
growth and development. One kind of food was given one day
and another the next. Previously the animals had been fed for
a considerable length of time on one kind of tissue and were then
transferred to another.

Experiments VIII and IX consider a thyroid-thymus diet only.
The tadpoles of Experiment VIII had been fed on muscle before
the thyroid-thymus treatment began, while those of Experiment
IX had starved. It may be well at present to record the notes

TABLE 12

Measurements in millimeters and diary notes

( 21.0-28.0

8 .5-10.5

13 .0-19 .0

| 4.5- 6.0

June 10........ | Hind legs appear

( 20.0-24.0

7.0- 8.0

June 12........ 4 12.0-16.0

4.5- 5.5

The frog shape is noticeable

14 .0-24.0

5.5- 8.0

9 .0-16 .0

4/8 ae eee } 3.5- 5.0

Very active; legs are very thin (fig. 2 m), but much better de-

veloped than in thyroid (Experiment IV, fig. 4p). Length
of the hind legs 2.5 to 3.5 mm; the abdomen is bloated.

June 19........ About 50% die *

14 .0-22.0

5.5- 7.5

8 .5-14.5

3.5- 5.0

Fore legs appear 10 days later than in the all-thyroid group
(Experiment IV).

14 .0-20 .0

5.5- 7.5

aUyantera tars <3 8 .5-12.5

3.5- 4.5

Last ones preserved

Juin bysn ease sae

==

RITUEV LD). = se

—v

462 J. F. GUDERNATSCH

from these experiments and later discuss them in connection
with Experiment X, in which several kinds of foods in various
combinations were applied.

TABLE 13

Measurements in millimeters and diary notes

|

CONTROL (VEGETABLE) THYMUS-THYROID
14 .0-22.0
JMNCIO es eee ce eanerelae
9 .0-14.0
| 3.0- 4.0
| 15 .0-25 .0* | 14 .0-21 .0
5 5.5- 8.5 | 5.0- 7.0
MINE V2 sd eee: 10 0-17.0 9 0-14.0
3.5- 4.5 3.0- 4.0
| Hind legs appear
ines ee ae | Manycdie
15 .5-26.0 14 .0-20.0
5 .5- 9.0 5.0- 7.0
10 .5-17 .0 9 .0-13.0
TONNE MS. peso oo as 3.5= 5.0 3.0- 4.0
| Fore legs appear
Very active
{ The abdomen is bloated
16 .0-26 .0 14.0-18.5
B 5.5- 9.0 5.0- 6.0
de Pe: cae 10.5-17.5 9.0-11.5
L 3.5- 5.5 3.0- 4.0
Gl A Atckeieesae s. date a: Hind leg buds
July glee Last one dies

‘Experiment VIII

Rana temporaria, Set II. Figure 2, k, 2, m. Hatched April 6 to
7, 1912 (Experiment IV), and fed on muscle until June 3. The largest
individuals of Set Il were selected for this experiment. The feeding
of thyroid-thymus began on June 5.

Experiment LX

Rana temporaria, Sets V to VII. Figure 6, a to d. Probably
three sets mixed. Hatched April 6 to 8, 1912, and not fed until June
5, when the experiment was started.

FEEDING EXPERIMENTS ON TADPOLES 463

Experiment X

Bufo vulgaris, Set III. Figure 9,ator. Brought into the laboratory
May 21, 1912, size 18 to 23 mm, age unknown. Experiment started
June 5. Thirteen groups of tadpoles, 150 individuals in each, were
given food as indicated below; one group was starved.

A part of this experiment corresponds to Experiment IV in
which several groups of tadpoles of Rana temporaria were fed
on thyroid for from 1 to 5 days respectively and afterwards put
on a vegetable diet. The Bufo tadpoles reacted very quickly to
the thyroid stimulus, but when the thyroid feeding was stopped,
they seemed to overcome the thyroid influence more readily than
any other tadpoles. The groups fed on thyroid from 3 to 5 days
(fig. 9, b to d) developed hind legs 5 days after the first feeding,
showing as in other experiments, that a 3 day feeding of thyroid
gland suffices to give the typical results. The 2 day group budded
the hind extremities on the 7th day and the 1 day group on the
11th day. These intervals approach close to those observed in
Experiment IV, which were 5, 6 and 14 days. The anterior ex-
tremities appear in the 5 day group 14 days after the first feeding
(in Experiment IV on the 10th day) and this group begins to
undergo metamorphosis on the 18th day. In the other, 1 to 4
day groups, the after-treatment with vegetable food seems to
check the hastened differentiation following the intake of thy-
roid tissue. They finally do not go much faster than tadpoles
which had not received thyroid. It might thus appear as if the
Bufo tadpoles had a stronger resistancy against the thyroid stimu-
lus than the Rana larvae. This point is not entirely clear, how-
ever, since the former had not received any food before the thy-
roid feedings began, while the latter had previously lived for 50
days on muscle. As has been stated above, any meat diet before
or after the thyroid treatment is apt to render the animals more
susceptible to the thyroid stimulus than does starvation.

The thyroid treated Bufo tadpoles do not reduce their size so
much as the Rana tadpoles, as before mentioned under Experi-
ment VII.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, No. 4

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"gz oun

“yz oune
“gz oun

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“ge oun

465

466 J. F. GUDERNATSCH

The main interest of Experiment X lies in the results following
the mixed feedings of two kinds of animal tissues or animal tis-
sue and plants.

It will be seen from the notes that, whenever thyroid tissue
was one of the two foods it exerted an accelerating influence on
development. The four groups fed on thyroid-thymus (fig. 9 h),
thyroid-muscle (fig. 9 f, 9), thyroid-hypophysis (fig. 97, 9 p)
and thyroid-plants (fig. 9 g, 9 q) developed their hind legs on the
5th day. The fore legs appeared in the third and fourth groups
on the 21st day, in the second group on the 22d day and in the
Ist group on the 24th day and the tadpoles of the several groups
began to metamorphose in the same order. These facts indicate
that the thymus was best able to counteract the thyroid, the
hypophysis and plants least. It is peculiar, however, that the or-
der of the last individual metamorphoses is exactly the reverse,
the thyroid-thymus group completing the change first, the thy-
roid-plant group last. It is doubtful whether this has any con-
nection with the fact that the greatest reduction in size was in
the first group and gradually less down to the fourth.

We may now compare these thyroid-thymus results with those
of Experiments VIII and IX. They are essentially alike, the
thymus always being able to check the thyroid influence to some
degree, but unable to suppress it entirely (fig. 2, 2 m, 6b, 6d,
9h).

The remaining three groups, thymus-hypophysis (fig. 9 7, 9 p),
thymus-plants (fig. 9k, 9q) and hypophysis-plants run much
slower than the corresponding thyroid groups. Their hind legs
appear from 16 to 19 days later than in the thyroid combination
feedings.

The hypophysis-plant group leads the three, the thymus-plant
group is a few days behind. This corresponds to the thyroid
groups, where the hypophysis was less able to counteract the thy-
roid acceleration than was the thymus. In the thymus-hypophy-
sis group (91, 97r) two retarding factors combine, therefore the
tadpoles fed on this diet never develop fore legs nor do they be-
gin to metamorphose. Yet they are very big healthy specimens. -

FEEDING EXPERIMENTS ON TADPOLES 467

It is thus clear that, whenever thyroid forms a part of the diet,
a precocious differentiation sets in; when thymus is added (thy-
mus-thyroid, thymus-hypophysis, thymus-plants), the opposite
influence is noticed.

One group (9 m) of Bufo tadpoles was starved to test again
whether hunger might cause differentiation, but no differentia-
tion was noted even 5 weeks after the start of the experiment.

Finally another group was fed on testicle. The testicle diet
had not been fully tested before. The development of these tad-
poles did not differ from the control and this group can therefore
serve for a further comparison with the groups given a mixed
diet.

Experiments were also begun on Triton alpestris and on Bufo
vulgaris tadpoles which had a part of their tails amputated, but
the time was too short to carry them out completely.

Feedings were started with Triton alpestris larvae on June 24.
On July 12 the outer gills of the thyroid-fed ones had disappeared
and the fore legs were well differentiated. The thymus-fed speci-
mens still possessed their outer gills and had only small buds of
their fore legs. The animals treated with other foods showed
intermediate conditions.

Bufo vulgaris tadpoles of 19 to 23 mm in length, tails 10 to 14
mm. long, had 5 mm of their tails amputated on June 24. The
next day separate groups were started on thyroid, thymus, mus-
cle and vegetable diets. The thyroid group developed hind legs
on July 3 (8th day) and fore legs on July 12 (17th day). The thy-
mus and muscle groups developed hind legs on July 9 (14th day).
Their measurements are given in table 15, page 468.

The thymus-fed ones had grown considerably by July 14 and
regained their former average length, since they had almost
completely regenerated their tails. The other groups had grown
and regenerated much less, the thyroid least of all, while in turn
it showed the fastest differentiation. ‘These observations cor-
respond with those of 1911. Romeis has studied extensively the ©

468 * J. F. GUDERNATSCH

TABLE 15

CONTROL

3
: ead
VEGETABLE RE ee THYMUS | MUSCLE

mm |

19 .0-23 .0 (14-19 mm after amputation)
7.0-9.0 |
June 24. ......- 10 .0-14.0 | ( 5- 9mm after amputation)
3.5- 52D) | |
13 .5-18 .0 110-140 ||» 20°0-22.0, |, 15. 0-18
Tuily 14 7.0- 9.5 5 .0- 6.5 9 .0-10.5 | 7.5- 9.5
Aah oe 6.0- 9.0 6.0-8.0 | 11.0-12.0 7.5- 9.0
6.5 4. &

450-75:.0 | 2.5- 4.0 5 .Q-

influence of different diets on the regeneration of the tail in
Rana esculenta tadpoles.

The experiments of 1911 and 1912 leave no doubt that the
mammalian thyroid gland contains an agens which, when the
gland is given as food, calls forth a rapid differentiation in a
developing vertebrate organism. This differentiation may be
brought about at any stage of development, before maturity is
reached. The differentiation may therefore be highly precocious
in cases where the treatment is begun on extremely young ani-
mals. Experiment I showed that tadpoles could be brought to
the point of metamorphosis within 18 days after hatching, while
normally they would require 10 to 12 weeks to reach such a stage.
The results of this premature metamorphosis are perfect frogs
of minute size, pygmy frogs, as the figures show (fig. 4 0, 4 p and
others). The thyroid influence is very decided and there is no
escape from it for any tadpole given a thyroid diet. All of the
individuals, even if thousands be employed as was the case in
several experiments, will react almost immediately to the thy-
roid stimulus, so that certain changes in their structure may be
observed after 24 hours, when only one application of thyroid
has been made. They will all react simultaneously so that any
individual differences in development will become unobservable.

The experiments further bring out the fact that the time of re-
action to the thyroid stimulus varies to a certain degree with the

FEEDING EXPERIMENTS ON TADPOLES 469

kind of food that had been given before the thyroid diet was
started. There is also a difference in response between previously
starved and fed tadpoles. The experiments with mixed diets
showed that the accelerating influence of the thyroid could be
checked to some extent and the rapid differentiation more or less
retarded. However, there is no complete counteraction against
the thyroid stimulus, so that sooner or later any tadpole, receiv-
ing thyroid after other or mixed with other diets, must respond to
its influence.

Experiments were carried out to determine the least amount of
thyroid food necessary to produce the typical reactions, and also
to determine whether or not the tadpoles could recover from the
thyroid shock if afterwards put on other food. A feeding of only
24 hours—that is to say, the thyroid was kept in the dish for
about 24 hours, though the animals did not feed on it continuously
—sufficed to cause a hastened differentiation. A feeding for three
days was enough to give the fastest rate of differentiation, a rate
that could not be increased by longer feedings.

When thyroid is applied too rapidly, the animals usually die
very soon after the appearance of their fore limbs and the simul-
taneous reduction of their tails.. By careful feedings at rather
long intervals and in all not more than four times, the animals may
be kept alive for several weeks. They will not undergo, however,
any further changes, except perhaps a continued reduction of
their tails, nor will they ever feed again. In 1911 I succeeded in
bringing some tadpoles to a complete absorption of their tails and
these thyroid frogs were kept alive on wet sand for from 2 to
4 days. In 1912 some Bufo tadpoles almost completely ab-
sorbed their tails under the thyroid treatment (fig. 9 e, 9 n), but
could not be kept alive for more than 24 hours.

A recovery from the thyroid influence is extremely rare. Only
5 individuals out of 400 in one experiment and 6 in another
were able to survive, and, although, they were never seen to feed,
began slowly to grow again after a standstill of several weeks.

470 J. F. GUDERNATSCH

Not one of the many thousand tadpoles fed on thymus in the
spring of 1912 could be brought to metamorphosis during the 15
weeks in which the animals were under observation, while tad-
poles of the same set, though fed on other substances, metamor-
phosed before that time (fig. 4 0, p, liver). In some experiments
the thymus fed tadpoles never succeeded in growing their hind
extremities before the controls completely metamorphosed. Yet
the thymus tadpoles would grow very rapidly, especially during
the first weeks of the experiments.

The tadpoles feed on spleen behaved in very much the same
way as the thymus-fed ones, though they always were somewhat
ahead of the latter and did not counteract the thyroid feedings
so strongly. Of the great number of spleen fed tadpoles also not
one could be caused to metamorphose.

We must therefore conclude that the thymus and to some ex-
tent the spleen also, and probably the lymphatic organs, when
given as food, will cause a rapid growth followed by a rather late
differentiation or none at all.

These experiments with various foods, except thyroid, thymus
and spleen, and the investigations of other workers show that the
animals must reach a certain constant minimum size before the
final metamorphosis can begin. There is on the other hand a
constant assimilation of food and a gradual increase in body size
up to a constant maximum, beyond which a normal animal may
not pass without the beginning of the final differentiation and
metamorphosis.

In the thyroid-fed tadpoles there is differentiation without
growth, in the thymus- (and spleen- ) fed tadpoles growth with-
out differentiation. ‘The experiments, therefore, emphasize the
fact that in development we deal with two entirely separate factors,
the factor of growth and the factor of differentiation.

The two naturally work simultaneously; but differentiation is
not the result of growth, otherwise there could be no thyroid differ-

FEEDING EXPERIMENTS ON TADPOLES 471

entiation without growth. Nor must growth necessarily be fol-
lowed by differentiation, as seen in the growing thymus tadpoles.

One might say that ordinary foods which are being assimilated
bring with them the two factors of growth and differentiation.
Then the assumption is necessary that the thyroid food lacks the
power of causing growth, while the thymus and spleen lack the
power of causing differentiation. But we can hardly assume that
all the various kinds of foods an animal may take in, with the only
exceptions of thyroid and thymus, contain these two factors in the
proper proportions.

The factors for growth and differentiation can only be located
within the organism itself.

We face the following propositions:

1. The thyroid has the power to excite differentiation, but it
lacks the power to cause growth.

The thyroid calls forth differentiation, whether the animals
be small or large, and without regard to the standard minimum
size, necessary for the final change. It must possess an agens for
stimulating differentiation which other foods do not possess.
That the thyroid also possesses a power which prevents growth is
not evident. The suppression of growth may merely be inci-
dental, for rapid differentiation does not allow growth.

2. The thymus has the power to stimulate growth, but lacks
the power to excite differentiation.

It has been stated above that tadpoles feeding on any food
(except thyroid) reach a maximum size, and when this maximum
is reached, differentiation begins independently. The thymus-fed
tadpoles reach this maximum size and differentiation should set in
of itself, even if the thymus lacked the necessary stimulus. But
differentiation does not begin; therefore the thymus (and spleen)
must exert an influence not possessed by the other foods which
suppresses differentiation. That the thymus possesses the power
to stimulate growth, is again not so evident, since the thymus
growth may be merely the normal result of the intake of food.
The thymus growth is rather rapid, but this may be attributed to
the better nutritive qualities of the thymus tissue. The thymus-
fed tadpoles may also grow beyond the normal as brought out in

472 J. F. GUDERNATSCH

1911, yet this again may be merely incidental. There is no be-
ginning of differentiation in thymus-fed tadpoles which would
check a further growth.

We can only say with reasonable safety:

1. The thyroid possesses a quality that stimulates differentia-
tion, not contained in any other food used.”

2. The thymus (and spleen) possess a quality that suppresses
differentiation not contained in any other food used.

Thus the thyroid and thymus must produce, or at least con-
tain, agents which, when passed into developing organisms, in the
one case stimulate, in the other suppress, differentiation. The
production of such substances to be thrown into the circulation
characterises the thyroid and thymus as glands with a positive
internal secretion. ‘That these two types of tissues may also be
capable of performing the reverse action, viz. the elimination of
certain substances from the circulation, as has been assumed
especially for the thyroid, is neither demonstrated nor denied by
these experiments. j

The other glands used in the studies may or may not contain
either the accelerating or the depressing power. However, the
macroscopic differences in the rate of growth and differentiation
of tadpoles fed on such glands and of the control animals are only
slight and might easily be attributed to differences in the nutri-
tive values of the various foods. The feeding experiments on tad-
poles are, therefore, not likely to reveal these factors, if at all
present in other glands, in a striking degree. Their study must
beleft to experiments of a different kind.

2 By ‘differentiation’ is meant merely the macroscopic changes, hind, fore-
limbs, metamorphosis. The microscopic differentiation will be discussed in a
later paper.

FEEDING EXPERIMENTS ON TADPOLES 473

LITERATURE CITED

BarFurti, D. 1887 a Versuche iiber die Verwandlung von Froschlarven. Arch.
f. mikr. Anat., Bd. 29, p. 1.
1887 b Der Hunger als férderndes Prinzip in der Natur. Arch. f.
mikr. Anat., Bd. 29, p. 28.

Bascu, K. 1906 Beitriige zur Physiologie und Pathologie der Thymus. Jahrb.
f. Kinderheilk., Bd. 64; 1908, Bd. 68.

Brepu, A. 1910 Innere Secretion, I. Auflage. Wien-Berlin.
1913 Innere Secretion, 2. Auflage. Wien-Berlin.

GupERNATsCH, J. F. 1912a Fiifterungsversuche an Kaulquappen. Demonstr.
Verh. Anat. Ges., Bd. 26. Vers. Miinchen.

1912 b Fiitterungsversuche an Kaulquappen. Vorl. Mitteil. Centralbl.
f. Physiol., Bd. 26, no. 7.

1912¢ Feeding experiments on tadpoles.I. Arch. f. Entwickel.-
Mech., Bd. 35, p. 457.

Hammar, I. A. 1910 Fiinfzig Jahre Thymusforschung. Ergebn. d. Anat. u.
Entwicklungsg., Bd. 19.

LIEBEN, 8. Uber die Wirkung von Extrakten chromaffinen Gewebes (Adre-
nalin) auf die Pigmentzellen. Centralbl. f. Physiol., Bd. 20.

Romets, B. 1913 Der Einfluss verschiedenartiger Ernihrung auf die Regenera-
tion bei Kaulquappen (Rana esculenta) I. Arch. f. Entwicklungs-
mech., Bd. 37, p. 183.

VINCENT, 8. 1912 Internal secretion and the ductless glands. London.

All illustrations taken from living tadpoles. All figures in natural size except
figures 10, a to7z and 11, a tof.

PLATE 1

EXPLANATION OF FIGURES

1,atoze Rana temporaria lI. Experiment I. a, liver; b, spleen; c, thymus;
d, muscle; e, ovary; f, adrenal cortex; g, adrenal medulla; h, brain; June 6, 1912.
i, liver; k, liver-thyroid; 1, spleen; m, spleen-thyroid; n, muscle; 0, muscle-thyroid;
p, Ovary; q, ovary-thyroid; 7, adrenal cortex; s, adrenal cortex-thyroid; t, adrenal
medulla; u, adrenal medulla-thyroid; v, brain; w, brain-thyroid; June 26, 1912.
In k, m, 0, q, 8, u, w, thyroid feeding had been started on June 17, 1912. x, liver;
y, brain; z, spleen-thyroid; za, muscle-thyroid; zb, ovary-thyroid; zc, adrenal cor-
tex-thyroid; zd, adrenal medulla-thyroid; ze, brain-thyroid, July 6, 1912. In z
to ze, thyroid feeding had been started on June 17, 1912.

2,a to n Rana temporaria II. Experiment IV. a, original size, June4,
1912. 6, control; c, thyroid-muscle, thyroid given 1 day; d, thyroid-muscle,
thyroid given 3 days; e, thyroid-muscle, thyroid given 5 days; June 17, 1912. f,
thyroid-plants; thyroid given 1 day; g, thyroid-plants, thyroid given 2 days; h,
thyroid-plants, thyroid given 4 days; 7, thyroid-plants, thyroid given 5 days; k,
thyroid-thymus; /, thyroid emulsion; June 17, 1912. m, thyroid-thymus, n, thy-
roid-muscle; thryoid given 4 days; June 26.

3,a ton. Rana temporaria III. Experiment III. a, thymus; b, muscle; c¢,
thymus-thyroid I; d, thymus-thyroid II; e, thymus-thyroid III; June 1, 1912. f,
thymus-thyroid II; g, thymus-thyroid III; h, thymus-thyroid IV; June 17, 1912.
i, thymus; k, thymus-thyroid V; /, muscle; m, muscle-thyroid; June 26, 1912. n,
muscle-thyroid; July 6, 1912.

5,atoe Rana temporaria V to VII. Experiment VI. a, control; 6b, glandu-
lar hypophysis; June 26. c, control; d, glandular; e, neural hypophysis; July 6,
1912.

6,atod Ranatemporaria V to VII. Experiment IX. a, control; b, thymus-
thyroid; June 17, 1912. c, control; d, thymus-thyroid; June 26, 1912.

474

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PLATE 2
EXPLANATION OF FIGURES

4,atop RanatemporarialV. ExperimentII. a, control; b, thymus; c, spleen;
d, muscle (thyroid under 4, 0 and p); e, thymus-thyroid IT; f, thymus-thyroid IIT;
g, thymus-thyroid IV; June 1, 1912. A, thymus-thyroid IV; 7, thymus-thyroid V;
June 17, 1912. k, muscle; /, muscle-thyroid; m, spleen; n, spleen-thyroid; June
26, 1912. o to p, 1 frog fed on liver, metamorphosing on July 12, and 5 pigmy
frogs, fed on thyroid metamorphosing on April 22, 81 days earlier. 0, dorsal; p,
ventral view.

7,atob Ranatemporaria VIII to XI. Experiment V. a, spleen; b, adrenal;
July 6, 1912.

8,atod Bufo vulgarisI. Experiment VII. a, control; b, muscle; c, thymus;
d, thyroid; June 6, 1912.

9,a to r Bufo vulgaris II]. Experiment X. a, original size; June 5, 1912.
b, control; c, thyroid-plants, thyroid given 1 day; d, thyroid-plants, thyroid given
3 days; e, thyroid-plants, thyroid given 5 days; June 26, 1912. f, thyroid-muscle;
g, thyroid-plants (alternately); h, thyroid-thymus; 7, thyroid-hypophysis; k,
thymus-plants; J, thymus-hypophysis; June 26, 1912. m, control; n, thyroid-
muscle; 0, thyroid-plants; p, thyroid-hypophysis; q, thymus-plants; 7, thymus-
hypophysis; July 6, 1912.

10,a to i Rana temporaria IV. Experiment II; compare figure 4, a to p.
Thyroid-fed frogs, 16 to 18 days old, at the time of metamorphosis, figure 4, J.

l11,a tof. Tails of tadpoles fed on different substances, to show pigmenta-
tion; figure 4, /. a thymus; b, liver; c, spleen; d, muscle; e, adrenal cortex; /,
adrenal medulla.

FEEDING EXPERIMENTS ON TADPOLES PLATE 2
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ORIGIN AND EARLY HISTORY OF THE PRIMORDIAL
GERM-CELLS IN THE CHICK

CHARLES H. SWIFT
From the Hull Laboratory of Anatomy, University of Chicago

FIFTEEN FIGURES

CONTENTS
introduction and review of the’ literature en. 42... . 2.2... eis oh bade ce ee 483
Materralsan dim et hod Strzissy jsceyserer eran eee ae oe) alee «SG SSIS oe yh ees ee 489
Origin) and history of the primordial werm-cells (3... 3... . .. wh. Pn een 49]
(Ee Stinuctune-otethe genm=Ccell sear meme: etter yeas or.) 50's Says a, Wetene ee eee 491
ye Ma eration ofthesrerm-Cellsae sate ay way ey 20 Palossieiete als ols sonora ena 496
SeOrieingon the: cerm-Cell sven (pin ihian One eee, ord boo SaiaWo-vranie wae ee 510
Siam diay fan GO CONCLUSIONS: 1c stews ek te eae AM ew wi Sis. tyoyn'es Av ems pee 514
Bolo omar Layee rariey cenctnarct se SRR OLA ETRE Occ cl oS, SEES a ecatacie Uahetace megeeheS 515

INTRODUCTION AND REVIEW OF THE LITERATURE

There are several theories held as to the origin of primordial
germ-cells. The theory that they arise from a differentiated por-
tion of the coelomic epithelium over the Wolffian body has pri-
ority and should be mentioned first. Waldeyer (’70) was the
first to advance this idea as to an origin from a so-called germinal
epithelium. His opinion advanced in the work ‘“‘Eierstock und
Ei,” remained unchallenged for a long time. It has had a num-
ber of supporters, and even at the present day, the theory that
the primordial germ-cells arise in the germinal epithelium, is the
only one found in a majority of text-books. That this theory
should have a long life is only natural. The early investigators
did not possess the finished methods or perfected instruments of
today and so were placed at a serious disadvantage when working
with the younger stages. Also, as we now know, the germ cells
in the earlier stages of development, are not grouped, but may
be widely scattered, and for this reason, if for no other, they

483

484 CHARLES H. SWIFT

would be much more difficult of demonstration than when col-
lected into a relatively circumscribed area like the germinal epithe-
lium or developing gonad. In the older embryos, however, they
were able to see the typical primordial germ-cells in the modified
coelomic epithelium, and so naturally thought that they arose
by a series of transformations from the epithelial cells. Semon
(87) claimed to have seen a number of cells undergoing this
change of form, but since that time no other investigator has
ever been able to see any intermediate stages between the cells
of the germinal epithelium and the primordial germ-cells. _D’Hol-
lander (’04) described in the chick embryo of ten days, however,
the production of oogonia from a superficial indifferent layer of
cells—another term for germinal epithelium. According to d’Hol-
lander, the germinal epithelium gives rise to epithelial buds, which
grow down into the ovarian stroma. From these epithelial, germi-
native buds, by a process of differentiation, the oogonia and
primordial follicular cells arise. This origin of oogonia at such
a late period and from the germinal epithelium must be second-
ary to the production of primordial germ-cells, which, according
to all authorities, make their appearance in the gonad at a much
earlier period of development than ten days.

Another view as to the origin of primordial germ-cells may be
called the gonotome theory. The supporters of this theory agree
with the advocates of the germinal epithelium origin in so far
that the primordial germ-cells are found at one time in the modi-
fied coelomic epithelium. They, however, claim that the pri-
mordial germ-cells do not arise in the germinal epithelium, but
from a portion of the segmental mesoderm, and reach the epithe-
lium at a later period either by migration or by tissue growth.
Rickert (88) advanced this idea, and called that portion of the
segmental mesoderm, from which he supposed they took origin,
the gonotome. That certain of the primordial germ-cells are, at
times, found in the region of the segmental mesoderm there can
be no doubt, but, their occurrence there can be explained in an-
other and better way than by supposing that they take origin
there. This theory has had few advocates, and is of importance
now, mainly because of the historical interest attached to it.

ORIGIN OF PRIMORDIAL GERM CELLS 485

The supporters of the third theory, that of early segregation,
agree with the exponents of the other two, that the primordial
germ-cells are at one time in their history found in the germinal
epithelium. They do not, however, believe that they arise in
situ, nor from the gonotome, but that they have a much longer
history dating back to an early embryonic stage. It may be
possible, at some future time, to trace the primordial germ-cells
back to the very early divisions of the segmenting egg, if not to
the fertilized egg itself. In vertebrates, the work of Eigenmann
(97), demonstrated an origin of primordial germ-cells within five
divisions of the fertilized egg; that is, he found that the young-
est germ-cells possessed a size equivalent to blastomeres of the
fifth cleavage. If this last is correct, that the germ-cells have
an independent existence from a very early period, and are not
differentiated from somatic cells, then the idea of germ-plasm
continuity has received valuable support. This idea of early and
extraregional origin is borne out by all recent research, and is be-
coming more firmly established with each succeeding year.

The names of Hoffmann (’93) Eigenmann (’97), Nussbaum (’01)
and Beard (’04) are associated with the early years of this early
segregation theory of germ-cell origin.

Hoffman (93) was the first to bring forward any evidence
which cast doubt on the theory of origin of primordial germ-
cells from the germinal epithelium. He employed in his work
twelve species of birds, in three of which, Haematopus ostralegus,
Sterna paradisea, and Gallinula chloropus, there was sufficient
evidence brought out to prove that some, if not all the primor-
dial germ-cells, did not originate in the modified coelomic epi-
thelium. In the three species mentioned above, he found at the
proper time, numbers of primordial ova in the germinal epithe-
lium. But, in addition he found cells—supposedly primordial ova,
because of their resemblance to those found later in the germinal
epithelium—in embryos of 23 somites. An embryo of 23 so-
mites does not possess the so-called germinal epithelium, the coe-
lomic epithelium over the Wolffian body not having been modi-
fied at this age, yet in these he found primordial germ-cells far
removed from the site of the future sex-gland, in the splanchnic

486 CHARLES H. SWIFT

plate of mesoderm, in the region between splanchnic mesoderm
and entoderm and in the entoderm.

Eigenmann (’97) was one of the first to prove that primordial
germ-cells may have an extra-regional origin. His work on the
viviparous teleost Cymatogaster showed that the primordial germ-
cells in that form, have an origin from cells having the size of
fifth cleavage blastomeres.

Beard (’04) discovered in Raja batis that the primordial germ-
cells have an origin from a group of cells in the anterior part of
the blastoderm, beneath the embryonic anlage, and that these
cells then migrate in towards the region of the future sex-glands
along a very definite path, through the hypoblast of the gut and
the line between hypoblast and unsegmented splanchnic meso-
derm. These early extra-embryonic germ-cells, which he called
megaspheres, differ in no particular, except size, from the germ-
cells, which are found in the older embryos.

Nussbaum (’01), Rubaschkin (’07), and von Berenberg-Gossler
(12), in addition to Hoffmann (’93) have investigated the pri-
mordial germ-cells in bird embryos. ‘The first three worked with
the chick and were able to find in embryos of 22 to 23 somites
typical germ-cells in the entoderm and splanchnic mesoderm lat-
eral to the coelomic angle, some of which in older embryos passed
into the germinal epithelium. The 22-somite embryo is the
youngest in which primordial germ-cells have been found in the
chick.

Allen (06), (09), (09), investigated this question of primordial
germ-cell origin in Chrysemys, Amia and Lepidosteus and dis-
covered that the primordial germ-cells in these forms had an en-
todermal origin, in the case of Chrysemys from the entoderm
near the margin of the area pellucida in a zone extending on
either side from the anterior extremity of the pronephros to a
point behind the embryo. The primordial germ-cells of Lepi-
dosteus have a not very different origin, while in Amia they were
shown to come from the entoderm of the roof and margin of the
floor of the sub-germinal cavity.

Allen has shown that in these forms there is a definite migra-
tion. In the youngest embryos the primordial germ-cells have

ORIGIN OF PRIMORDIAL GERM CELLS 487

an amoeboid form and a lateral position in the hypoblast; in
older stages these cells still have an amoeboid form, but have a
more median position, which they have attained by a centripetal
movement along the hypoblast. In the still older embryos these
cells are found to have lost their amoeboid character and to be
in the entoderm under the notochord or in the mesoderm in that
immediate neighborhood. Thence, according to the evidence of
the still older embryos, they pass into the mesentery and finally
assume a position which corresponds to that of the future sex-
gland.

Woods (’02), working on Acanthias, found that the primordial
germ-cells were first evident in the entoderm and in the periblast
and thence migrated to the sex-gland anlage.

Jarvis (08), in Phrynosoma, found that the germ-cells ap-
peared first in the entoderm of the vascular area of the blastoderm,
where they were cephalad, caudad, and laterad to the embryo.
Then by a definite migration path they reached the germinal
anlage.

Dodds (10), investigating the question of germ origin and his-
tory in the teleost, Lophius, was able to recognize the primordial
germ-cells in the primitive entoblast, when the blastoderm had
not quite half covered the yolk. He, however, believed that
they were set apart at a time earlier still in embryonic history.

Finally, as far as vertebrates are concerned, Fuss (711), in a
human embryo of four weeks, with about 33 somites, demon-
strated extra-regional primordial germ-cells in the mesentery di-
rectly under the coelomic epithelium.

Among invertebrates, in many phyla, the primordial germ-
cells have been observed at a distance from the site of the future
sex-gland, that is, in an extra-regional position, and in several
cases have been traced back to their true origin. Indeed, Bal-
biani’s (’85) work with Chironomus was one of the very earliest
in which a definite origin was proven for the primordial germ-
cells. He found that they originated from cells differentiated |
very early in the history of the segmenting egg.

The most remarkable known case of early differentiation of
the primordial germ-cells is that of Ascaris, in which Boveri

488 CHARLES H. SWIFT

(92) was able to follow them back directly through all the cleav-
age stages to the two-cell stage.

Haecker (’97), in Cyclops, and Hegner (’09), in some Chry-
somelid beetles, were able also to trace the primordial germ-cells
back to early segmentation stages.

It may be said, then, that in many groups of vertebrates, as
well as invertebrates, all the primordial germ-cells do not origi-
nate in a differentiated portion of the coelomic epithelium called
the germinal epithelium. In many of the cases cited the exact
origin of the primordial germ-cells has not been found, but even
in these it has been definitely established that the germ-cells
arise in the early history of the embryo long before the appear-
ance of a germinal epithelium. So, with the evidence at hand,
it would seem that Nussbaum was correct when in 1880 he form-
ulated the hypothesis that ‘“‘the sexual cells do not come from
any cells that have given up their embryonic character or have
gone into building any part of the body, nor do sexual cells ever
go into body formation.”

The bird groups still offer an excellent opportunity for research
into the history of the primordial germ-cells, for, notwithstand-
ing the amount of work which has been done by Nussbaum, Hoff-
mann, Rubaschkin, von Berenberg-Gossler, and others, the ori-
gin of the primordial germ-cells still remains unrevealed. The
stage of 22 or 23 somites is the youngest in which germ-cells
have been described among birds.

In carrying out this research I have received constant aid and
advice from Prof. R. R. Bensley. I wish to thank him for this,
as well as for a training which has made possible whatever in
my results may be considered of value. I also wish to thank
Dr. E. V. Cowdry and Dr. G. W. Bartelmez for their assist-
ance as well as kindness in allowing me to use their excellent
preparations. I am also indebted to Mr. A. B. Streedain for the
drawings which illustrate this article.

ORIGIN OF PRIMORDIAL GERM CELLS 489
MATERIAL AND METHODS

In the selection of a fixative for the germ-cells, the yolk ma-
terial, mitochondria, and attraction-sphere, in addition to the
nucleus and general cytoplasm, have to be taken into considera-
tion. The same fixing fluid cannot be relied upon in all stages,
but in order to meet the changing cell content, a variation is
necessary. For instance, the yolk-laden germ-cell of the very
young embryos requires very different treatment from the almost
yolk-free cell of the later stages, if equally good results in both
cases are desired. So that in the case of embryos over 20 somites
in development, in which the germ-cells contain only a moderate
amount of the primary vitellus, a fixing fluid containing osmic
acid may be used to advantage. Osmic acid resembles potassium
bi-chromate, with which it is frequently combined, in being an ex-
cellent cytoplasmic preservative. It has its drawbacks, however,
in being a poor penetrant, amd in blackening the yolk of the germ-
cells. This last property of osmic acid is a decided disadvantage
in the early stages, in which the cytoplasm of the germ-cells is
stuffed with yolk spheres, and an advantage in the case of the
older embryos, in which the yolk is reduced in amount, since the
blackening of the vitellus facilitates identification.

Fixatives such as Benda’s, Meves’ modification of Flemming’s
fluid, and Bensley’s acetic-osmic-bichromate mixture, all of which
contain osmic acid, were found to be best adapted for use in the
case of older embryos, in which there is little yolk material re-
maining. They preserve such cytoplasmic structures as mito-
chondria and the attraction-sphere excellently, and, thanks to
their osmice acid content, bring out the yolk spheres plainly.
However, in the younger embryos, from the primitive streak
stage to 20 somites, they were found to be not nearly so efficient.
In these stages the yolk content of the germ-cells is so great that
the value of any fixative containing osmic acid is seriously di-
minished. In these young embryos a mixture of equal parts of
5 per cent trichloracetic acid and 5 per cent sublimate was found
to be excellent. The mitochondria are not preserved by this

490 CHARLES H. SWIFT

method, but the obscuring of the nucleus and cytoplasm by the
large, blackened yolk spheres is avoided.

An ideal fixing fluid should not only be an excellent cytoplas-
mic preservative but should also have the ability to penetrate
rapidly. Unfortunately osmic acid and potassium bichromate
are lacking in this last quality. Acetic acid is frequently com-
bined with them because of its penetrating powers. However,
too much acetic acid is worse than none at all, since cytoplasm
and its contained organs, like mitochondria, are very sensitive
to this agent. For this reason Zenker’s fluid is of no value in the
preservation of mitochondria. The acetic-osmic-bichromate mix-
ture of Bensley contains these fixing agents in about the proper
proportions, and yet even this leaves something to be desired.
It is of value as a fixative only when small pieces are used. It
really preserves, as in the natural state, only the outer surface of
a piece of tissue. In the center of a block of any size the mito-
chondria are found to be globular, instead of rod-like, or even
to have disappeared altogether. The value of thé acetic-osmic-
bichromate mixture depends upon all the elements in it. They
do not all penetrate with equal speed; the acetic acid is much
the more rapid in this regard. Near the periphery of a piece of
tissue they act in unison, as is intended, but the acetic acid soon
forges ahead and acts in an injurious manner upon the cyto-
plasm and especially upon the mitochondria,

The following staining methods were employed. Following the
Benda fixaton the Benda staining method was used. This pro-
cedure is the one commonly employed for demonstrating mito-
chondria. It was first published by Benda in 1901.

Following Bensley’s acetic-osmic-bichromate mixture two gen-
eral staining methods were used, namely, the anilin acid fuchsin-
methyl green method and Bensley’s copper-chrome-hematoxylin
technique. There were, however, several modifications of the
first. In place of methyl green as a counterstain, toluidin blue
or Wright’s blood stain may be substituted. Bensley (11) de-
scribed these methods in detail, so there is no need to recapitulate
here.

ORIGIN OF PRIMORDIAL GERM CELLS 491

All these procedures are reliable and a beautiful result is usu-
ally obtained, especially at the periphery of a section.

Following Meves’ modification of Flemming’s fluid the iron-
hematoxylin stain was employed. This method was found to
be more reliable in the moderately young embryos—15 somites
to 25 somites—than the acetic-osmic-bichromate method.

After the 5 per cent trichloracetic acid and 5 per cent subli-
mate fixation iron-hematoxylin and acid fuchsin gave excellent
results. The cytoplasm is well preserved and the attraction-
sphere especially so. The centrosomes were more frequently
seen after this procedure than after any other.

In all cases where embryos of relatively advanced age were
employed, the anterior body wall, amnion and viscera were re-
moved, thus exposing directly to the fixative the Wolffian body
and gonad. All sections were cut 4 micra thick.

Table 1 will show at a glance the number of embryos employ-
ed, their age, together with the methods used in fixation and
staining.

ORIGIN AND HISTORY OF THE PRIMORDIAL GERM-CELLS

1. Structure of the germ-cells

It will be better to describe the form and structure of the pri-
mordial germ-cells in the chick before proceeding to a descrip-
tion of their origin and history. In this way the necessity of de-
scribing them with each stage of development will be avoided.
There is all the more reason for this since more than one portrayal
of them would be a repetition. The change in the germ-cells,
from their origin to the time when they pass into the indifferent
gonad, is limited to one or two structures. These variations
will be taken up at the stage of development at which they ap-
pear most evident.

Size and shape. The most noticeable attributes of these pri-
mordial germ-cells, the characters, which first attract attention
in examining a section containing them, are their size and shape.
Isolated in the general mesenchymal tissue they appear immense
(figs. 2 and 8). Their size, which is much greater than that of

SWIFT

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the neighboring cells, is due to an increase in volume of both
nucleus and cytoplasm. Their shape also is different from that
of the surrounding cells. The latter are frequently stellate, al-
though they may be round, oval, flattened, or elongated, while
the germ-cells are always round or oval (figs. 2, 6, and 10). The
germ-cells have an average diameter of about 16 micra. They
may have a diameter as small as 14 micra, while in exceptional
cases this may reach 20 or even 22 micra.

Nucleus. The large nucleus, which has a diameter of 8 to 12
micra, is spherical and vesicular, and is nearly always eccentric-
ally placed (figs. 7, 8 and 13). It is surrounded by a definite
nuclear membrane. On comparing the nuclei of the primordial
germ-cells, with those of the neighboring cells, in addition to the
disparity in size, it is noticed that the germ-cell nuclei are clearer.
The germ-cell nuclei seem to contain about the same amount of
chromatin as the nuclei of neighboring cells, but owing to the in-
crease in size of the former it appears greatly reduced in amount.
The germ-cell nucleus, at one pole, is separated from the cell
- membrane by a thin layer of cytoplasm, while the attraction
sphere generally occupies the other pole, where a wider expanse
of cytoplasm separates the nucleus from the membrane. The
clear appearance of the nucleus of the germ-cell is not confined
to the chick alone but is common to many species.

The chromatin within the nucleus generally has a definite ar-
rangement, being grouped in two masses (figs. 2,4, and 8). This
holds in nearly all stages except the youngest where the chroma-
tin has the appearance of a reticulum. The chromatin in the
two masses, mentioned above, appears in the granular form.
The two masses themselves may be quite distinct, being sepa-
rated by a wide clear interval, or they may approach each other
closely. In some cases they are connected by granular threads.
This definite arrangement of the chromatin does not appear after
one fixation only, but can be observed after acetic-osmic-bichro-
mate, equal parts of 5 per cent trichloracetic acid and 5 per cent
sublimate as well as Meves’ modification of Flemming’s fluid.

The attraction-sphere. The attraction sphere is the most char-
acteristic organ of the primordial germ-cell in the chick, and,

494 CHARLES H. SWIFT

oddly enough, its importance has been overlooked by all workers
except Rubaschkin (’08) and von Berenberg-Gossler (’12). It is
present in all stages, and, from the origin of the germ-cell to its
entrance into the indifferent gonad, does not undergo any change.
It appears as a condensed or flattened sphere of cytoplasm rest-
ing usually on the nuclear membrane, on that side of the nucleus,
which is the farthest removed from the cell membrane (figs. 2,
5and9). It may rest on the nuclear membrane directly, in which
case, it has a concavity towards the nucleus, or it may be separated
from the nucleus by some distance. It has an average diameter
of 3 to 4 micra, but in some cases where it is flattened against
the nucleus, its long axis may measure 6 micra. At times it is
very discrete and seems to be surrounded by a definite mem-
brane. The cytoplasm immediately around it appears less dense
than the rest, so that the attraction-sphere seems to be situated
in a vacuole. In all stages and with all stains the sphere is
prominent and unless obscured by yolk spheres is easily seen.

The centrosomes are best seen after trichloracetic acid fixa-
tion and iron-hematoxylin staining, when they appear as black
dots. With this fixation cytoplasmic radiations may be ob-
served radiating out from the attraction sphere.

Attraction-spheres are, of course, not confined to the primor-
dial germ-cells, but in the other cells they are not nearly so large,
distinct or constant as in the germ-cells. Because of its size,
discreteness and constancy, it serves as the best criterion for
identification of germ-cells in the chick.

Yolk. Yolk material is a very characteristic constituent of
the primordial germ-cells in the chick. Its abundance in the
young stages has been recorded by Nussbaum (’01), Rubaschkin
(08) and von Berenberg-Gossler (’12). The very young germ-
cells, just after their origin, are simply loaded with the yolk which
has the form of spheres; the cytoplasm is so crowded that prac-
tically nothing but yolk can be seen (figs. 12 and 14). Most
abundant in the younger germ-cells, it gradually decreases in
amount as the embryo increases in age. In embryos having
about 20 somites and thereafter, there is a great variation in the
quantity of yolk in the germ-cells. In some embryos the germ-

ORIGIN OF PRIMORDIAL GERM CELLS 495

cells may all have no yolk, while in others, some will have yolk
while the remainder will have none at all.

In the embryos of moderate age—20 to 26 somites—there is
evidence that the yolk is undergoing digestion, as is shown by
the fact that in the same primordial germ-cell fixed with osmic
acid some of the yolk spheres will be intensely black while others
will be brown or yellowish. The yolk spheres which are not black
in these cells are frequently surrounded by a clear ring.

In the embryos of about 20 to 26 somites, the yolk spheres
reach their greatest size (fig. 7). In one case one had a diameter
of 8micra. In the younger germ-cells the yolk spheres are smaller
having an average diameter of 2 to 3 micra. In embryos with
33 somites to four and one-half days the yolk content varies a
great deal; usually there is only a small quantity present, which
is confined to 5 to 10 small spheres, which usually show signs of
digestion.

In the very young primordial germ-cells the yolk is scattered
evenly throughout the germ-cell, usually entirely surrounding the
nucleus, which may have a central rather than an eccentric po-
sition. As the germ-cell grows older the yolk diminishes in
amount and retreats to one side of the nucleus, usually the cell
pole which is occupied by the attraction-sphere (figs. 7 and 9).
As the yolk decreases still more in amount it is confined to the
region between the attraction-sphere and the cell membrane.
At this stage the yolk spheres are apt to be arranged in a horse-
shoe shaped manner, with the concavity turned towards the
attraction-sphere (fig. 6).

The yolk remains in the primordial germ-cells long after it
has disappeared from the tissue cells. The presence of the yolk
in the germ-cells is probably related to the absence of mitoses In
these cells.

Mitochondria. The mitochondria in the primordial germ-cells
of the chick are not at all characteristic. They resemble the
mitochondria of the somatic cells (figs. 5, 8 and 11), and at the
same time retain the same appearance, whether in the primitive
streak stage or four and one-half day chick. They are seen usu-
ally to be in the form of short rods of a length varying from 1 to

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 4

496 CHARLES H. SWIFT

3 micra. These red-like mitochondria do not as a rule have a
homogenous structure, but are beaded or granular rods; that is,
beads or granules closely approximated and arranged in a row or
rod. At the same time in all stages discrete granules may be
present, but they are never seen to the exclusion of the rod form
unless the fixation has been faulty.

This is at variance with the work of Rubaschkin (10) and
Tschaschin (10), who found, the former in mammals and the lat-
ter in birds, that the primordial germ-cells are characterized by
granular mitochondria, while those of the somatic cells are
rodlike in character.

Size of the primordial germ-cell, nucleus and attraction-sphere.
The size of the germ-cell varies little, if any, from its origin, to its
entrance into the indifferent. gonad. The average diameter of
nearly every one is found between the figures 14 and 18 micra.
The majority approach the larger figures.

The nucleus undergoes a little variation in size. That of the
germ-cells of the primitive streak stage to 9 somites has a diameter
of 8 to 10 micra. In germ-cells of embryos possessing more than
12 somites the nuclear diameter varies between 10 to 12 micra.

The attraction-sphere has a constant long diameter of 3 to 4
micra. This relatively constant size of the primordial germ-cell,
and of its organs, the nucleus and attraction-sphere, together
with the retention of yolk, after its disappearance from the so-
matic cells, may be taken as an evidence that the germ-cells do
not divide during their migration history.

2. Migration of the germ-cells

In investigating the history of the primordial germ-cells of
the chick it was found to be expedient to commence the study on
older embryos. The advantages of this method of study are
obvious; the older embryos are larger and therefore much easier
to handle than the younger ones, and in addition the germ-cells
have been observed down to embryos of about 22 somites and
described and studied by several able investigators—notably
Hoffman (’93), Nussbaum (’01), Rubaschkin (’07), Tschaschin
C10) and von Berenberg-Gossler (’12). Therefore, in begin-

ORIGIN OF PRIMORDIAL GERM CELLS 497

ning with these older forms, one can become familiar with the
germ-cells in regions where they have been seen and studied be-
fore, and acquire a more perfect technique, before following them
back through an unknown migration to an equally unknown and
obscure origin.

In an embryo of four and one-half days incubation the germi-
nal epithelium is found to be developed along the medial surface
of the Wolffian body. It is composed of cells which differ from
those of the general coelomic epithelium in being cuboidal or
columnar. Owing to the greater height of these cells a ridge-
like appearance is produced on the medial surface of the Wolffian
body, which may simulate a developing gonad. The gonad itself
is forming only at the level of the anterior half of the Wolffian
body, where stroma is beginning to appear beneath the germinal
epithelium. The developing mesentery, springing from between
the Wolffian bodies, has attained a length easily appreciated by
the unaided eye.

Scattered here and there in the stroma of the dee eloping gonad,
in the root of the mesentery, both in the mesenchymal tissue and
in the coelomic epithelium near the coelomic angle, and in the
germinal epithelium, clothing the gonad, are certain cells which
are sharply marked off from the neighboring cells. They are the
primordial germ-cells which have previously been described in
this article, and which have been noticed and studied at this
stage and this region by all investigators of germ-cell history in
the chick since the publication of Waldeyer’s “‘ Kierstock und Ei.”

By far the greater number of the primordial germ-cells in the
four and one-half day embryo are found in the gonad, and in the
root of the mesentery near the coelomic angle, but some are to
be seen in other situations, namely, in the mesentery at a dis-
tance from its point of attachment to the body, in the wall of
the gut, in the mesenchyme behind the aorta and in front of the
notochord and even an occasional one dorsal and lateral to the
notochord.

The germinal epithelium in the chick of four and one-half days
of incubation is well developed. It consists of a strip of cuboidal
to columnar cells, without any basement membrane, which on

498 CHARLES H. SWIFT

either side merge into the flatter cells of the general coelomic
epithelium. Scattered among the germinal epithelial cells an
ocasional primordial germ-cell is to be seen. In observing these
cells the question naturally arises as to whether they have arisen
in situ or have reached that position through a migration after
having originated elsewhere. Waldeyer (’70), it will be remem-
bered, formulated, the former idea. The researches of Hoffman
(93), Nussbaum (’01), Rubaschkin (’07), and others have cast
rather serious doubts upon the correctness of this theory. In
fact it may be said to have been disproven as regards some of
the germ-cells. The fact that typical germ-cells are found in the
root of the mesentery and in the coelomic epithelium before the
differentiation of a portion of the coelomic epithelium into ger-
minal epithelium certainly is proof enough that some of them at
least do not arise there. However, the origin of some of the
germ-cells from the germinal epithelium has never been posi-
tively dispproven. Semon (’87) in supporting Waldeyer’s (’70)
idea, claims to have seen transitions between cells of the germi-
nal epithelium and primordial germ-cells. In that case, in these
chick embryos fixed after an incubation of four and one-half days,
one would expect to see certain of the epithelial cells with the
large attraction-sphere which is so constant and characteristic
of the primordial germ-cells. Also, it would naturally be ex-
pected that some of them should possess yolk granules, and the
large vesicular nucleus and typical chromatin arrangement, while
yet possessing the moderate size and cuboidal shape, which is
typical of the epithelial cells. On the contrary, however, there
seem to be no cells present which could in any way be classed
as intermediate between the germinal epithelial cells and the
true primordial germ-cells. They are all either germ-cells or
epithelial cells, and, as far as the four and one-half day chick
is concerned, one is forced to the conclusion, either that the
germ-cells in the germinal epithelium do not arise in situ but
reach that position through tissue growth or migration, or, if any
have arisen there through a transformation, it occurred at an ear-
lier stage. All recent investigation seems to favor the former
alternative.

ORIGIN OF PRIMORDIAL GERM CELLS 499

In chick embryos possessing 31 to 33 pairs of somites, the gonad
has not commenced to develop, nor has the germinal epithelium
been differentiated, as yet, from the coelomic epithelium. At
this stage the primordial germ-cells are found in the root of the
forming mesentery, in the coelomic epithelium, in the neigh-
borhood of the coelomic angle, and, in the medial and posterior

Fig. 1 Portion of a transverse section through the twenty-second somite of a
33 somite embryo; same embryo as figure 2, for which this figure is the key-plate.
Bensley’s acetic-osmic-bichromate fixation and Bensley’s anilin acid fuchsin-
Wright’s stain. X 120. This figure is intended to give an idea as to the usual
position of the primordial germ-cells in the 33 somite chick. pr.o., primordial
germ-cells; ent., entoderm; sp.m., splanchnic plate of mesoderm; ao., aorta; c.a.
coelomic angle.

region of the splanchnic plate of mesoderm, just where it is pass-
ing into the developing mesentery (fig. 1). The vast majority
of the germ-cells are found in these regions, but an occasional
one is located elsewhere; far out in the splanchnic mesoderm, and
at times in a more dorsal situation in the loose tissue around the
aortae.

500 CHARLES H. SWIFT

The primordial germ-cells, at the stage of 31 to 33 somites, are
frequenty found in groups of three or four, and are typical germ-
cells in all respects (fig. 2). Only two facts relating to them are
worthy of special attention. The yolk material found in them is
subject to great variation and is undergoing some change, which
is evidenced by the varying reaction to the osmic acid and stain.
In some germ-cells the yolk is black, in others brown or yellow,
while in others it is stained by the dye used rather than the fixing
agent. In some cases all these changes are shown in a single
germ-cell. The yolk material is probably being used up in cell
metabolism, or being transported elsewhere.

The amoeboid appearance of the primordial germ-cells at this
stage is also worthy of mention. This property was not evident
in the germ-cells of the four and one-half day chick, owing to
the fact that active migration had ceased. However, the germ-
cells at 31 to 33 somites are highly amoeboid. Nearly all pos-
sess an oval shape, while many are drawn out into a tapering
process, which extends between the somatic cells. This process
is single and is usually on the side of the cell opposite to the
attraction-sphere.

Embryos with 29 and 26 pairs of somites may be classed to-
gether, since there is no difference worthy of mention as far as
the position of the germ-cells is concerned. In these embryos
of this age the primordial germ-cells are found in the splanchnic
mesoderm at no great distance from the point where splanchno-
pleure and somatopleure become continuous, that is, near the
coelomic angle. In the embryo possessing 29 somites 110 germ-
cells were counted. Of this number the vast majority (98) were
found to be in the splanchnic mesoderm while only 12 were
counted in the somatopleuric side of the angle of the coelom.

The primordial germ-cells in both these embryos are present
either in the mesodermal portion of the splanchnopleure or be-
tween the mesoderm and entoderm. In not a single instance is
one to be seen in the entoderm. The germ-cells are usually
found singly, although, as was mentioned before, they may oc-
cur in groups of two to four. They are all situated behind the
level of the 20th mesoblastic somite.

ORIGIN OF PRIMORDIAL GERM CELLS 501

The next embryo to be studied possesses 25 pairs of somites
and an approximate age of forty-four hours.

Down to this point the situation in which the primordial germ-
cells are found agrees with that given by all who have done any
investigation on this subject. Hoffman (93) and, in fact, all
who have worked upon the subject of primordial germ-cell his-
tory in the chick, have described the indifferent gonad and the
presence of the sex-cells in the splanchnic mesoderm. No one,

Fig. 2. Portion of a transverse section through the twenty-second somite of a
33 somite embryo; this figure is drawn from the same embryo as figurel. Bensley’s
acetic-osmic-bichromate fixation and Bensley’s anilin acid fuchsin-Wright’s
stain. X 787. This figure shows the position, form and grouping of the primor-
dial germ-cells in the 33 somite chick embryo. pr.o., primordial germ-cells; ent.,
entoderm; sp.m., splanchnic plate of mesoderm; c.a., coelomic angle.

however, has been able to trace them back to a stage earlier than
that of 22 somites. This inability to find them in younger em-
bryos is surprising but nevertheless can be explained. The stu-
dents of germ-cell history and migration in the chick have, no
doubt, had constantly in mind the work of Beard, Allen, Woods
and Jarvis, who investigated this question in forms, in which a
definite migration occurred through the entoderm or splanchnic
mesoderm. They have expected this same kind of migration to

502 CHARLES H. SWIFT

occur in the chick, and, therefore, have studied with care the en-
toderm and splanchnic mesoderm, and in so doing have neglected
the key to the situation, the blood vessels.

The embryo with 25 somites may be called a transition stage,
for the primordial germ-cells are present both in the blood ves-
sels (figs. 3 and 4) and in the splanchnic mesoderm. The embryo
with 21 somites may also be placed in this category. Were it
not for the presence of primordial germ-cells in the blood ves-
sels, this embryo with 25 somites could be passed over rapidly,
because the position of those in the tissues coincides exactly with
their situation in the older embryos. That is to say, they are
found in the splanchnic mesoderm near its junction with the
somatic mesoderm at the coelomic angle.

In the blood vessels the primordial germ-cells are found in two
situations, in the small vessels of the medial portion of the splanch-
nic mesoderm (figs. 3 and 4) caudad to the 19th somite in certain
vessels of the head (fig. 5).

The primordial germ-cells found in the latter situation—that
is, in the vessels of the head region of this 25 somite embryo
are remarkably disposed. Twelve embryos of about this age—
between 22 and 29 somites—were examined, yet in only one, that
possessing 25 somites, were the germ-cells massed. And, in-
deed, in no embryo studied was a like condition encountered.
The arrangement, then, of the germ-cells is probably abnormal,
but nevertheless, merits a description. The germ cells are pres-
ent in the radicles of the anterior cardinal veins (fig. 5). In this
region, as was mentioned before, the germ cells are not founda
singly as in the vessels of the splanchnic mesoderm, but in masses
or lumps. ‘These masses are four in number; a large group on
either side of the forebrain and two smaller groups. These col-
lections of germ-cells seem to entirely occlude the vessels in which
they are contained and remind one of emboli (fig. 5). Mixed in
with the germ-cells are the smaller blood cells (fig. 5). In one
of the large groups four of the germ-cells are dividing; this
presence of mitotic figures is worthy of notice, since in only
one primordial germ-cell (fig. 10), these excepted, was division
observed, from their origin until they pass into the gonad. To

Fig. 3 Fortion of a transverse section through the twentieth somite of a 25
somite embryo; same embryo as figure 4, for which this figure is the key-plate.
Meves’ fixation and Meves’ iron-hematoxylin stain. X 120. pro., primordial
germ-cell in small blood vessel of splanchnic mesoderm; nuc., nucleus of primor-
dial germ-cell; at.sp., attraction-sphere of primordial germ-cell; bl.c., blood cell
in blood vessel; sp.m., splanchnic layer of mesoderm; so.m., somatic layer of
mesoderm; ao., aorta; ent., entoderm; ect., ectoderm; Wd., Wolffian duct; neph.,
nephrotome.

Fig. 4 Portion of a transverse section through the twentieth somite of a 25
somite embryo; this figure is drawn from the same embryo as figure 3 and is a higher
magnification of the marked off region. Meves’ fixation and Meves’ iron-hema-
toxylin stain. X 1125. pr.o., primordial germ-cell; nuc., nucleus of germ-cell;
at.sp., attraction-sphere of germ-cell containing a centrosome; bl.c., blood-cell;
ent., er toderm; sp.m., splanchnic mesoderm.

503

)

504 CHARLES H. SWIFT

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Fig.5 Portion of a transverse section through the head region of an 2»mbryo
with 25 somites. Meves’ fixation and Meves’ iron-hematoxylin stain. 787.
pr.o., primordial germ-cell; pr.o’., primordial germ-cell dividing; at.sp., attraction-
sphere of germ-cell; bl.c., blood cell; f.b., fore-brain.

ORIGIN OF PRIMORDIAL GERM CELLS 505

repeat, then, this massing of the primordial germ-cells in the
vessels is something out of the ordinary for no evidence of it 1s
to be seen in either older or younger embryos.

The largest mass (fig. 5) In proximity to the fore brain con-
tains 35 germ-cells and extends through a dozen 4 micra sections.

In the former situation in this embryo—that is in the splanch-
nic mesoderm—there are 56 germ-cells in all. These are distri-
buted as follows—48 in the tissues and 8 in the vessels. The
cells in the blood vessels are in some cases larger than average
caliber of the vessel so that its walls are pushed out noticeably on
either side (fig. 4).

As regards the number of primordial germ-cells found in the
embryos, in which they are present in the blood vessels, it may
be said here that the figures given are only approximately cor-
rect. This applies to embryos with 25 somites, and especially to
those which are younger, for in the latter, as will be seen, the
germ-cells are found in the larger blood vessels, and the loss due
to staining and dehydrating is greater than in the case of the
older embryos, in which the primordial germ-cells are present
in the smaller vessels only.

That the large cells within the blood vessels are primordial
germ-celis, and that they pass out into the tissues, there can be
no doubt. In the first place, they are identical in appearance
with the germ-cells described in the splanchnic mesoderm of older
embryos (compare figs. 2 and 4). This similarity is best ob-
served in the embryo of 25 somites and the next younger one
studied, which possesses 21 somites. In these two embryos,
which have been called transition stages, because the primordial
germ-cells are in the vessels as well as in the tissues, in neighbor-
ing sections germ-cells may be observed in both situations (figs.
7, 8,9 and 11). Thus, it will be noted that the germ-cells in
both situations have the same size and shape (figs. 8 and 9).
The mitochondria are similar, nor is there any nuclear difference.
Cells within and without the vessels have the same amount of
yolk material and the same conspicuous attraction-sphere (figs.
8 and 9).

506 CHARLES H. SWIFT

Add to this similarity the facts (1) that, in embryos younger
than 21 somites these large characteristics cells are all in the ves-
sels; (2) that in embryos with 21 and 25 somites respectively they
are in the tissues as well as in the vessels (figs. 4, 6, 7, 8 and 10);
and (3) that, in embryos older than this they are found in the
tissues of the splanchnic mesoderm alone; so that there can be
no question as to the fact that the large, characteristic cells in
the blood vessels are primordial germ-cells and are identical with
the germ-cells found in the splanchnic mesoderm.

Fig. 6 Portion of a transverse section through the head region of a 21 somite
embryo. Meves’ fixation and Meves’ iron-hematoxylin stain. X 787. pr.o.,
primordial germ-cell near fore-brain; pr’.o’., primordial germ-cell in a blood vessel
near optic cup; vit., yolk sphere; /.b., fore-brain; 0.c., optic cup; ep., epidermis.

In the embryo with 21 mesoblastic somites there are present
altogether 73 primordial germ-cells. Of these, 13 are outside the
blood-vessels, nearly all in the splanchnic mesoderm, and 60 are
contained within vessels throughout the embryos and area vas-
culosa. Of these 60, in the vessels of the splanchnic mesoderm,
there are 30, while in vessels of the head region 25 are to be seen
(fig. 6). The distribution of the germ-cells in the splanchnic
mesoderm is of interest in that all except two or three are caudad

ORIGIN OF PRIMORDIAL GERM CELLS 507

to the 18th somite. This applies to cells both in and without the
vessels. In the splanchnic mesoderm region of this embryo two
cells are present which are partly within and partly without the
vessel (fig. 11).

In regard to the embryos with 25 and 21 somites several in-
teresting facts should be recapitulated. In both, germ-cells are
both within and without the blood vessels. In the younger em-
bryo the cells in the vessels are more numerous, while in the older
embryo this state of affairs is reversed. Also, let 1t be remem-
bered that all investigators hitherto have failed to find the germ-
cells in chick embryos with less than 22 somites.

In one series of an embryo with 19 mesoblastic somites there
are present in all 82 primordial germ-cells. The distribution of
these is markedly different from anything described hitherto. All,
with the exception of 6, are in the blood-vessels. They are found
in the large (fig. 13) as well as small channels both of the area
vasculosa and embryo proper. They are present in the heart
and aortae as well as in the capillaries. They are never found
in groups and in only one case could two be seen in a single field.

The germ-cells in this embryo as well as those still younger are
characterized by an immense amount of yolk (figs. 12 and 13).
This yolk material consists of spheres, having a diameter of 2-4
micra, and is massed principally in that portion of the cell in
which is located the attraction-sphere. However, this yolk is
not confined only to the pole of the cell occupied by the attrac-
tion-sphere, but usually surrounds the nucleus completely (fig.
12). This yolk is so abundant, that in preparations fixed with
a fluid containing osmic acid, the other cell contents are seriously
masked.

In embryos of this age the primordial germ-cells possess, in
nearly all cases, amoeboid processes.

The stages of 16, 12 and 10 somites respectively may be passed
over rapidly since they present nothing essentially different from
the 19 somite embryo as far as either the distribution or form of
the germ-cells is concerned. Thus, the latter are found in the
area vasculosa and in the developing vascular structures of the
embryo proper. In the embryo possessing 12 somites, one germ-

508 CHARLES H. SWIFT

cell in particular is found in the heart. It must have moved in
through the vessels by its own amoeboid properties, since the
heart, in an embryo of this age, has not as yet begun to propel
the blood. In fact this cell has a marked amoeboid appearance.

In embryos with 9 and 6 somites respectively the primordial
germ-cells are all anterior and antero-lateral in respect to the em-
bryo proper. Most of the germ-cells are present in the space
between germ-wall entoderm and mesoderm. The few present in
the mesodermal spaces have reached that position by migration
from the former position. Some are present in the space between
ectoderm and mesoderm; they have either passed through the
mesoderm from the space between entoderm and mesoderm or
have been left in that position by the outgrowth of mesoderm.

These germ-cells at this age have the large attraction-sphere
so often referred to, as well as the abundant yolk. The nucleus
is not so large as in older germ-cells and the chromatin does
not exist in the two definite masses. Instead it is more evenly
scattered throughout the nucleus and in some is reticular in
" appearance.

The germ-cells are very conspicuous, as, either isolated or in
groups of two or three, they lie in the space between entoderm
and mesoderm.

Fig.7 Portion of a transverse section through a 21 somite chick embryo at the
level of the 20th somite. Meves’ fixation and Meves’ iron-hematoxylin stain.
1125. pr.o., primordial germ-cell; vit., sphere of vitellus or yolk material; sp.m.,
splanchnic mesoderm; bl.v., lumen of blood vessel.

Fig. 8 Portion of a transverse section through a 21 somite chick at the level of
of the 18th somite. Meves’ fixation and Meves’ iron-hematoxylin stain. X 1125.
pr.o., primordial germ-cell; nuc., nucleus of primordial germ-cell; sp.m., splanchnic
mesoderm; bl.v., lumen of blood vessel; end., nucleus of endothelial cell.

Fig. 9 Portionof a transverse section through a2l somite chick embryo at the
level of the 21st somite. Meves’ fixation and Meves’ iron-hematoxylin stain. XX
1125. pr.o., primordial germ-cell; sp.m., splanchnic mesoderm; bl.v., lumen of
blood vessel; ent., entoderm.

Fig. 10 Portion of a transverse section through a 21 somite chickembryo at the
level of the 19th somite. Meves’ fixation and Meves’ iron-hematoxylin stain. X
1125. pr.o., primordial germ-cell; pr.’o.’, primordial germ-cell dividing; sp.m.,
splanchnic mesoderm; bl.v., lumen of blood vessel; end., nucleus of endothelial
cell; ent., entoderm.

ORIGIN OF PRIMORDIAL GERM CELLS 509

ent. ea e

—_ =e

AB.Streedain de!

Fig. 11 Portion of transverse section through a 21 somite chick embryo.
Meves’ fixation and Meves’ iron-hematoxylin stain. X 1125. pr.o., primordial
germ-cell; sp.m., splanchnic mesoderm; bl.v., lumen of blood vessel.

Fig. 12 Portion of a transverse section through a 19 somite chick embryo.
Meves’ fixation and Meves’ iron-hematoxylin stain. 1125. pr.o., primordial
germ-cell; bl.c., blood cell; bl.v., lamen of blood vessel.

510 CHARLES H. SWIFT

Fig. 13 Portion of a transverse section through a 19 somite chick embryo.
Meves’ fixation and Meves’ iron-hematoxylin stain. 1125. pr.o., primordial
germ-cell; at.sp., attraction-sphere of germ-cell; bl.c., blood cell; bl.v., lumen of
blood vessel.

3. Origin of the germ-cells

Dantschakoff (08) has described certain cells in the chick em-
bryo, which arise anterior and lateral to the primitive streak and
young embryo. They originate, she states, from the margin of
the germ-wall entoderm. They begin to appear during the prim-
itive streak stage and continue to be produced until the embryo
has 3 pairs of somites. They are large, have a large round nu-
cleus and an abundance of yolk, and are especially character-
ized by their amoeboid properties. Because of their amoeboid

ORIGIN OF PRIMORDIAL GERM CELLS oll

tendencies she called them entodermal wander-cells. They ap-
pear at first in groups, between the germ wall entoderm and ecto-
derm, anterior to the forming embryo, before mesodermal tissue
has grown out into that region. They then enter the mesoderm
and forming blood-vessels, and, either by their own amoeboid
powers, or aided by the vascular circulation penetrate into every
part of the area vasculosa and embryo. Although for some time
in the vascular system yet they are not blood cells as is evidenced
by their appearance, origin and fate. Some of these entodermal
wander-cells enter the embryonic tissues, leaving the blood-vessels
by their power of diapedesis, and degenerate. Others undergo
a like fate in the blood-vessels themselves. The remainder dis-
appear from the blood-vessels and by the time the embryo has
22 somites all have gone. Although the fate of those which do
not degenerate is unknown, yet Mlle. Dantschakoff believes that
they have no share in tissue formation.

These entodermal wander-cells of Dantschakoff are in reality
the primordial germ-cells of the chick. There is a close agreement
as far as origin is concerned, for I also find that the germ-cells
originate from certain cells of the germ-wall entoderm near its
junction with the area pellucida. I find also that they are pro-
duced during the primitive streak stage and in the embryo pos-
sessing at least 3 somites.

These germ-cells arise in a region of the germ-wall anterior to
the forming embryo and also antero-lateral. This region, just at
the junction of area pellucida and germ-wall, has roughly the
shape of a crescent, the concavity being turned towards the em-
bryo; the horns extend caudalward on either side (fig. 15). In
this crescent shaped region many of the germ-wall entodermal
cells resemble the germ-cells (fig. 14). They have the round nu-
cleus, great quantity of yolk and the large conspicuous attrac-
tion-sphere.

That these particular germ-wall entodermal cells are producing
the primordial germ-cells, is proven by the fact that in the primi-
tive streak stage, before the appearance of mesoderm anterior
to the embryo, the germ-cells are grouped in the space between
entoderm and ectoderm in this immediate neighborhood; the

THE AMERICAN JOURNAL O# ANATOMY, VOL. 15, No. 4

5 CHARLES H. SWIFT

germ-cells cytologically are very similar to the cells of the germ-
wall near its border (fig. 14); mitoses are also seen in these cells
(fig. 14).

When the mesoderm reaches this region anterior to the embryo,
which it does relatively late, the germ-cells enter the mesoderm
and the forming blood-vessels.

at. sp.

g.wW.

AG foie dain ctf!

Fig. 14 Portion of a transverse section through an embryo of primitive streak
stage development. Meves’ fixation and Meves’ iron-hematoxylin stain. > 1125.
pr.o., primordial germ-cell; a., cell of germ-wall, which resembles primordial
germ-cell; a’., cell of germ-wall dividing; at.sp., attraction-sphere of germ-wall
cell; g.w., germ-wall entoderm; ect., ectoderm.

As regards the history of the entodermal wander-cells in the
blood there is also a close agreement with that of the germ-cells.
However, a majority of the germ-cells do not degenerate in the
blood stream or in the tissues. Only a few of the germ-cells
leave the vessels before a stage of 21 somites is reached and only
a few degenerate.

ORIGIN OF PRIMORDIAL GERM CELLS IL;

The statement made by Dantschakoff, that the entodermal
wander-cells leave the blood entirely by the time the embryo has
22 somites, is of interest in view of the fact that at that stage the

Fig. 15 Surface view of a primitive streak stage of a chick embryo, viewed by
transmitted light (semi-diagrammatic). Zeiss ocular 2, Zeiss objective A3. This
figure is intended to show the point at which the primordial germ-cells arise.
The circles a, at the junction of germ wall and area pellucida, indicate this region.
a., region in which the primordial germ-cells arise; h.p., head process; pr.kn.,
primitive knot; a.op., area opaca; a.pe., area pellucida. .

germ-cells are leaving the vessels of the splanchnic mesoderm
and passing into the tissues of the splanchnic mesoderm near
the coelomic angle.

514 CHARLES H. SWIFT
SUMMARY AND CONCLUSIONS

1. The primordial germ-cells arise anterior and antero-lateral
to the embryo in a specialized region of germ-wall entoderm just
at the margin of the area pellucida. This region has roughly
the shape of a crescent and the germ-cells arise during the primi-
tive streak stage and until the embryo has about 3 somites.
The concavity of this crescent is towards the embryo and the
horns extend caudalward on either side.

2. Owing to the late appearance of mesoderm in this region,
the primordial germ-cells at first are in the space between ento-
derm and ectoderm. Later, thanks to their amoeboid power,
they enter the mesoderm and the forming blood vessels of the
mesoderm.

5. They are at first carried by their own movement, and later
by that of the blood to all parts of the embryo and vascular area.
They remain generally distributed in this way until the embryo
has about 20 somites.

4. In embryos with about 20 to 22 pairs of somites, the primor-
dial germ-cells, while generally distributed throughout the em-
bryo in the blood-vessels, are becoming relatively more numerous
in the vessels of the splanchnic mesoderm. This increase in the
number of the germ-cells in the vessels of the splanchic mesoderm
may be in part only apparent, that is, a degeneration of some
may have occurred elsewhere, or, it may be a real increase due
to some influence, probably of a chemotactic nature, exerted in
the region of the future gonad. At this period the great major-
ity of the cells are found in the vessels, but a few, chiefly in the
splanchnic mesoderm, are present in the tissues. In some cases
they are present in the wall of the vessel, as if fixed in the act
of leaving the vessel for the tissues (fig. 11).

5. In embryos with about 23 to 25 pairs of somites the major-
ity of the primordial germ-cells are found in the mesodermal tis-
sue of the splanchnic mesoderm near the angle of the coelom.
A few cells are still present in the blood-vessels; at the 25th somite
stage, in one embryo, in addition to the few found in the vessels
of the splanchnic mesoderm, four masses of germ-cells are in the

ORIGIN OF PRIMORDIAL GERM CELLS als

vessels of the head. This is an abnormal condition, since the
germ-cells in all the other embryos studied, are never grouped
in this way. The embryo with 25 somites is the oldest in which
germ-cells are found in the vessels.

The embryo with about 22 to 23 segments is the youngest in
which the primordial germ-cells have hitherto been described in
the bird. Nussbaum (’80) and Keibel and Abraham (’00) in
the chick, and Hoffmann (’93) in Gallinula, Sterna and Haema-
topus described the germ-cells at 23 somites in the splanchno-
pleure.

6. In embryos possessing about 26 to 29 somites the primor-
dial germ-cells are found in the splanchnic mesoderm near the
radix mesenteri.

7. In embryos with 30 to 33 somites the primordial germ-cells
are in the radix mesenterii and coelomic epithelium on both sides
of the coelomic angle. They remain in this position until the
formation of the gonad begins when they gradually pass into
that organ.

BIBLIOGRAPHY

ALLEN, B. M. 1905 The origin of the sex-cells of Chrysemis. Anat. Anz., Bd.
29.
1909 The origin of the sex-cells of Amia and Lepidosteus. Anat. Rec.,
vol. 3.

Bearp, J. 1904 The germ-cells. Part1. Journ. Anat. and Phys., vol. 38.

Benstey, R. R. 1911 The pancreas of the guinea-pig. Amer. Jour. Anat., vol.
12, pp. 297-388.

VON BERENBERG-GOSSLER 1912 Die Urgeschlechtszellen des Hiihnerembryos
aus 3 and 4 Bebriitungstage. Arch. Mik. Anat., Bd. 81.

Baxrrant, E.G. 1885 Contribution al’etude de la formation des organes sexuels
chez les insectes. Recueil Zoologique, Suisse.

DantscHakorr, W. 1908 Entwicklung des Blutes bei den Végeln. Anat. Hefte,
Bd. 37, 8. 471.

d’Hotuanper, F. 1904 Recherches sur l’oogenese et le noyau vitellin de Bal-
biani chez les oiseaux. Arch. d’Anatomie Mic., T. 7.

Dopps, G. 8. 1910 Segregation of the germ-cells of the teleost Lophius. Jour.
Morph. vol. 21, p. 563.

EIGENMANN, C. H. 1892 On the precocious segregation of the sex-cells in Cyma-
togaster. Journ. Morph., no. 5, p. 481.
1897 Sex differentiation in the viviparous teleost Cymatogaster. Arch.
Arch. f. Entw’mech., Bd. 4, p. 125.

516 CHARLES H. SWIFT

Fevix, W. 1906 Die Entwicklung der Keimdriisen and ihrer Ausfiihrungsgange.
Hand. d. Entw. von O. Hertwig.

Fuss, A. Uber die Geschlechtszellen des Menschen und der Siugetiere. Arch. fiir
Mikros. Anat., Bd. 81, Heft. 1.

1911 Uber Extraregionare Geschlechtszellen bei einen Menschlichen
Embryo von vier Wochen. Anat. Anz., Bd. 39.

Haecker, V. 1897 Die Keimbahn von Cyclops. Arch. f. Mik. Anat., Bd. 49.

Hecner, R. W. 1909 The origin and early history of the germ-cells in some
chrysomelid beetles. Jour. Morph., no. 20.

HorrMann, C.K. 1893 Etude sur le developpement de l’appareil urogenital des
oiseaux. Verhand. der Koninklyte Akademie von Wetenschoppen,
Amsterdam, Tweedie Sectie, vol. 1.

Jarvis, Miss 1908 The segregation of the germ-cells of Phrynosoma cornutum:
Preliminary note. Biol. Bulletin, vol. 15, p. 119.

Nusssaum, M. 1901 Zur Entwicklung der Geschlechts beim Huhn. Anat. Anz.

RupascukKin, W. 1907a Uber das erste Auftreten und Migration der Keimzellen
bei Végelembryonen. Anat. Hefte., Bd. 39.

1907 Zur Frage von der Entstehung der Keimzellen bei Siugetierem-
bryonen. Anat. Anz., Bd. 31.

1910 Chondriosomen und Differenzierungsprozesse bei Siugetierem-
bryonen. Anat. Hefte, Bd. 41.

Semon, R. 1887 Die indifferente Anlage der Keimdriisen beim Hiihnchen und
ihre Differenzierung zum Hoden. Jena Zeitschr. Naturwiss., Bd. 21.

Tscuascurn, 8S. 1910 Uber die Chondriosomen der Urgeschlechtszellen bei Vogel-

‘embryonen. Anat. Anz., vol. 37.

Waupeyer, W. 1870 Eierstock und Ei. Leipzig, Englemann.

Woops, F.A. 1902 Origin and migration of the germ-cells in Acanthias. Amer.
JounsAnate. volsL, p. 307.

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